US20090176217A1 - Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis - Google Patents

Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis Download PDF

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US20090176217A1
US20090176217A1 US12/089,051 US8905106A US2009176217A1 US 20090176217 A1 US20090176217 A1 US 20090176217A1 US 8905106 A US8905106 A US 8905106A US 2009176217 A1 US2009176217 A1 US 2009176217A1
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seq id
amino acid
acid sequence
preferably
gt
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US12/089,051
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Osnat Sella-Tavor
Sarah Pollock
Gad S. Cojocaru
Amit Novik
Lily Bazak
Elena Tsypkin
Shira Wallach
Shirley Sameah-Greenwald
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Compugen Ltd
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Compugen Ltd
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Priority to US72240005P priority Critical
Priority to US73582505P priority
Priority to IL172297A priority patent/IL172297A/en
Priority to IL172297 priority
Priority to US74292905P priority
Application filed by Compugen Ltd filed Critical Compugen Ltd
Priority to PCT/IL2006/001154 priority patent/WO2007039903A2/en
Priority to US12/089,051 priority patent/US20090176217A1/en
Assigned to COMPUGEN LTD reassignment COMPUGEN LTD ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: POLLOCK, SARAH, SAMEAH-GREENWALD, SHIRLEY, SELLA-TAVOR, OSNAT, BAZAK, LILY, COJOCARU, GAD S., NOVIK, AMIT, TSYPKIN, ELENA, WALLACH, SHIRA
Publication of US20090176217A1 publication Critical patent/US20090176217A1/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/52Predicting or monitoring the response to treatment; Prognosis

Abstract

Novel splice variants, amino acid sequences and nucleotide sequences thereof, and methods of using same.

Description

    FIELD OF THE INVENTION
  • The present invention is related to novel nucleotide and protein sequences, and assays and methods of use thereof.
  • BACKGROUND OF THE INVENTION
  • Diagnostic markers are important for early diagnosis of many diseases, as well as predicting response to treatment, monitoring treatment and determining prognosis of such diseases.
  • Serum markers are examples of such diagnostic markers and are used for diagnosis of many different diseases. Such serum markers typically encompass secreted proteins and/or peptides; however, some serum markers may be released to the blood upon tissue lysis, such as from myocardial infarction (for example Troponin-I). Serum markers can also be used as risk factors for disease (for example base-line levels of CRP, as a predictor of cardiovascular disease), to monitor disease activity and progression (for example, determination of CRP levels to monitor acute phase inflammatory response) and to predict and monitor drug response (for example, as shedded fragments of the protein Erb-B2).
  • Immunohistochemistry (IHC) is the study of distribution of an antigen of choice in a sample based on specific antibody-antigen binding, typically on tissue slices. The antibody features a label which can be detected, for example as a stain which is detectable under a microscope. The tissue slices are prepared by being fixed. IHC is therefore particularly suitable for antibody-antigen reactions that are not disturbed or destroyed by the process of tissue fixation.
  • 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:
  • 1) the histological type of the tissue sample
    2) the presence of specific cell types within the sample
    3) information on the physiological and/or pathological state of cells (e.g. which phase of the cell-cycle they are in)
    4) the presence of disease related changes within the sample
    5) differentiation between different specific disease subtypes where it is already known the tissue is of disease state (for example, the differentiation between different tumor types when it is already known the sample was taken from cancerous tissue).
  • 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 only an estimation (on a scale of 1-4) of how prevalent the antigen on target is.
  • SUMMARY OF THE INVENTION
  • The present invention provides, in different embodiments, many novel amino acid and nucleic acid sequences, which may optionally be used as diagnostic markers.
  • In some embodiments, the present invention provides a number of different variants of known proteins, which are expressed in serum and may optionally be used as diagnostic markers, which in some embodiments, are serum markers, or in other embodiments, are 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. In one embodiment, tissue specific markers are identifible in serum or plasma. In some embodiments, a simple blood test can provide qualitative and/or quantitative indicators for expression of a desired marker, for example, serving as an indicator for various diseases and/or pathological conditions. The markers presented in the present invention can also potentially be used for in-vivo imaging applications.
  • The present invention also provides, in some embodiments, a number of different variants, which serve as IHC markers or indicators, which in some embodiments, serve as diagnostic markers, for example as serum markers, or 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. For example, serum markers require only a simple blood test and their result is typically a scientifically measured number. As IHC markers, 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 as diagnostic markers are described in greater detail below. The variants themselves are described by “cluster” or by gene, as these variants are splice variants of known proteins. In some embodiments, the term “marker-detectable disease” refers to a disease that may be detected by a particular marker, with regard to the description of such diseases below. In some embodiments, 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 relates, in some embodiments, to diagnostic assays for disease detection, which in some embodiments, utilizes a biological sample taken from a subject (patient), which for example may comprise a 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, secretions of the breast ductal system (and/or lavage thereof), broncho alveolar lavage, lavage of the reproductive system, lavage of any other part of the body or system in the body, stool or a tissue sample, or any combination thereof. In some embodiments, the term encompasses 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.
  • In some embodiments of the present invention, nucleic acids, or polypeptides, having a sequence as described herein, or homologues thereof.
  • In some embodiments, the terms “homology”, “homologue” or “homologous”, in any instance, indicate that the sequence referred to, whether an amino acid sequence, or a nucleic acid sequence, exhibits, in one embodiment at least 70% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 72% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 75% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 77% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 80% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 82% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 85% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 87% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 90% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 92% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 95% or more correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits 95%-100% correspondence to the indicated sequence. Similarly, in some embodiments, the reference to a correspondence to a particular sequence includes both direct correspondence, as well as homology to that sequence as herein defined.
  • In some embodiments, this invention provides an isolated polynucleotide comprising a nucleic acid having a sequence corresponding to, or homologous to that set forth in SEQ ID NOs: 1-15, 61-64, 96-98, 114-126, 189-195, 211-214, 235, 244, 152-253, 305-306, 340-344.
  • In some embodiments, this invention provides an isolated polynucleotide comprising a nucleic acid having a sequence corresponding to, or homologous to that set forth in SEQ ID NOs: 32-60, 71-95, 103-113, 139-188, 196-219, 220-234, 237-243, 246-251, 256-304, 309-339, 350-358, 502-530.
  • In some embodiments, this invention provides an isolated protein or polypeptide having an amino acid sequence corresponding to, or homologous to that set forth in SEQ ID NOs:16-31, 65-70, 99-102, 127-138, 215-219, 236, 245, 254-255, 307-308.
  • In some embodiments, the proteins or polypeptides of this invention comprise chimeric protein or polypeptides.
  • In some embodiments, the terms “chimeric protein or polypeptide”, or “chimera” refers to an assembly or a string of amino acids in a particular sequence, or nucleotides encoding the same, respectively, which does not correspond to the sequence of the known (wild type) polypeptide or protein, or nucleic acid, respectively. In some embodiments, the variants of this invention are derived by the by the assembly or stringing of amino acids or polynucleotides encoding the same, from two exons, or an exon and an intron, or fragments thereof, or segments having sequences with the indicated homology.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that set forth in SEQ ID NO:16 (HSFLT_P6).
  • In some embodiments, such isolated chimeric proteins or polypeptides may comprise an amino acid sequence corresponding to or homologous to that set forth in HSFLT_P6 (SEQ ID NO:16), 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 MTAP (SEQ ID NO: 459) corresponding to amino acids 1-4 of HSFLT_P6 (SEQ ID NO:16), and a second amino acid sequence being at least 90% homologous to amino acids 172-1338 of VGR1_HUMAN_V1 (SEQ ID NO: 575), which also corresponds to amino acids 5-1171 of HSFLT_P6 (SEQ ID NO:16), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide comprising a head portion of an HSFLT_P6 (SEQ ID NO:16), 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 MTAP (SEQ ID NO: 459) of HSFLT_P6 (SEQ ID NO:16).
  • In some embodiments, such isolated chimeric proteins or polypeptides may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P6 (SEQ ID NO:16), 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 MTAP (SEQ ID NO: 459) corresponding to amino acids 1-4 of HSFLT_P6 (SEQ ID NO:16), a second amino acid sequence being at least 90% homologous to amino acids 172-656 of P17948-2 (SEQ ID NO:360), which also corresponds to amino acids 5-489 of HSFLT_P6 (SEQ ID NO:16), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGIILGPGSSTLFIERVTEEDEGVYHCK ATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDE QCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAVKMLKEGATASEYKALMTELK ILTMGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQG KKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNI LLSENNVVKICDFGLARDIYKNDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPG VQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAI LTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLL ASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPP DYNSVVLYSTPPI (SEQ ID NO: 460) corresponding to amino acids 490-1171 of HSFLT_P6 (SEQ ID NO:16), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HSFLT_P6 (SEQ ID NO:16), 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 MTAP (SEQ ID NO: 459) of HSFLT_P6 (SEQ ID NO:16).
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P6 (SEQ ID NO:16), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTILDCHANGVPEPQITWFKNKIQQEPGIILGPGSSTLFIERVTEEDEGVYHCK ATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDE QCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAVKMLKEGATASEYKALMTELK ILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQG KKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNI LLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPG VQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAI LTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLL ASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPP DYNSVVLYSTPPI (SEQ ID NO: 460) of HSFLT_P6 (SEQ ID NO:16).
  • In some embodiments, such isolated chimeric proteins or polypeptides may comprise an amino acid sequence corresponding to or homologous to that set forth in HSFLT_P7 (SEQ ID NO:17).
  • In some embodiments, this invention provides an isolated chimeric polypeptide as set forth in HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) corresponding to amino acids 1-6 of HSFLT_P7 (SEQ ID NO:17), and a second amino acid sequence being at least 90% homologous to amino acids 172-1338 of VGR1_HUMAN_V1 (SEQ ID NO: 575), which also corresponds to amino acids 7-1173 of HSFLT_P7 (SEQ ID NO:17), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) of HSFLT_P7 (SEQ ID NO:17).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that, as set forth in HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) corresponding to amino acids 1-6 of HSFLT_P7 (SEQ ID NO:17), and a second amino acid sequence being at least 90% homologous to amino acids 172-1338 of NP002010_V1 (SEQ ID NO: 574), which also corresponds to amino acids 7-1173 of HSFLT_P7 (SEQ ID NO:17), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) of HSFLT_P7 (SEQ ID NO:17).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) corresponding to amino acids 1-6 of HSFLT_P7 (SEQ ID NO:17), a second amino acid sequence being at least 90% homologous to amino acids 172-656 of P 7948-2 (SEQ ID NO:360), which also corresponds to amino acids 7-491 of HSFLT_P7 (SEQ ID NO:17), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGIILGPGSSTLFIERVTEEDEGVYHCK ATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDE QCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAVKMLKEGATASEYKALMTELK ILTUIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKKEPGLEQG KKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEFLSSRKCMIRDLAARNI LLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPG VQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAI LTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLL ASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPP DYNSVVLYSTPPI (SEQ ID NO: 460) corresponding to amino acids 492-1173 of HSFLT_P7 (SEQ ID NO:17), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) of HSFLT_P7 (SEQ ID NO:17).
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P7 (SEQ ID NO:17), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNIQQEPGIILGPGSSTLFIERVTEEDEGVYHCK ATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDE QCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAVKMLKEGATASEYKALMTELK ILTMIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQG KKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNI LLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPG VQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAI LTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLL ASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPP DYNSVVLYSTPPI (SEQ ID NO: 460) of HSFLT_P7 (SEQ ID NO:17).
  • In some embodiments, such isolated chimeric proteins or polypeptides may comprise an amino acid sequence corresponding to or homologous to that set forth in HSFLT_P10 (SEQ ID NO:18).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P10 (SEQ ID NO:18), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-705 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-705 of HSFLT_P10 (SEQ ID NO:18), 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 ELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 462) corresponding to amino acids 706-733 of HSFLT_P10 (SEQ ID NO:18), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P10 (SEQ ID NO:18), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 462) of HSFLT_P10 (SEQ ID NO:18).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P10 (SEQ ID NO:18), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-656 of P17948-2 (SEQ ID NO:360), which also corresponds to amino acids 1-656 of HSFLT_P10 (SEQ ID NO:18), 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 DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 463) corresponding to amino acids 657-733 of HSFLT_P10 (SEQ ID NO:18), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P10 (SEQ ID NO:18), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 463) of HSFLT_P10 (SEQ ID NO:18).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P10 (SEQ ID NO:18), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-705 of NP002010 (SEQ ID NO: 531), which also corresponds to amino acids 1-705 of HSFLT_P10 (SEQ ID NO:18), 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 ELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 462) corresponding to amino acids 706-733 of HSFLT_P10 (SEQ ID NO:18), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, such isolated chimeric proteins or polypeptides may comprise an amino acid sequence corresponding to or homologous to that set forth in HSFLT_P11 (SEQ ID NO:19).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P11 (SEQ ID NO:19), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-706 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-706 of HSFLT_P11 (SEQ ID NO:19), 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 SANTAVNKKTEI (SEQ ID NO: 464) corresponding to amino acids 707-718 of HSFLT_P11 (SEQ ID NO:19), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P11 (SEQ ID NO:19), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SANTAVNKKTEI (SEQ ID NO: 464) of HSFLT_P11 (SEQ ID NO:19).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P11 (SEQ ID NO:19), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-656 of P17948-2 (SEQ ID NO:360), which also corresponds to amino acids 1-656 of HSFLT_P111 (SEQ ID NO:19), 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 DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGSANTAVNKKTEI (SEQ ID NO: 465) corresponding to amino acids 657-718 of HSFLT_P11 (SEQ ID NO:19), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P11 (SEQ ID NO:19), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGSANTAVNKKTEI (SEQ ID NO: 465) of HSFLT_P11 (SEQ ID NO:19).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P11 (SEQ ID NO:19), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-706 of NP002010 (SEQ ID NO: 531), which also corresponds to amino acids 1-706 of HSFLT_P11 (SEQ ID NO:19), 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 SANTAVNKKTEI (SEQ ID NO: 464) corresponding to amino acids 707-718 of HSFLT_P11 (SEQ ID NO:19), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, such isolated chimeric proteins or polypeptides may comprise an amino acid sequence corresponding to or homologous to that set forth in HSFLT_P13 (SEQ ID NO:20).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P13 (SEQ ID NO:20), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-706 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-706 of HSFLT_P13 (SEQ ID NO:20), 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 KRLFFLPFIISHLSSAPLSLNSPVTCFQYV (SEQ ID NO: 466) corresponding to amino acids 707-736 of HSFLT_P13 (SEQ ID NO:20), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P13 (SEQ ID NO:20), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence KRLFFLPFIISHLSSAPLSLNSPVTCFQYV (SEQ ID NO: 466) of HSFLT_P13 (SEQ ID NO:20).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P13 (SEQ ID NO:20), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-656 of P17948-2 (SEQ ID NO:360), which also corresponds to amino acids 1-656 of HSFLT_P13 (SEQ ID NO:20), 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 DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGKRLFFLPFIISHLSSAPLSLNSPVTCFQYV (SEQ ID NO: 467) corresponding to amino acids 657-736 of HSFLT_P13 (SEQ ID NO:20), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P13 (SEQ ID NO:20), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGKRLFFLPFIISHLSSAPLSLNSPVTCFQYV (SEQ ID NO: 467) of HSFLT_P13 (SEQ ID NO:20).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P13 (SEQ ID NO:20), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-706 of NP002010 (SEQ ID NO: 531), which also corresponds to amino acids 1-706 of HSFLT_P13 (SEQ ID NO:20), 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 KRLFFLPFIISHLSSAPLSLNSPVTCFQYV (SEQ ID NO: 466) corresponding to amino acids 707-736 of HSFLT_P13 (SEQ ID NO:20), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P14 (SEQ ID NO:21), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-517 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-517 of HSFLT_P14 (SEQ ID NO:21), 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 YLDIRTEEQIFSFIQKTQTLKLTVSCKAAF (SEQ ID NO: 468) corresponding to amino acids 518-547 of HSFLT_P14 (SEQ ID NO:21), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P14 (SEQ ID NO:21), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence YLDIRTEEQIFSFIQKTQTLKLTVSCKAAF (SEQ ID NO: 468) of HSFLT_P14 (SEQ ID NO:21).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P15 (SEQ ID NO:22), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-329 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-329 of HSFLT_P15 (SEQ ID NO:22), 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 GKHSSALPTHAMLSNHCRCLCSLNKSVFCWPRVTLS (SEQ ID NO: 469) corresponding to amino acids 330-365 of HSFLT_P15 (SEQ ID NO:22), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P15 (SEQ ID NO:22), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GKHSSALPTHAMLSNTICRCLCSLNKSVFCWPRVTLS (SEQ ID NO: 469) of HSFLT_P15 (SEQ ID NO:22).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P16 (SEQ ID NO:23), comprising an amino acid sequence being at least 90% homologous to amino acids 906-1338 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-433 of HSFLT_P16 (SEQ ID NO:23).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P16 (SEQ ID NO:23), comprising an amino acid sequence being at least 90% homologous to amino acids 906-1338 of NP002010 (SEQ ID NO: 531), which also corresponds to amino acids 1-433 of HSFLT_P16 (SEQ ID NO:23).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P17 (SEQ ID NO:24), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-171 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-171 of HSFLT_P17 (SEQ ID NO:24), 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 VNLNTAILSILSLQISIMKFYSFYLSGIISLQTPGLLSGLSCN (SEQ ID NO: 470) corresponding to amino acids 172-214 of HSFLT_P17 (SEQ ID NO:24), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P17 (SEQ ID NO:24), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VNLNTAILSILSLQISIMKFYSFYLSGIISLQTPGLLSGLSCN (SEQ ID NO: 470) of HSFLT_P17 (SEQ ID NO:24).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P18 (SEQ ID NO:25), comprising an amino acid sequence being at least 90% homologous to amino acids 996-1338 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-343 of HSFLT_P18 (SEQ ID NO:25).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P18 (SEQ ID NO:25), comprising an amino acid sequence being at least 90% homologous to amino acids 996-1338 of NP002010 (SEQ ID NO: 531), which also corresponds to amino acids 1-343 of HSFLT_P18 (SEQ ID NO:25).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P19 (SEQ ID NO:26), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-129 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-129 of HSFLT_P19 (SEQ ID NO:26), 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 GKTSIFYILFAFALQMSHKSTLIHKGCFPSEYERNGLGKRFHPSCRHFRGCQF (SEQ ID NO: 471) corresponding to amino acids 130-183 of HSFLT_P19 (SEQ ID NO:26), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides isolated polypeptide encoding for an edge portion of HSFLT_P19 (SEQ ID NO:26), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GKTSIFYILFAFALQMSHKSTLIHWKGCFPSEYERNGLGKRFHPSCRHFRGCQF (SEQ ID NO: 471) of HSFLT_P19 (SEQ ID NO:26).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P20 (SEQ ID NO:27), comprising an amino acid sequence being at least 90% homologous to amino acids 1133-1338 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-206 of HSFLT_P20 (SEQ ID NO:27).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P21 (SEQ ID NO:28), comprising an amino acid sequence being at least 90% homologous to amino acids 1220-1338 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-119 of HSFLT_P21 (SEQ ID NO:28).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P41 (SEQ ID NO:29), 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 LWAACPAQACSGNAGQERGGLQSAAGLPSQPSCFLQTGVGLANQ (SEQ ID NO: 577) corresponding to amino acids 1-44 of HSFLT_P41 (SEQ ID NO:29), and a second amino acid sequence being at least 90% homologous to amino acids 903-1338 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 45-480 of HSFLT_P41 (SEQ ID NO:29), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides isolated polypeptide encoding for a head of HSFLT_P41 (SEQ ID NO:29), 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 LWAACPAQACSGNAGQERGGLQSAAGLPSQPSCFLQTGVGLANQ (SEQ ID NO: 577) of HSFLT_P41 (SEQ ID NO:29).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P48 (SEQ ID NO:30), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-517 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-517 of HSFLT_P48 (SEQ ID NO:30), 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 LPPANSSFMLPPTSFSSNYFHFLP (SEQ ID NO: 472) corresponding to amino acids 518-541 of HSFLT_P48 (SEQ ID NO:30), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides isolated polypeptide encoding for an edge portion of HSFLT_P48 (SEQ ID NO:30), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LPPANSSFMLPPTSFSSNYFHFLP (SEQ ID NO: 472) of HSFLT_P48 (SEQ ID NO:30).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSFLT_P49 (SEQ ID NO:31), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-553 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-553 of HSFLT_P49 (SEQ ID NO:31), 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 ELSNFECLHPCSQE (SEQ ID NO: 473) corresponding to amino acids 554-567 of HSFLT_P49 (SEQ ID NO:31), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSFLT_P49 (SEQ ID NO:31), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ELSNFECLHPCSQE (SEQ ID NO: 473) of HSFLT_P49 (SEQ ID NO:31).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P5 (SEQ ID NO:65), comprising a first amino acid sequence being at least 90% homologous to amino acids 35-68 of IL1X_HUMAN (SEQ ID NO: 372), which also corresponds to amino acids 1-34 of HSI1RA_P5 (SEQ ID NO:65), 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 GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 35-64 of HSI1RA_P5 (SEQ ID NO:65), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSI1RA_P5 (SEQ ID NO:65), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) of HSI1RA_P5 (SEQ ID NO:65).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P5 (SEQ ID NO:65), comprising a first amino acid sequence being at least 90% homologous to amino acids 17-50 of NP000568 (SEQ ID NO: 532), which also corresponds to amino acids 1-34 of HSI1RA_P5 (SEQ ID NO:65), 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 GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 35-64 of HSI1RA_P5 (SEQ ID NO:65), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P5 (SEQ ID NO:65), comprising a first amino acid sequence being at least 90% homologous to amino acids 35-68 of NP776214 (SEQ ID NO: 534), which also corresponds to amino acids 1-34 of HSI1RA_P5 (SEQ ID NO:65), 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 GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 35-64 of HSI1RA_P5 (SEQ ID NO:65), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P5 (SEQ ID NO:65), comprising a first amino acid sequence being at least 90% homologous to amino acids 38-71 of NP776213 (SEQ ID NO: 533), which also corresponds to amino acids 1-34 of HSI1RA_P5 (SEQ ID NO:65), 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 GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 35-64 of HSI1RA_P5 (SEQ ID NO:65), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P6 (SEQ ID NO:66), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-68 of IL1X_HUMAN (SEQ ID NO: 372), which also corresponds to amino acids 1-68 of HSI1RA_P6 (SEQ ID NO:66), 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 GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 69-98 of HSI1RA_P6 (SEQ ID NO:66), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P6 (SEQ ID NO:66), 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 MEICRGLRSHLITLLLFLFHS (SEQ ID NO: 578) corresponding to amino acids 1-21 of HSI1RA_P6 (SEQ ID NO:66), a second amino acid sequence being at least 90% homologous to amino acids 4-50 of P18510-2 (SEQ ID NO:373), which also corresponds to amino acids 22-68 of HSI1RA_P6 (SEQ ID NO:66), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 69-98 of HSI1RA_P6 (SEQ ID NO:66), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • An isolated polypeptide encoding for a head of HSI1RA_P6 (SEQ ID NO:66), 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 MEICRGLRSHLITLLLFLFHS (SEQ ID NO: 578) of HSI1RA_P6 (SEQ ID NO:66).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P6 (SEQ ID NO:66), 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 MEICRGLRSHLITLLLFLFHSETICRPSGRKSSK (SEQ ID NO: 579) corresponding to amino acids 1-34 of HSI1RA_P6 (SEQ ID NO:66), a second amino acid sequence being at least 90% homologous to amino acids 1-34 of P18510-4 (SEQ ID NO:375), which also corresponds to amino acids 35-68 of HSI1RA_P6 (SEQ ID NO:66), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 69-98 of HSI1RA_P6 (SEQ ID NO:66), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • An isolated polypeptide encoding for a head of HSI1RA_P6 (SEQ ID NO:66), 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 MEICRGLRSHLITLLLFLFHSETICRPSGRKSSK (SEQ ID NO: 579) of HSI1RA_P6 (SEQ ID NO:66).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P13 (SEQ ID NO:67), 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 MAL (SEQ ID NO: 580) corresponding to amino acids 1-3 of HSI1RA_P13 (SEQ ID NO:67), a second amino acid sequence being at least 90% homologous to amino acids 22-68 of IL1X_HUMAN (SEQ ID NO: 372), which also corresponds to amino acids 4-50 of HSI1RA_P13 (SEQ ID NO:67), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 51-80 of HSI1RA_P13 (SEQ ID NO:67), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P13 (SEQ ID NO:67), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-50 of NP000568 (SEQ ID NO: 532), which also corresponds to amino acids 1-50 of HSI1RA_P13 (SEQ ID NO:67), 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 GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 51-80 of HSI1RA_P13 (SEQ ID NO:67), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P13 (SEQ ID NO:67), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-3 of P18510-3 (SEQ ID NO:374), which also corresponds to amino acids 1-3 of HSI1RA_P13 (SEQ ID NO:67), a second amino acid sequence being at least 90% homologous to amino acids 25-71 of P18510-3 (SEQ ID NO:374), which also corresponds to amino acids 4-50 of HSI1RA_P13 (SEQ ID NO:67), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 51-80 of HSI1RA_P13 (SEQ ID NO:67), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated chimeric polypeptide encoding for an edge portion of HSI1RA_P13 (SEQ ID NO:67), 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 LE, having a structure as follows: a sequence starting from any of amino acid numbers 3−x to 3; and ending at any of amino acid numbers 4+((n−2)−x), in which x varies from 0 to n−2.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P13 (SEQ ID NO:67), 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 MAL (SEQ ID NO: 580) corresponding to amino acids 1-3 of HSI1RA_P13 (SEQ ID NO:67), a second amino acid sequence being at least 90% homologous to ETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE corresponding to amino acids 22-68 of NP776214 (SEQ ID NO: 534), which also corresponds to amino acids 4-50 of HSI1RA_P13 (SEQ ID NO:67), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 51-80 of HSI1RA_P13 (SEQ ID NO:67), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P13 (SEQ ID NO:67), 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 MALETICRPSGRKSSK (SEQ ID NO: 581) corresponding to amino acids 1-16 of HSI1RA_P13 (SEQ ID NO:67), a second amino acid sequence being at least 90% homologous to amino acids 1-34 of P18510-4 (SEQ ID NO:375), which also corresponds to amino acids 17-50 of HSI1RA_P13 (SEQ ID NO:67), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GEWLPGKPMYVGITSLCPSVCSSMACLHKP (SEQ ID NO: 474) corresponding to amino acids 51-80 of HSI1RA_P13 (SEQ ID NO:67), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • An isolated polypeptide encoding for a head of HSI1RA_P13 (SEQ ID NO:67), 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 MALETICRPSGRKSSK (SEQ ID NO: 581) of HSI1RA_P13 (SEQ ID NO:67).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P14 (SEQ ID NO:68), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-24 of NP776213 (SEQ ID NO: 533), which also corresponds to amino acids 1-24 of HSI1RA_P14 (SEQ ID NO:68), 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 GGL (SEQ ID NO: 477) corresponding to amino acids 25-27 of HSI1RA_P14 (SEQ ID NO:68), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSI1RA_P14 (SEQ ID NO:68), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GGL (SEQ ID NO: 477) of HSI1RA_P14 (SEQ ID NO:68).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P16 (SEQ ID NO:69), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-68 of IL1X_HUMAN (SEQ ID NO: 372), which also corresponds to amino acids 1-68 of HSI1RA_P16 (SEQ ID NO:69), 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 DRCGTH (SEQ ID NO: 478) corresponding to amino acids 69-74 of HSI1RA_P16 (SEQ ID NO:69), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSI1RA_P16 (SEQ ID NO:69), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DRCGTH (SEQ ID NO: 478) of HSI1RA_P16 (SEQ ID NO:69).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P16 (SEQ ID NO:69), 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 MEICRGLRSHLITLLLFLFHS (SEQ ID NO: 578) corresponding to amino acids 1-21 of HSI1RA_P16 (SEQ ID NO:69), a second amino acid sequence being at least 90% homologous to amino acids 4-50 of P18510-2 (SEQ ID NO:373), which also corresponds to amino acids 22-68 of HSI1RA_P16 (SEQ ID NO:69), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DRCGTH (SEQ ID NO: 478) corresponding to amino acids 69-74 of HSI1RA_P16 (SEQ ID NO:69), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • An isolated polypeptide encoding for a head of HSI1RA_P16 (SEQ ID NO:69), 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 MEICRGLRSHLITLLLFLFHS (SEQ ID NO: 578) of HSI1RA_P16 (SEQ ID NO:69).
  • An isolated chimeric polypeptide as set forth in HSI1RA_P16 (SEQ ID NO:69), 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 MEICRGLRSHLITLLLFLFHS (SEQ ID NO: 578) corresponding to amino acids 1-21 of HSI1RA_P16 (SEQ ID NO:69), a second amino acid sequence being at least 90% homologous to amino acids 25-71 of NP776213 (SEQ ID NO: 533), which also corresponds to amino acids 22-68 of HSI1RA_P16 (SEQ ID NO:69), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DRCGTH (SEQ ID NO: 478) corresponding to amino acids 69-74 of HSI1RA_P16 (SEQ ID NO:69), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P16 (SEQ ID NO:69), 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 MEICRGLRSHLITLLLFLFHSETICRPSGRKSSK (SEQ ID NO: 579) corresponding to amino acids 1-34 of HSI1RA_P16 (SEQ ID NO:69), a second amino acid sequence being at least 90% homologous to amino acids 1-34 of P18510-4 (SEQ ID NO:375), which also corresponds to amino acids 35-68 of HSI1RA_P16 (SEQ ID NO:69), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DRCGTH (SEQ ID NO: 478) corresponding to amino acids 69-74 of HSI1RA_P16 (SEQ ID NO:69), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HSI1RA_P16 (SEQ ID NO:69), 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 MEICRGLRSHLITLLLFLFHSETICRPSGRKSSK (SEQ ID NO: 579) of HSI1RA_P16 (SEQ ID NO:69).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P16 (SEQ ID NO:69), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-68 of IL1X_HUMAN (SEQ ID NO: 372), which also corresponds to amino acids 1-68 of HSI1RA_P16 (SEQ ID NO:69), 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 DRCGTH (SEQ ID NO: 478) corresponding to amino acids 69-74 of HSI1RA_P16 (SEQ ID NO:69), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P16 (SEQ ID NO:69), 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 MEICRGLRSHLITLLLFLFHS (SEQ ID NO: 578) corresponding to amino acids 1-21 of HSI1RA_P16 (SEQ ID NO:69), a second amino acid sequence being at least 90% homologous to amino acids 4-50 of P18510-2 (SEQ ID NO:373), which also corresponds to amino acids 22-68 of HSI1RA_P16 (SEQ ID NO:69), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DRCGTH (SEQ ID NO: 478) corresponding to amino acids 69-74 of HSI1RA_P16 (SEQ ID NO:69), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P16 (SEQ ID NO:69), 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 MEICRGLRSHLITLLLFLFHS (SEQ ID NO: 578) corresponding to amino acids 1-21 of HSI1RA_P16 (SEQ ID NO:69), a second amino acid sequence being at least 90% homologous amino acids 25-71 of NP776213 (SEQ ID NO: 533), which also corresponds to amino acids 22-68 of HSI1RA_P16 (SEQ ID NO:69), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DRCGTH (SEQ ID NO: 478) corresponding to amino acids 69-74 of HSI1RA_P16 (SEQ ID NO:69), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P16 (SEQ ID NO:69), 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 MEICRGLRSHLITLLLFLFHSETICRPSGRKSSK (SEQ ID NO: 579) corresponding to amino acids 1-34 of HSI1RA_P16 (SEQ ID NO:69), a second amino acid sequence being at least 90% homologous to amino acids 1-34 of P18510-4 (SEQ ID NO:375), which also corresponds to amino acids 35-68 of HSI1RA_P16 (SEQ ID NO:69), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DRCGTH (SEQ ID NO: 478) corresponding to amino acids 69-74 of HSI1RA_P16 (SEQ ID NO:69), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P17 (SEQ ID NO:70), comprising a first amino acid sequence being at least 90% homologous to amino acids 35-68 of IL1X_HUMAN (SEQ ID NO: 372), which also corresponds to amino acids 1-34 of HSI1RA_P17 (SEQ ID NO:70), 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 DRCGTH (SEQ ID NO: 478) corresponding to amino acids 35-40 of HSI1RA_P17 (SEQ ID NO:70), wherein said, first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P17 (SEQ ID NO:70), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-34 of P18510-4 (SEQ ID NO:375), which also corresponds to amino acids 1-34 of HSI1RA_P17 (SEQ ID NO:70), 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 DRCGTH (SEQ ID NO: 478) corresponding to amino acids 35-40 of HSI1RA_P17 (SEQ ID NO:70), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P17 (SEQ ID NO:70), comprising a first amino acid sequence being at least 90% homologous to amino acids 17-50 of NP000568 (SEQ ID NO: 532), which also corresponds to amino acids 1-34 of HSI1RA_P17 (SEQ ID NO:70), 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 DRCGTH (SEQ ID NO: 478) corresponding to amino acids 35-40 of HSI1RA_P17 (SEQ ID NO:70), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P17 (SEQ ID NO:70), comprising a first amino acid sequence being at least 90% homologous to amino acids 35-68 of NP776214 (SEQ ID NO: 534), which also corresponds to amino acids 1-34 of HSI1RA_P17 (SEQ ID NO:70), 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 DRCGTH (SEQ ID NO: 478) corresponding to amino acids 35-40 of HSI1RA_P17 (SEQ ID NO:70), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSI1RA_P17 (SEQ ID NO:70), comprising a first amino acid sequence being at least 90% homologous to amino acids 38-71 of NP776213 (SEQ ID NO: 533), which also corresponds to amino acids 1-34 of HSI1RA_P17 (SEQ ID NO:70), 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 DRCGTH (SEQ ID NO: 478) corresponding to amino acids 35-40 of HSI1RA_P17 (SEQ ID NO:70), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSPLGF1_P4 (SEQ ID NO:99), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-140 of P49763-2 (SEQ ID NO: 536), which also corresponds to amino acids 1-140 of HSPLGF1_P4 (SEQ ID NO:99), 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 SHLVLTLGLLQEETQGQGEEEEREAETHRLPPVRRCCSPEVTHPLEERDPAPGSCIYYRHTLQ (SEQ ID NO: 480) corresponding to amino acids 141-203 of HSPLGF1_P4 (SEQ ID NO:99), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HSPLGF1_P4 (SEQ ID NO:99), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SHLVLTLGLLQEETQGQGEEEEREAETHRLPPVRRCCSPEVTBPLEERDPAPGSCIYYRHTLQ (SEQ ID NO: 480) of HSPLGF1_P4 (SEQ ID NO:99).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HSPLGF1_P13 (SEQ ID NO:101), comprising a amino acid sequence being at least 90% homologous to amino acids 1-141 of P49763-2 (SEQ ID NO: 536), which also corresponds to amino acids 1-141 of HSPLGF1_P13 (SEQ ID NO:101).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P3 (SEQ ID NO:127), 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 MHQAGYPGCRGA (SEQ ID NO: 582) corresponding to amino acids 1-12 of HUMSP18A_P3 (SEQ ID NO:127), a second amino acid sequence being at least 90% homologous to amino acids 1-285 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 13-297 of HUMSP18A_P3 (SEQ ID NO:127), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SEPTAPSLAQCLLSSSPYPATA (SEQ ID NO: 481) corresponding to amino acids 298-319 of HUMSP18A_P3 (SEQ ID NO:127), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HUMSP18A_P3 (SEQ ID NO:127), 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 MHQAGYPGCRGA (SEQ ID NO: 582) of HUMSP18A_P3 (SEQ ID NO:127).
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMSP18A_P3 (SEQ ID NO:127), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SEPTAPSLAQCLLSSSPYPATA (SEQ ID NO: 481) of HUMSP18A_P3 (SEQ ID NO:127).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P20 (SEQ ID NO:128), 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 MDEMGQVGLVGSCMCLGVLCWPLPKRTSPLELGASPTHVSSTLGPLPPQ (SEQ ID NO: 583) corresponding to amino acids 1-49 of HUMSP18A_P20 (SEQ ID NO:128), and a second amino acid sequence being at least 90% homologous to amino acids 66-381 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 50-365 of HUMSP18A_P20 (SEQ ID NO:128), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HUMSP18A_P20 (SEQ ID NO:128), 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 MDEMGQVGLVGSCMCLGVLCWPLPKRTSPLELGASPTHVSSTLGPLPPQ (SEQ ID NO: 583) of HUMSP18A_P20 (SEQ ID NO:128).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P22 (SEQ ID NO:129), 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 MDEMGQVGLVGSCMCLGVLCWPLPKRTSPLELGASPTHVSSTLGPLPPQ (SEQ ID NO: 583) corresponding to amino acids 1-49 of HUMSP18A_P22 (SEQ ID NO:129), a second amino acid sequence being at least 90% homologous to amino acids 66-131 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 50-115 of HUMSP18A_P22 (SEQ ID NO:129), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRAASSPPACLPTQAPVPTHGEPHTQHPSQPDTHTHTHTHTAPKPARHKHTAPQPAGHTHTHTHNTPAGRTHT HTVPQLAGHTHTQHPIQTHTHTQYPSQLETHTHTALHPDTYPHSTPASQTHTHTHTHTQHTHSTPAGHTHTH THPVHKGPRKLRALQPCTRPWAPRFRCTRWACTLTHPYTLTLHMLTHLFILTYMLMLIHTQSRPPALKSPHSPI FAFCPPT (SEQ ID NO: 482) corresponding to amino acids 116-344 of HUMSP18A_P22 (SEQ ID NO:129), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMSP18A_P22 (SEQ ID NO:129), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRAASSPPACLPTQAPVPTHGEPHTQHPSQPDTHTHTHTHTAPKPARHKHTAPQPAGHTHTHTHNTPAGRTHT HTVPQLAGHTHTQHPIQTHTHTQYPSQLETHTHTALHPDTYPHSTPASQTHTHTHTHTHTQHTHSTPAGHTHTH THPVHKGPRKLRALQPCTRPWAPRFRCTRWACTLTHPYTLTLTHMLTHLFILTYMLMLIHITQSRPPALKSPHSPI FAFCPPT (SEQ ID NO: 482) of HUMSP18A_P22 (SEQ ID NO:129).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P38 (SEQ ID NO:130), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-285 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 1-285 of HUMSP18A_P38 (SEQ ID NO:130), 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 SEPTAPSLAQCLLSSSPYPATA (SEQ ID NO:481) corresponding to amino acids 286-307 of HUMSP18A_P38 (SEQ ID NO:130), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMSP18A_P38 (SEQ ID NO:130), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SEPTAPSLAQCLLSSSPYPATA (SEQ ID NO: 481) of HUMSP18A_P38 (SEQ ID NO:130).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P39 (SEQ ID NO:131), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-334 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 1-334 of HUMSP18A_P39 (SEQ ID NO:131), 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 LAPVC (SEQ ID NO: 484) corresponding to amino acids 335-339 of HUMSP18A_P39 (SEQ ID NO:131), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMSP18A_P39 (SEQ ID NO:131), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LAPVC(SEQ ID NO:484) of HUMSP18A_P39 (SEQ ID NO:131).
  • {In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P41 (SEQ ID NO:132), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-224 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 1-224 of HUMSP18A_P41 (SEQ ID NO:132), 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 VRHPGPHRAQEHTTCSSLQLPPLSQLTPPSGPSWLPEVRRGESRLCIAPTQGTLGLRLRPGRCQAYSSCNKH (SEQ ID NO: 485) corresponding to amino acids 225-297 of HUMSP18A_P41 (SEQ ID NO:132), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMSP18A_P41 (SEQ ID NO:132), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRHPGPHRAQEHITHTCSSLQLPPLSQLTPPSGPSWLPEVRRGESRLCIAPTQGTLGLRLRPGRCQAYSSCNKH (SEQ ID NO:485) of HUMSP18A_P41 (SEQ ID NO:132).
  • {In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P43 (SEQ ID NO:133), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-131 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 1-131 of HUMSP18A_P43 (SEQ ID NO:133), 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 VRAASSPPACLPTQAPVPTHGEPHTQHPSQPDTHTHTHTHTAPKPARHKHTAPQPAGHTHTUTHNTPAGRTHT HTVPQLAGHTHTQHPIQTHTHQYPSQLETHTHTALHPDTYPHSTPASQTHTHTHTHTHTQHHTHSTPAGHTHTH TBPVHKGPRKLRALQPCTRPWAPRFRCTRWACTLTTIPYTLTLTHMLTBLFILTYMLMLIHTQSRPPALKSPHSPI FAFCPPT (SEQ ID NO: 482) corresponding to amino acids 132-360 of HUMSP18A_P43 (SEQ ID NO:133), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMSP18A_P43 (SEQ ID NO:133), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRAASSPPACLPTQAPVPTHGEPHTQBPSQPDHTHFHTHTTAPKPARHKHTAPQPAGHETHTHNTPAGRTHT HTVPQLAGHTHTQBPIQTHTHTQYPSQLE-fHTHTALHPDTYPHSTPASQHTHTQHTHSTPAGH-FHTH THPVHKGPRKLRALQPCTRPWAPRFRCTRWACTLTHPYTLTLTHMLTBLFILTYMLMLIHTQSRPPALKSPHSPI FAFCPPT (SEQ ID NO: 482) of HUMSP18A_P43 (SEQ ID NO:133).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P45 (SEQ ID NO:134), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-65 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 1-65 of HUMSP18A_P45 (SEQ ID NO:134), 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 RTSPLELGASPTHVSSTLGPLPPQ (SEQ ID NO: 487) corresponding to amino acids 66-89 of HUMSP18A_P45 (SEQ ID NO:134), and a third amino acid sequence being at least 90% homologous to amino acids 66-381 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 90-405 of HUMSP18A_P45 (SEQ ID NO:134), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMSP18A_P45 (SEQ ID NO:134), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence RTSPLELGASPTHVSSTLGPLPPQ (SEQ ID NO: 487) of HUMSP18A_P45 (SEQ ID NO:134).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P48 (SEQ ID NO:135), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-225 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 1-225 of HUMSP18A_P48 (SEQ ID NO:135), 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 RRQENGCRETLSATSACP (SEQ ID NO: 488) corresponding to amino acids 226-243 of HUMSP18A_P48 (SEQ ID NO:135), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMSP18A_P48 (SEQ ID NO:135), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence RRQENGCRETLSATSACP (SEQ ID NO: 488) of HUMSP18A_P48 (SEQ ID NO:135).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P49 (SEQ ID NO:136), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-361 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 1-361 of HUMSP18A_P49 (SEQ ID NO:136), 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 KKTPSFKVLQYGQTWWLTPAIPAP (SEQ ID NO: 489) corresponding to amino acids 362-385 of HUMSP18A_P49 (SEQ ID NO:136), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMSP18A_P49 (SEQ ID NO:136), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence KKTPSFKVLQYGQTWWLTPAIPAP (SEQ ID NO: 489) of HUMSP18A_P49 (SEQ ID NO:136).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P50 (SEQ ID NO:137), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-194 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 1-194 of HUMSP18A_P50 (SEQ ID NO:137), and a second amino acid sequence being at least 90% homologous to amino acids 225-381 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 195-351 of HUMSP18A_P50 (SEQ ID NO:137), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated chimeric polypeptide encoding for an edge portion of HUMSP18A_P50 (SEQ ID NO:137), 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 QG, having a structure as follows: a sequence starting from any of amino acid numbers 194−x to 194; and ending at any of amino acid numbers 195+((n−2)−x), in which x varies from 0 to n−2.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMSP18A_P53 (SEQ ID NO:138), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-89 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 1-89 of HUMSP18A_P53 (SEQ ID NO:138), and a second amino acid sequence being at least 90% homologous to amino acids 132-381 of PSPB_HUMAN (SEQ ID NO:406), which also corresponds to amino acids 90-339 of HUMSP18A_P53 (SEQ ID NO:138), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated chimeric polypeptide encoding for an edge portion of HUMSP18A_P53 (SEQ ID NO:138), 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 QD, having a structure as follows: a sequence starting from any of amino acid numbers 89−x to 89; and ending at any of amino acid numbers 90+((n−2)−x), in which x varies from 0 to n−2.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in F05068_P6 (SEQ ID NO:193), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-83 of ADML_HUMAN (SEQ ID NO:413), which also corresponds to amino acids 1-83 of F05068_P6 (SEQ ID NO:193), 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 CLSSPSPRPQQSGCRPHPSQALPPEHEQLPGPPELWLPLRDVHGAEAGTPDLPVHR (SEQ ID NO: 490) corresponding to amino acids 84-139 of F05068_P6 (SEQ ID NO:193), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of F05068_P6 (SEQ ID NO:193), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence CLSSPSPRPQQSGCRPHPSQALPPEHEQLPGPPELWLPLRDVHGAEAGTPDLPVHR (SEQ ID NO: 490) of F05068_P6 (SEQ ID NO:193).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in F05068_P9 (SEQ ID NO:194), comprising a amino acid sequence being at least 90% homologous to amino acids 1-33 of ADML_HUMAN (SEQ ID NO:413), which also corresponds to amino acids 1-33 of F05068_P9 (SEQ ID NO:194).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in F05068_P10 (SEQ ID NO:195), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-82 of ADML_HUMAN (SEQ ID NO:413), which also corresponds to amino acids 1-82 of F05068_P10 (SEQ ID NO:195), and an amino acid R, wherein said first amino acid sequence and said amino acid are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P6 (SEQ ID NO:215), comprising an amino acid sequence being at least 90% homologous to amino acids 86-178 of IL10_HUMAN (SEQ ID NO:423), which also corresponds to amino acids 1-93 of HUMIL10_P6 (SEQ ID NO:215).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P6 (SEQ ID NO:215), comprising a first amino acid sequence being at least 90% homologous to amino acids 86-126 of Q6FGS9_HUMAN (SEQ ID NO: 545), which also corresponds to amino acids 1-41 of HUMIL10_P6 (SEQ ID NO:215), a bridging amino acid H corresponding to amino acid 42 of HUMIL10_P6 (SEQ ID NO:215), and a second amino acid sequence being at least 90% homologous to amino acids 128-178 of Q6FGS9_HUMAN (SEQ ID NO: 545), which also corresponds to amino acids 43-93 of HUMIL10_P6 (SEQ ID NO:215), wherein said first amino acid sequence, bridging amino acid and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P9 (SEQ ID NO:216), 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 MMPPACPLSVMDMELEARTNTFSFLPQ (SEQ ID NO: 584) corresponding to amino acids 1-28 of HUMIL10_P9 (SEQ ID NO:216), and a second amino acid sequence being at least 90% homologous to amino acids 127-178 of IL10_HUMAN (SEQ ID NO:423), which also corresponds to amino acids 29-80 of HUMIL10_P9 (SEQ ID NO:216), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HUMIL10_P9 (SEQ ID NO:216), 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 MMPPACPLSVMDMELEARITNTFSFLPQ (SEQ ID NO: 584) of HUMIL10_P9 (SEQ ID NO:216).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P9 (SEQ ID NO:216), 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 MMPPACPLSVMDMELEARITNTFSFLPQ (SEQ ID NO: 584) corresponding to amino acids 1-28 of HUMIL10_P9 (SEQ ID NO:216), and a second amino acid sequence being at least 90% homologous to amino acids 109-160 of Q71UZ1_HUMAN (SEQ ID NO: 542), which also corresponds to amino acids 29-80 of HUMIL10_P9 (SEQ ID NO:216), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P9 (SEQ ID NO:216), 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 MMPPACPLSVMDMELEARITNTFSFLPQH (SEQ ID NO: 585) corresponding to amino acids 1-29 of HUMIL10_P9 (SEQ ID NO:216), and a second amino acid sequence being at least 90% homologous to amino acids 128-178 of Q6FGS9_HUMAN (SEQ ID NO: 545), which also corresponds to amino acids 30-80 of HUMIL10_P9 (SEQ ID NO:216), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P10 (SEQ ID NO:217), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-50 of IL10_HUMAN (SEQ ID NO:423), which also corresponds to amino acids 1-50 of HUMIL10_P10 (SEQ ID NO:217), and a second amino acid sequence being at least 90% homologous to QMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHR FLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYEAYMTMKIRN (SEQ ID NO: 491) corresponding to amino acids 56-178 of IL10_HUMAN (SEQ ID NO:423), which also corresponds to amino acids 51-173 of HUMIL10_P10 (SEQ ID NO:217), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated chimeric polypeptide encoding for an edge portion of HUMIL10_P10 (SEQ ID NO:217), 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 RQ, having a structure as follows: a sequence starting from any of amino acid numbers 50−x to 50; and ending at any of amino acid numbers 51+((n−2)−x), in which x varies from 0 to n−2.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P10 (SEQ ID NO:217), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-50 of Q6FGW4_HUMAN (SEQ ID NO: 543), which also corresponds to amino acids 1-50 of HUMIL10_P10 (SEQ ID NO:217), and a second amino acid sequence being at least 90% homologous to QMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHR FLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 491) corresponding to amino acids 56-178 of Q6FGW4_HUMAN (SEQ ID NO: 543), which also corresponds to amino acids 51-173 of HUMIL10_P10 (SEQ ID NO:217), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P10 (SEQ ID NO:217), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-50 of Q6FGS9_HUMAN (SEQ ID NO: 545), which also corresponds to amino acids 1-50 of HUMIL10_P10 (SEQ ID NO:217), a second amino acid sequence being at least 90% homologous to amino acids 56-126 of Q6FGS9_HUMAN (SEQ ID NO: 545), which also corresponds to amino acids 51-121 of HUMIL10_P10 (SEQ ID NO:217), a bridging amino acid H corresponding to amino acid 122 of HUMIL10_P10 (SEQ ID NO:217), and a third amino acid sequence being at least 90% homologous to amino acids 128-178 of Q6FGS9_HUMAN (SEQ ID NO: 545), which also corresponds to amino acids 123-173 of HUMIL10_P10 (SEQ ID NO:217), wherein said first amino acid sequence, second amino acid sequence, bridging amino acid and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P10 (SEQ ID NO:217), 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 MHSSALLCCLVLLTGVRA (SEQ ID NO: 586) corresponding to amino acids 1-18 of HUMIL10_P10 (SEQ ID NO:217), a second amino acid sequence being at least 90% homologous to amino acids 1-32 of Q71UZ1_HUMAN (SEQ ID NO: 542), which also corresponds to amino acids 19-50 of HUMIL10_P10 (SEQ ID NO:217), and a third amino acid sequence being at least 90% homologous to QMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHR FLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFNYIEAYMTMKIRN (SEQ ID NO: 491) corresponding to amino acids 38-160 of Q71UZ1_HUMAN (SEQ ID NO: 542), which also corresponds to amino acids 51-173 of HUMIL10_P10 (SEQ ID NO:217), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HUMIL10_P10 (SEQ ID NO:217), 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 MHSSALLCCLVLLTGVRA (SEQ ID NO: 586) of HUMIL10_P10 (SEQ ID NO:217).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P10 (SEQ ID NO:217), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-50 of Q6LBF4_HUMAN (SEQ ID NO: 546), which also corresponds to amino acids 1-50 of HUMIL10_P10 (SEQ ID NO:217), 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 QMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHR FLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 491) corresponding to amino acids 51-173 of HUMIL10_P10 (SEQ ID NO:217), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMIL10_P10 (SEQ ID NO:217), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence QMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHR FLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 491) of HUMIL10_P10 (SEQ ID NO:217).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P12 (SEQ ID NO:218), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-126 of IL10_HUMAN (SEQ ID NO:423), which also corresponds to amino acids 1-126 of HUMIL10_P12 (SEQ ID NO:218), and a second amino acid sequence being at least 90% homologous to LQEKGIYKAMSEFDIFINYIEAYMTMKIRN corresponding to amino acids 149-178 of IL10_HUMAN (SEQ ID NO:423), which also corresponds to amino acids 127-156 of HUMIL10_P12 (SEQ ID NO:218), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated chimeric polypeptide encoding for an edge portion of HUMIL10_P12 (SEQ ID NO:218), 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 CL, having a structure as follows: a sequence starting from any of amino acid numbers 126−x to 126; and ending at any of amino acid numbers 127+((n−2)−x), in which x varies from 0 to n−2.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P12 (SEQ ID NO:218), 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 MHSSALLCCLVLLTGVRA (SEQ ID NO: 586) corresponding to amino acids 1-18 of HUMIL10_P12 (SEQ ID NO:218), a second amino acid sequence being at least 90% homologous to amino acids 1-108 of Q71UZ1_HUMAN (SEQ ID NO: 542), which also corresponds to amino acids 19-126 of HUMIL10_P12 (SEQ ID NO:218), and a third amino acid sequence being at least 90% homologous to LQEKGIYKAMSEFDIFINYIEAYMTMKIRN corresponding to amino acids 131-160 of Q71UZ1_HUMAN (SEQ ID NO: 542), which also corresponds to amino acids 127-156 of HUMIL10_P12 (SEQ ID NO:218), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HUMIL10_P12 (SEQ ID NO:218), 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 MHSSALLCCLVLLTGVRA (SEQ ID NO: 586) of HUMIL10_P12 (SEQ ID NO:218).
  • In some embodiments, this invention provides an isolated chimeric polypeptide encoding for an edge portion of HUMIL10_P12 (SEQ ID NO:218), 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 CL, having a structure as follows: a sequence starting from any of amino acid numbers 126−x to 126; and ending at any of amino acid numbers 127+((n−2)−x), in which x varies from 0 to n−2.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P12 (SEQ ID NO:218), comprising a first amino acid sequence being at least 90% homologous to MHSSALLCCLVLLTGVRASPGQGTQSENSCTBFPGNLPNMLRDLRDAFSRVKTFF corresponding to amino acids 1-55 of Q6LBF4_HUMAN (SEQ ID NO: 546), which also corresponds to amino acids 1-55 of HUMIL10_P12 (SEQ ID NO:218), 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 QMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCLQ EKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 492) corresponding to amino acids 56-156 of HUMIL10_P12 (SEQ ID NO:218), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide as set forth in a tail of HUMIL10_P12 (SEQ ID NO:218), 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 QMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCLQ EKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 492) of HUMIL10_P12 (SEQ ID NO:218).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P13 (SEQ ID NO:219), 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 MPPACPLSVMDMELEARITNTFSFLPQ (SEQ ID NO: 587) corresponding to amino acids 1-27 of HUMIL10_P13 (SEQ ID NO:219), and a second amino acid sequence being at least 90% homologous to amino acids 127-178 of IL10_HUMAN (SEQ ID NO:423), which also corresponds to amino acids 28-79 of HUMIL10_P13 (SEQ ID NO:219), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HUMIL10_P13 (SEQ ID NO:219), 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 MPPACPLSVMDMELEARITNTFSFLPQ (SEQ ID NO: 587) of HUMIL10_P13 (SEQ ID NO:219).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMIL10_P13 (SEQ ID NO:219), 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 MPPACPLSVMDMELEARITNTFSFLPQ (SEQ ID NO: 587) corresponding to amino acids 1-27 of HUMIL10_P13 (SEQ ID NO:219), and a second amino acid sequence being at least 90% homologous to amino acids 109-160 of Q71UZ1_HUMAN (SEQ ID NO: 542), which also corresponds to amino acids 28-79 of HUMIL10_P13 (SEQ ID NO:219), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in AA336074_P30 (SEQ ID NO:236), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-158 of KLK4_HUMAN (SEQ ID NO:430), which also corresponds to amino acids 1-158 of AA336074_P30 (SEQ ID NO:236), 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 DAVIAIQSQTVGGWECEKLSQPWQGCTISATSSARTSCCILTGCSLLLTASPGVEIRRDSAGCSHMIKEGPELGV TPDPS (SEQ ID NO: 493) corresponding to amino acids 159-238 of AA336074_P30 (SEQ ID NO:236), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of AA336074_P30 (SEQ ID NO:236), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DAVIAIQSQTVGGWECEKLSQPWQGCTISATSSARTSCCILTGCSLLLTASPGVEIRRDSAGCSHMIKEGPELGV TPDPS (SEQ ID NO: 493) of AA336074_P30 (SEQ ID NO:236).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z39737_P9 (SEQ ID NO:307), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-136 of SPO2_HUMAN_V1, which also corresponds to amino acids 1-136 of Z39737_P9 (SEQ ID NO:307), 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 FLQQGCPPSPGVPTGFPGASYSATMWEFHHHRDLSGSSGSYVETRNSSP (SEQ ID NO: 494) corresponding to amino acids 137-185 of Z39737_P9 (SEQ ID NO:307), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of Z39737_P9 (SEQ ID NO:307), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence FLQQGCPPSPGVPTGFPGASYSATMWEFHHHRDLSGSSGSYVETRNSSP (SEQ ID NO: 494) of Z39737_P9 (SEQ ID NO:307).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z25299_P1 (SEQ ID NO:345), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-131 of ALK1_HUMAN (SEQ ID NO:443), which also corresponds to amino acids 1-131 of Z25299_P1 (SEQ ID NO:345), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GKQGMRAH (SEQ ID NO: 495) corresponding to amino acids 132-139 of Z25299_P1 (SEQ ID NO:345), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of Z25299_P1 (SEQ ID NO:345), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GKQGMRAH (SEQ ID NO: 495) of Z25299_P1 (SEQ ID NO:345).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z25299_P4 (SEQ ID NO:346), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-131 of ALK1_HUMAN (SEQ ID NO:443), which also corresponds to amino acids 1-131 of Z25299_P4 (SEQ ID NO:346), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GCFSPSISPSHFFTMSSISTFSAVLRTSASSLSACVLPATHQMRSGEEFSTFGFMLVLK (SEQ ID NO: 496) corresponding to amino acids 132-190 of Z25299_P4 (SEQ ID NO:346), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of Z25299_P4 (SEQ ID NO:346), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GCFSPSISPSHFFTMSSISTFSAVLRTSASSLSACVLPATHQMRSGEEFSTFGFMLVLK (SEQ ID NO: 496) of Z25299_P4 (SEQ ID NO:346).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z25299_P5 (SEQ ID NO:347), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-131 of ALK1_HUMAN (SEQ ID NO:443), which also corresponds to amino acids 1-131 of Z25299_P5 (SEQ ID NO:347), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GEKRHHKQLRDQEVDPLEMRRHSAG (SEQ ID NO: 497) corresponding to amino acids 132-156 of Z25299_P5 (SEQ ID NO:347), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of Z25299_P5 (SEQ ID NO:347), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GEKRHHKQLRDQEVDPLEMRRHSAG (SEQ ID NO: 497) of Z25299_P5 (SEQ ID NO:347).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z25299_P6 (SEQ ID NO:348), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-81 of NP003055 (SEQ ID NO: 550), which also corresponds to amino acids 1-81 of Z25299_P6 (SEQ ID NO:348), 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 RGSLGSAQ (SEQ ID NO: 498) corresponding to amino acids 82-89 of Z25299_P6 (SEQ ID NO:348), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of Z25299_P6 (SEQ ID NO:348), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence RGSLGSAQ (SEQ ID NO: 498) of Z25299_P6 (SEQ ID NO:348).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z25299_P8 (SEQ ID NO:349), comprising a amino acid sequence being at least 90% homologous to amino acids 1-82 of ALK1_HUMAN (SEQ ID NO:443), which also corresponds to amino acids 1-82 of Z25299_P8 (SEQ ID NO:349), wherein said and first amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated chimeric polypeptide encoding for an edge portion of Z25299_P8 (SEQ ID NO:349), 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 T, having a structure as follows: a sequence starting from any of amino acid numbers 82−x to 82; and ending at any of amino acid numbers 82+((n−2)−x), in which x varies from 0 to n−2. In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z22012_P41 (SEQ ID NO:254), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-125 of L3BP_HUMAN (SEQ ID NO: 441), which also corresponds to amino acids 1-125 of Z22012_P41 (SEQ ID NO:254), 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 GAPTPWTSPGSSRRPLARSLTASGAATCPSA (SEQ ID NO: 499) corresponding to amino acids 126-156 of Z22012_P41 (SEQ ID NO:254), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of Z22012_P41 (SEQ ID NO:254), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GAPTPWTSPGSSRRPLARSLTASGAATCPSA (SEQ ID NO: 499) of Z22012_P41 (SEQ ID NO:254).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z22012_P41 (SEQ ID NO:254), 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 MTPPRLFWVWLLVAGTQGVNDGDMRLADGGATNQGRVEIFYRGQWGTVCDNLWDLTDASVVCRALGFENA TQALGRAAFGQGSGPIMLDEVQCTGTEASLADCKSLGWLKSNCRBERDAGVVCTNGAPTPWTSPGSSRRPLA RSLTASGAATCPSA (SEQ ID NO: 589) corresponding to amino acids 126-156 of Z22012_P41 (SEQ ID NO:254), and a second amino acid sequence being at least 90% homologous to amino acids 1-125 of NP005558 (SEQ ID NO: 551), which also corresponds to amino acids 1-125 of Z22012_P41 (SEQ ID NO:254), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of Z22012_P41 (SEQ ID NO:254), 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 MTPPRLFWVWLLVAGTQGVNDGDMRLADGGATNQGRVEIFYRGQWGTVCDNLWDLTDASVVCRALGFENA TQALGRAAFGQGSGPIMLDEVQCTGTEASLADCKSLGWLKSNCRHERDAGVVCTNGAPTPWTSPGSSRRPLA RSLTASGAATCPSA (SEQ ID NO: 589) of Z22012_P41 (SEQ ID NO:254).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z22012_P42 (SEQ ID NO:255), 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 MTPPRLFWVWLLVAGTQGVNDGDMRLADGGATNQGRVEIFYRGQWGTVCDNLWDLTDASVVCRALGFENA TQALGRAAFGQGSGPIMLDEVQCTGTEASLADCKSLGWLKSNCRHERDAGVVCTNGTSTPEGLTSPCRQSSAS TSWPLPMGPGSCRATAQASLPSSSPRTPRSRCPWTCMPMQWPQGTPCWRSSAYSSWPGTSRP (SEQ ID NO: 590) corresponding to amino acids 126-205 of Z22012_P42 (SEQ ID NO:255), and a second amino acid sequence being at least 90% homologous to amino acids 1-125 of L3BP_HUMAN (SEQ ID NO: 441), which also corresponds to amino acids 1-125 of Z22012_P42 (SEQ ID NO:255), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of Z22012_P42 (SEQ ID NO:255), 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 MTPPRLFWVWLLVAGTQGVNDGDMRLADGGATNQGRVEIFYRGQWGTVCDNLWDLTDASVVCRALGFENA TQALGRAAFGQGSGPIMLDEVQCTGTEASLADCKSLGWLKSNCRHERDAGVVCTNGTSTPEGLTSPCRQSSAS TSWPLPMGPGSCRATAQASLPSSSPRTPRSRCPWTCMPMQWPQGTPCWRSSAYSSWPGTSRP (SEQ ID NO: 590) of Z22012_P42 (SEQ ID NO:255).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in Z22012_P42 (SEQ ID NO:255), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-125 of NP005558 (SEQ ID NO: 551), which also corresponds to amino acids 1-125 of Z22012_P42 (SEQ ID NO:255), 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 GTSTPEGLTSPCRQSSASTSWPLPMGPGSCRATAQASLPSSSPRTPRSRCPWTCMPMQWPQGTPCWRSSAYSS WPGTSRP (SEQ ID NO: 500) corresponding to amino acids 126-205 of Z22012_P42 (SEQ ID NO:255), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of Z22012_P42 (SEQ ID NO:255), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GTSTPEGLTSPCRQSSASTSWPLPMGPGSCRATAQASLPSSSPRTPRSRCPWTCMPMQWPQGTPCWRSSAYSS WPGTSRP (SEQ ID NO: 500) of Z22012_P42 (SEQ ID NO:255).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMTREFAC_P9 (SEQ ID NO:245), comprising a first amino acid sequence being at least 90% homologous to amino acids 1-28 of TFF3_HUMAN (SEQ ID NO:440), which also corresponds to amino acids 1-28 of HUMTREFAC_P9 (SEQ ID NO:245), 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 QQGLWQLTGLCLGQLQTSVPCQPRTGWTAATPMSPPRSATTGAAALTPGSLECLGVSSPCRKQNAPSEAPPAA PGRGMRGSEBPCPAVIAARHCSSQLFCPFAPGKRFC (SEQ ID NO: 501) corresponding to amino acids 29-137 of HUMTREFAC_P9 (SEQ ID NO:245), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for an edge portion of HUMTREFAC_P9 (SEQ ID NO:245), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence QQGLWQLTGLCLGQLQTSVPCQPRTGWTAATPMSPPRSATTGAAALTPGSLECLGVSSPCRKQNAPSEAPPAA PGRGMRGSEHPCPAVIAARHCSSQLFCPFAPGKRFC (SEQ ID NO: 501) of HUMTREFAC_P9 (SEQ ID NO:245).
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMTREFAC_P9 (SEQ ID NO:245), 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 MAARALCMLGLVLALLSSSSAEEYVGLSQQGLWQLTGLCLGQLQTSVPCQPRTGWTAATPMSPPRSATTGAA ALTPGSLECL (SEQ ID NO: 591) corresponding to amino acids 29-137 of HUMTREFAC_P9 (SEQ ID NO:245), and a second amino acid sequence being at least 90% homologous to ANQCAVPAKDRVDCGYPHVTPKE corresponding to amino acids 51-78 of Q96NX0_HUMAN (SEQ ID NO: 554), which also corresponds to amino acids 1-28 of HUMTREFAC_P9 (SEQ ID NO:245), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, this invention provides an isolated polypeptide encoding for a head of HUMTREFAC_P9 (SEQ ID NO:245), 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 MAARALCMLGLVLALLSSSSAEEYVGLSQQGLWQLTGLCLGQLQTSVPCQPRTGWTAATPMSPPRSATTGAA ALTPGSLECL (SEQ ID NO: 591) of HUMTREFAC_P9 (SEQ ID NO:245).
  • In some embodiments, this invention provides an isolated chimeric polypeptide encoding for an edge portion of HUMTREFAC_P9 (SEQ ID NO:245), 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 137−x to 137; and ending at any of amino acid numbers 1+((n−2)−x), in which x varies from 0 to n−2.
  • In some embodiments, the isolated chimeric proteins or polypeptides of the invention may comprise an amino acid sequence corresponding to or homologous to that as set forth in HUMTREFAC_P9 (SEQ ID NO:245), 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 MAARALCMLGLVLALLSSSSAEEYVGLSQQGLWQLTGLCLGQLQTSVPCQPRTGWTAATPMSPPRSATTGAA ALTPGSLECLGVSSPCRKQNAPSEAPPAAPGRGMRGSEEPCPAVIAARHCSSQLFCPFAPGKRFC (SEQ ID NO: 592) corresponding to amino acids 29-137 of HUMTREFAC_P9 (SEQ ID NO:245), and a second amino acid sequence being at least 90% homologous to amino acids 51-78 of NP003217 (SEQ ID NO: 555), which also corresponds to amino acids 1-28 of HUMTREFAC_P9 (SEQ ID NO:245), wherein said, first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • In some embodiments, the term “polypeptide” is to be understood to refer to a molecule comprising from at least 2 to several thousand or more amino acids. The term “polypeptide” is to be understood to include, inter alia, native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides), peptidomimetics, such as peptoids and semipeptoids or peptide analogs, which may comprise, for example, any desirable modification, including, inter alia, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells, or others as will be appreciated by one skilled in the art. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, residue modification, or others. Inclusion of such peptides within the polypeptides of this invention may produce a polypeptide sharing identity with the polypeptides described herein, for example, those provided in the sequence listing.
  • Methods for preparing, isolating, deriving, etc., the polypeptides of this invention are well known are well known in the art. In some embodiments, the polypeptides of this invention comprise variants of known proteins. For example, and in some embodiments, the polypeptides of this invention comprise splice variants of native proteins expressed in a given subject. In some embodiments, the polypeptides may be obtained through known protein evolution techniques available in the art. In some embodiments, the polypeptides of this invention may be obtained via rational design, based on a particular native polypeptide sequence.
  • In some embodiments, this invention provides for antibodies or antibody fragments specifically interacting with or recognizing a polypeptide of this invention.
  • In one embodiment, the antibody recognizes one or more epitopes (antigen determinants) contained within the polypeptides of this invention. In some embodiments, reference to the antibody property of “specific interaction” or “recognition” is to be understood as including covalent and non-covalent associations, and with a variance of affinity over several orders of magnitude. Such terms are to be understood as relative, with respect to an index molecule, for which the antibody is though to have little to no specific interaction or recognition.
  • In one embodiment, the antibodies will specifically interact or recognize a particular antigen determinant. In some embodiments, the antibodies or antibody fragments of this invention will recognize or interact with a polypeptide or protein of the invention, and will not substantially recognize or interact with other molecules, even when present in the same sample, such as a biological sample. In some embodiments, the antibodies of this invention have a specificity such that the specific interaction with or binding to the antigen is at least about 2, or in some embodiments, at least about 5, or in some embodiments, at least about 10-fold greater than interaction or binding observed under the same reaction conditions with a molecule that does not include the antigenic determinant.
  • The antibodies may be useful, in some embodiments, in detecting qualitative and/or quantitative changes in expression of the polypeptides or polynucleotides of this invention. In some embodiments, changes in expression are associated with a particular disease or disorder, such that detection of such changes comprises a diagnostic method of this invention.
  • In one embodiment, this invention provides a diagnostic kit for detecting a disease, comprising reagents which detect qualitative and/or quantitative changes in expression of a polypeptide or polynucleotide of this invention.
  • Optionally, the kit comprises a NAT-based technology; optionally and preferably, the kit further comprises at least one nucleotide probe or primer, alternatively and optionally this kit comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein; alternatively and optionally, said kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence according to any of the above claims.
  • Alternatively and optionally, the kit comprises an antibody according to any of the above claims (optionally and preferably, the kit further comprises at least one reagent for performing an ELISA or a Western blot.
  • In some embodiments, this invention provides a diagnostic method, for example, a method of detection of a polypeptide or polynucleotide of this invention, whereby expression, or relative changes in expression of the polypeptide or polynucleotide herald the onset, severity, or prognosis of an individual with regard to a particular disease, disorder or condition.
  • In some embodiments, such detection may comprise detection of specific expression of a splice variant, or other polypeptide or polynucleotide of this invention, via any means known in the art, and as described herein. In some embodiments, detection of the following genes and/or their products is part of the diagnostic methods of this invention: HSFLT, HSI1RA, HSPLGF, HUMSP18A, F05068, HUMIL10, or any combination thereof. In some embodiments, detection of these genes, or relative changes in expression of the genes, their products or certain variants thereof herald the onset, severity or prognosis of cardiovascular disease in a subject. In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in the treatment, diagnosis or prognosis assessment of any cardiovascular disease, including, inter alia, myocardial infarct, acute coronary syndrome, coronary artery disease, angina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure or any type of heart failure, reinfarction, assessment of thrombolytic therapy, assessment of myocardial infarct size, differential diagnosis between heart-related versus lung-related conditions (such as pulmonary embolism), the differential diagnosis of Dyspnea, cardiac valves related conditions, vascular disease, or any combination thereof.
  • The polypeptides and/or polynucleotides of this invention may serve as markers or indicators of disease initiation, severity and/or response to treatment, for the indicated disease, disorder or condition, and their use as such is to be considered part of this invention, and part of the methods of this invention.
  • In some embodiments, detection of SFLT, HSI1RA, HSPLGF, HUMSP18A, F05068, HUMIL10, or any combination thereof, or relative changes in expression of the genes, their products or certain variants thereof herald the onset, severity or prognosis of cerebrovascular disease in a subject. In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in the treatment, diagnosis or prognosis assessment of any cerebrovascular disease, including, inter alia, stroke, including any type of stroke or neural tissue injury, or any type of cerebrovascular accident, ischemic stroke, hemorrhagic stroke or transient ischemic attacks, thrombotic, embolic, lacunar or hypoperfusion types of strokes, brain trauma, etc. In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in the establishment of the timing of stroke; the type of stroke; the extent of tissue damage as a result of the stroke; response to immediate treatments that are meant to alleviate the extent of stroke and brain damage, when available, or any combination thereof.
  • In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in the diagnosis of stroke and indication if an ischemic stroke has occurred; or a hemorrhagic stroke has occurred; or prognosis of a subsequent cerebral vasospasm; etc.
  • In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in identifying a patient at risk for cerebral vasospasm. Such methods preferably comprise comparing an amount of one or more marker(s) predictive of a subsequent cerebral vasospasm in a test sample from a patient diagnosed with a subarachnoid hemorrhage. Such markers may be one or more markers related to blood pressure regulation, markers related to inflammation, markers related to apoptosis, and/or specific markers of neural tissue injury.
  • In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in the diagnosis, treatment or assessment of the prognosis of a subject with cardiomyopathy and/or myocarditis, and/or related conditions as described herein. In some embodiments, markers utilized in this context are polynucleotides encoding or polypeptides comprising HSFLT, HSI1Ra, HSPLGF, HUMSP18A, F05068 and/or HUMIL10 clusters, or variants thereof, or combinations thereof.
  • In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in the diagnosis, treatment or assessment of the prognosis of a subject with acute and chronic inflammation, and/or CVS diseases, and in some embodiments, the marker comprises one or more of HSFLT variants, HSI1Ra variants, HSPLGF variants, HUMSP18A variants, F05068 variants and/or HUMIL10 variants, including for a spectrum of diseases where an inflammatory process plays a substantial role. In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in the diagnosis, treatment or assessment of the prognosis of a subject with hypercholesterolemia, diabetes, atherosclerosis, inflammation that involves blood vessels—whether acute or chronic including but not limited to the coronary arteries and blood vessels of the brain, myocardial infarction, cerebral stroke, peripheral vascular disease, vasculitis, polyarteritis nodosa, ANCA associated small vessel vasculitis, Churg-Strauss syndrome, Henoch-Schonlein purpura, scleroderma, thromboangiitis obliterans, temporal arteritis, Takayasu's arteritis, hypersensitivity vasculitis, Kawasaki disease, Behçet syndrome, and their complications including but not limited to coronary disease, angina pectoris, deep vein thrombosis, renal disease, diabetic nephropathy, lupus nephritis, renal artery thrombosis, renal artery stenosis, atheroembolic disease of the renal arteries, renal vein thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, arteriolar nephrosclerosis, preeclampsia, eclampsia, albuminuria, microalbuminuria, glomerulonephritis, renal failure, hypertension, uremia, cerebrovascular disease, peripheral vascular disease, intermittent claudication, abdominal angina; rheumatic/autoimmune diseases that involve systemic immune reaction including but not limited to rheumatoid arthritis, scleroderma, mixed connective tissue disease, Sjogren syndrome, ankylosing spondylitis, spondyloarthropathy, psoriasis, psoriatic arthritis, myositis and systemic lupus erythematosus; acute and/or chronic infective processes that involve systemic immune reaction including but not limited to pneumonia, bacteremia, sepsis, pyelonephritis, cellulitis, osteomyelitis, meningitis and viral hepatitis; malignant and idiopathic processes that involve systemic immune reaction and/or proliferation of immune cells including but not limited to granulomatous disorders, Wegener's granulomatosis, lymphomatoid granulomatosis/polymorphic reticulosis, idiopathic midline granuloma, multiple myeloma, Waldenstrom's macroglobulinemia, Castleman's disease, amyloidosis, lymphoma, histiocytosis, renal cell carcinoma and paraneoplastic syndromes; conditions where CRP was shown to have a positive correlation with the presence of the condition including but not limited to weight loss, anorexia-cachexia syndrome, extent of disease, recurrence in advanced cancer, diabetes (types 1 & 2), obesity, hypertension, preterm delivery; conditions which have similar symptoms, signs and complications as the conditions above and where the differential diagnosis between them and the conditions above is of clinical importance including but not limited to: other (non vascular) causes of heart disease, renal disease and cerebral disease; other (non rheumatic) causes of arthropathy and musculoskeletal pain; other causes of non-specific symptoms and signs such as fever of unknown origin, loss of appetite, weight loss, nonspecific pains, breathing difficulties, anxiety, or any combination thereof, or any disease disorder or condition associated with inflammation.
  • In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in the diagnosis, treatment or assessment of the prognosis of a subject with congestive heart failure (CHF), and in some embodiments, the marker comprises a marker optionally selected from the group consisting of one or more variants in HSFLT variants, HSI1Ra variants, HSPLGF variants, HUMSP18A variants, F05068 variants and/or HUMIL10 variants or combinations thereof. In some embodiments, the polypeptides, polynucleotides and/or methods of this invention may be useful in the diagnosis, treatment or assessment of the prognosis of a subject with sudden cardiac death, from arrhythmia or any other heart related reason; rejection of a transplanted heart; conditions that lead to heart failure including but not limited to myocardial infarction, angina, arrhythmias, valvular diseases, atrial and/or ventricular septal defects; conditions that cause atrial and or ventricular wall volume overload, including but not limited to systemic arterial hypertension, pulmonary hypertension and pulmonary embolism; conditions which have similar clinical symptoms as heart failure and as states that cause atrial and or ventricular pressure-overload, where the differential diagnosis between these conditions to the latter is of clinical importance including but not limited to breathing difficulty and/or hypoxia due to pulmonary disease, anemia or anxiety.
  • Each polypeptide or polynucleotide of the present invention described herein may be used as a potential marker for cardiovascular conditions, might optionally be used alone or in combination with one or more other variant markers described herein, and or in combination with known markers for cardiovascular conditions, including but not limited to Heart-type fatty acid binding protein (H-FABP), Angiotensin, C-reactive protein (CRP), myeloperoxidase (MPO), and/or in combination with the known protein(s) for the variant marker as described herein. Each variant marker of the present invention described herein as potential marker for cerebrovascular conditions, might optionally be used alone or in combination with one or more other variant markers described herein, and or in combination with known markers for cerebrovascular conditions, including but not limited to CRP, S100b, BNGF, CD40, MCP1, N-Acetyl-Aspartate (NAA), N-methyl-d-aspartate (NMDA) receptor antibodies (NR2Ab), and/or in combination with the known protein(s) for the variant marker as described herein.
  • In some embodiments, the phrase “marker-detectable disease is a particular cluster marker detectable disease” refers to the fact that the any polynucleotides and/or polypeptides of this invention can be used to detect the indicated disease, or assess the parameters of the disease, etc., as described herein. In some embodiments, a particular cluster will be useful for the diagnosis, assessment and prognostic indications regarding the indicated disease disorder or condition.
  • In one embodiment, the marker-detectable disease is a cluster HSFLT marker-detectable disease and is a cancer, including but not limited to colon cancer, breast cancer, ovarian cancer, prostate cancer, or lung cancer.
  • In one embodiment, the marker-detectable disease is a cluster Z25299 marker-detectable disease and is a cancer including but not limited to colon cancer, breast cancer, ovarian cancer, lung cancer; and colon, breast, ovarian, and lung cancer invasion and metastasis.
  • In one embodiment, the marker-detectable disease is a cluster AA336074 marker-detectable disease and is a cancer, including but not limited to breast cancer, lung cancer; and breast and lung cancer invasion and metastasis.
  • In one embodiment, the marker-detectable disease is a cluster HSPLGF marker-detectable disease and is a variety of cancers, including but not limited to colon cancer, lung cancer; and colon and lung cancer invasion and metastasis.
  • In one embodiment, the marker-detectable disease is a cluster HSI1RA, cluster HUMSP18A, cluster F05068 marker-detectable disease and is a variety of cancers, including but not limited to lung cancer and lung cancer invasion and metastasis.
  • In one embodiment, the marker-detectable disease is a cluster Z22012, cluster HUMTREFAC, or cluster Z39737 marker-detectable disease and is a prostate cancer.
  • With regard to lung cancer, the disease (and/or diagnostic method to be performed) comprises, in some embodiments, one or more of invasive or metastatic lung cancer; squamous cell lung carcinoma, lung adenocarcinoma, carcinoid, small cell lung cancer or non-small cell lung cancer; detection of overexpression in lung metastasis (vs. primary tumor); detection of overexpression in lung cancer, for example non small cell lung cancer, for example adenocarcinoma, squamous cell cancer or carcinoid, or large cell carcinoma; identification of a metastasis of unknown origin which originated from a primary lung cancer; assessment of a malignant tissue residing in the lung that is from a non-lung origin, including but not limited to: osteogenic and soft tissue sarcomas; colorectal, uterine, cervix and corpus tumors; head and neck, breast, testis and salivary gland cancers; melanoma; and bladder and kidney tumors; distinguishing between different types of lung cancer, therefore potentially affect treatment choice (e.g. small cell vs. non small cell tumors); analysis of unexplained dyspnea and/or chronic cough and/or hemoptysis; differential diagnosis of the origin of a pleural effusion; diagnosis of conditions which have similar symptoms, signs and complications as lung cancer and where the differential diagnosis between them and lung cancer is of clinical importance including but not limited to: non-malignant causes of lung symptoms and signs, including but not limited to: lung lesions and infiltrates, wheeze, stridor, tracheal obstruction, esophageal compression, dysphagia, recurrent laryngeal nerve paralysis, hoarseness, phrenic nerve paralysis with elevation of the hemidiaphragm and Horner syndrome; or detecting a cause of any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, hypophosphatemia, hyponatremia, syndrome of inappropriate secretion of antidiuretic hormone, elevated ANP, elevated ACTH, hypokalemia, clubbing, neurologic-myopathic syndromes and thrombophlebitis.
  • In some embodiments, the polypeptides and/or polynucleotides of this invention may be useful as potential markers for lung cancer, alone or in combination with one or more alternative polynucleotides or polypeptides described herein, and/or in combination with known markers for lung cancer, including but not limited to CEA, CA15-3, Beta-2-microglobulin, CA19-9, TPA, and/or in combination with the known protein(s) for the variant marker as described herein.
  • With regard to breast cancer, the disease (and/or diagnostic method to be performed) the polypeptides, polynucleotides may be useful in determining a probable outcome in breast cancer; detecting breast cancer in patients with age above 55 and/or patients with an age below 45; identification of a metastasis of unknown origin which originated from a primary breast cancer tumor; assessing lymphadenopathy, and in particular axillary lymphadenopathy; distinguishing between different types of breast cancer, therefore potentially affect treatment choice (e.g. as HER-2); differentially diagnosing between a benign and malignant breast mass; as a tool in the assessment of conditions affecting breast skin (e.g. Paget's disease) and their differentiation from breast cancer; differential diagnosis of breast pain or discomfort resulting from either breast cancer or other possible conditions (e.g. mastitis, Mondors syndrome); non-breast cancer conditions which have similar symptoms, signs and complications as breast cancer and where the differential diagnosis between them and breast cancer is of clinical importance including but not limited to: abnormal mammogram and/or nipple retraction and/or nipple discharge due to causes other than breast cancer, including but not limited to benign breast masses, melanoma, trauma and technical and/or anatomical variations; determining a cause of any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, paraneoplastic syndrome; or determining a cause of lymphadenopathy, weight loss and other signs and symptoms associated with breast cancer but originate from diseases different from breast cancer including but not limited to other malignancies, infections and autoimmune diseases.
  • Each variant marker of the present invention described herein as potential marker for breast cancer, might optionally be used alone or in combination with one or more other variant breast cancer described herein, and/or in combination with known markers for breast cancer, including but not limited to Calcitonin, CA15-3 (Mucin1), CA27-29, TPA, a combination of CA 15-3 and CEA, CA 27.29 (monoclonal antibody directed against MUC1), Estrogen 2 (beta), HER-2 (c-erbB2), and/or in combination with the known protein(s) for the variant marker as described herein.
  • With regard to prostate cancer, the disease (and/or diagnostic method to be performed) optionally and preferably comprises one or more of invasive or metastatic prostate cancer.
  • Each marker of the present invention described herein as potential marker for prostate cancer, might optionally be used alone or in combination with one or more other variant prostate cancer described herein, and/or in combination with known markers for prostate cancer, including but not limited to PSA, PAP (prostatic acid phosphatase), CPK-BB, PSMA, PCA3, DD3, and/or in combination with the known protein(s) for the variant marker as described herein.
  • It is to be understood that any polynucleotide or polypeptide of this invention may be useful as a marker for a disease, disorder or condition, and such use is to be considered a part of this invention.
  • With regard to colon cancer, the disease (and/or diagnostic method to be performed) optionally and preferably comprises one or more of invasive or metastatic colon cancer.
  • Each marker of the present invention described herein as potential marker for colorectal cancer, might optionally be used alone or in combination with one or more other variant colorectal cancer described herein, and/or in combination with known markers for colorectal cancer, including but not limited to CEA, CA19-9, CA50, and/or in combination with the known protein(s) for the variant marker as described herein.
  • With regard to ovarian cancer, the polypeptides and/or polynucleotide may be used in the diagnosis, treatment or prognostic assessment of invasive or metastatic ovarian cancer; correlating stage and malignant potential; identification of a metastasis of unknown origin which originated from a primary ovarian cancer, for example gastric carcinoma (such as Krukenberg tumor), breast cancer, colorectal carcinoma and pancreatic carcinoma; distinguishing between different types of ovarian cancer, therefore potentially affect treatment choice (e.g. discrimination between epithelial tumors and germ cell tumors); differential diagnosis between benign and malignant ovarian cysts; diagnosing a cause of infertility, for example differential diagnosis of various causes thereof; detecting of one or more non-ovarian cancer conditions that may elevate serum levels of ovary related markers, including but not limited to: cancers of the endometrium, cervix, fallopian tubes, pancreas, breast, lung and colon; nonmalignant conditions such as pregnancy, endometriosis, pelvic inflammatory disease and uterine fibroids; diagnosing conditions which have similar symptoms, signs and complications as ovarian cancer and where the differential diagnosis between them and ovarian cancer is of clinical importance including but not limited to: non-malignant causes of pelvic mass, including, but not limited to: benign (functional) ovarian cyst, uterine fibroids, endometriosis, benign ovarian neoplasms and inflammatory bowel lesions; determining a cause of any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, skeletal or abdominal pain, paraneoplastic syndrome, or ascites.
  • In some embodiments, the polypeptides and/or polynucleotides of this invention may be used in the diagnosis, treatment or prognostic assessment of ovarian cancer, alone or in combination with one or more polypeptides and/or polynucleotides of this invention, and/or in combination with known markers for ovarian cancer, including but not limited to CEA, CA125 (Mucin 16), CA72-4TAG, CA-50, CA 54-61, CA-195 and CA 19-9 in combination with CA-125, and/or in combination with the known protein(s) associated with the indicated polypeptide or polynucleotide, as described herein.
  • Detecting specific expression may in some embodiments be performed with a NAT-based technology (optionally comprising at least one nucleotide probe or primer), and/or with an immunoassay (optionally comprising an antibody according to any of the embodiments described herein).
  • In some embodiments, this invention provides a method of detecting, treating and/or assessing prognosis of a disease, disorder or condition, comprising detecting polypeptides and/or polynucleotides of this invention. In some embodiments, such methods are also referred to herein as methods of screening for variant-detectable disease, whereby the detection of variant expression serves as an indicator for the disease. In some embodiments, such detection may make use of a biomarker, antibody or any method or assay as described herein.
  • In some embodiments, this invention provides a method for screening for a disease, comprising detecting expression of:
      • a. a polypeptide having an amino acid sequence as set forth in SEQ ID NOs: 16-31, 65-70, 99-102, 127-138, 215-219, 236, 245, 254-255, 307-308 or a homologue or fragment thereof;
      • b. a polypeptide comprising a bridge, edge portion, tail, or head portion, wherein said polypeptide has an amino acid sequence as set forth in SEQ ID NOs:459-501, 576-592 or a homologue or fragment thereof;
      • c. a polynucleotide having a nucleic acid sequence as set forth in SEQ ID NOs: 1-15, 61-64, 96-98, 114-126, 189-195, 211-214, 235, 244, 152-253, 305-306, 340-344. or a homologue or fragment thereof;
      • d. a polynucleotide comprising a node having a nucleic acid sequence as set forth in SEQ ID NOs: 32-60, 71-95, 103-113, 139-188, 196-219, 220-234, 237-243, 246-251, 256-304, 309-339, 350-358, 502-530;
      • e. an antibody capable of specifically binding to at least one epitope of a polypeptide comprising an amino acid sequence as set forth in SEQ ID NOs: 16-31, 65-70, 99-102, 127-138, 215-219, 236, 245, 254-255, 307-308, 459-501, 576-592;
      • f. an oligonucleotide having a nucleic acid sequence as set forth in SEQ ID NOs:363, 383, 386, 389, 393, 396, 399, 407, 410, 414, 417, 420, 424, 427, 431, 434, 437, 444, 447, 452, 455, 458;
      • g. a primer pair, comprising a pair of isolated oligonucleotides capable of specifically hybridizing to at least a portion of a polynucleotide having a nucleic acid sequence as set forth in SEQ ID NOs: 363, 383, 386, 389, 393, 396, 399, 407, 410, 414, 417, 420, 424, 427, 431, 434, 437, 444, 447, 452, 455; or homologous thereto;
      • h. a primer pair, comprising a pair of isolated oligonucleotides capable of specifically hybridizing to at least a portion of a polynucleotide having a nucleic acid sequence as set forth in SEQ ID NOs:361-362; 384-385; 387-388; 390-391; 394-395; 397-398; 400-401; 408-409; 411-412; 415-416; 418-419; 421-422; 425-426; 428-429; 432-433; 435-436; 438-439; 445-446; 448-449; 453-454; 456-457,
        whereby qualitative or quantitative differences in expression as compared to an index sample is indicator for the treatment, diagnosis or assessment of prognosis of the disease, disorder or condition.
  • In some embodiments, a method of this invention may make use of a polynucleotide, polypeptide, vector, antibody, biomarker, or combination thereof, as described herein, including any embodiments thereof.
  • In some embodiments, the methods of this invention may be conducted on a cell or tissue or body fluid sample isolated from a subject having, predisposed to, or suspected of having the disease disorder or condition. In some embodiments, the methods are directed to the monitoring of disease progression and/or treatment efficacy and/or relapse of the indicated disease, disorder or condition.
  • In another embodiment, this invention provides methods for the selection of a particular therapy, or optimization of a given therapy for a disease, disorder or condition, the method comprising quantitatively and/or qualitatively determining or assessing expression of the polypeptides and/or polynucleotides, whereby differences in expression from an index sample, or a sample taken from a subject prior to the initiation of the therapy, or during the course of therapy, is indicative of the efficacy, or optimal activity of the therapy.
  • In some embodiments, for the polypeptides and/or polynucleotides of this invention are useful in applications in cardiac disease, as described, and provide for sensitive and accurate assessment. Biomolecular sequences (amino acid and/or nucleic acid sequences) uncovered using the methodology of the present invention and described herein can be utilized, in some embodiments, as tissue or pathological markers and/or as drugs or drug targets for treating preventing, diagnosing or assessing a disease.
  • In some embodiments, these markers are specifically released to the bloodstream under conditions of cardiac disease and/or cardiac pathology, as described herein. presenting some embodiments, this invention identified, or provides the means to identify clusters (genes) which are characterized in that their transcripts are differentially expressed in heart muscle tissue compared with other normal tissues, for example, in comparison to skeletal muscle tissue. In acute conditions under which heart muscle tissue experiences hypoxia (with or without necrosis), intracellular proteins that are not normally secreted can leak through the cell membrane to the extracellular space. Therefore, heart muscle tissue differentially expresses proteins, and analysis methods as described herein may herald acute heart damage, thereby serving as cardiac disease markers.
  • In some embodiments, the identification/detection of the polypeptides and/or polynucleotides of this invention signify leakage of intracellular content, which can occur in chronic damage to the heart muscle, therefore proteins selected according to this method are potential markers for chronic heart conditions. When a protein that is differentially expressed in heart muscle is secreted, it may prove useful as a chronic heart damage marker, since secretion implies that the protein has a physiological role exterior to the cell, and in some embodiments may be used by the heart muscle to respond to the chronic damage.
  • In some embodiments, the markers described herein are overexpressed in heart versus skeletal muscle.
  • In some embodiments this invention provides 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), involving detection, in some embodiments of expression in a sample taken from a subject (patient), which in some embodiments, is a blood sample.
  • In some embodiments, the polypeptides and polynucleotides and methods of this invention find application in various cardiovascular and cerebrovascular conditions, and in some embodiments, the conditions may also optionally include stroke and various cardiomyopathies.
  • In some embodiments, the marker-detectable disease involves cluster Z25299 and comprises a variety of cancers, including but not limited to colon cancer, breast cancer, ovarian cancer, lung cancer; and colon, breast, ovarian, and lung cancer invasion and metastasis.
  • In some embodiments, the marker-detectable disease involves cluster AA336074 and comprises a variety of cancers, including but not limited to breast cancer, lung cancer; and breast and lung cancer invasion and metastasis.
  • In some embodiments, the marker-detectable disease involves cluster HSPLGF and comprises a variety of cancers, including but not limited to colon cancer, lung cancer; and colon and lung cancer invasion and metastasis.
  • In some embodiments, the marker-detectable disease involves cluster HSI1RA, cluster HUMSP18A, cluster F05068 and comprises a variety of cancers, including but not limited to lung cancer and lung cancer invasion and metastasis.
  • In some embodiments, the marker-detectable disease involves cluster Z22012, cluster HUMTREFAC, or cluster Z39737 and comprises prostate cancer.
  • According to some embodiments of the present invention, any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, at least about 80%, at least about 90%, least about 95% homology to the polynucleotides herein described.
  • The nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate, in some embodiments, 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 the terms “oligonucleotide” and “polynucleotide”, or “peptide” and “polypeptide”, may optionally be used interchangeably.
  • All technical and scientific terms used herein should be understood to have the meaning commonly understood by a person skilled in the art to which this invention belongs, as well as any other specified description. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). All of these are hereby incorporated by reference as if fully set forth herein.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
  • In the drawings:
  • FIG. 1: Schematic description of the cancer biomarker selection engine;
  • FIG. 2: Schematic illustration, depicting grouping of transcripts of a given cluster based on presence or absence of unique sequence regions;
  • FIG. 3 shows the schematic summary of quantitative real-time PCR analysis;
  • FIG. 4 shows the structure of the HSFLT variants mRNA and protein. Exons are represented by white boxes, while introns are represented by two headed arrows. Proteins are shown in boxes with upper right to lower left fill. The unique regions are represented by white boxes with dashed frame.
  • FIG. 5 shows expression of Homo sapiens fins-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) (FLT1) transcripts detectable by or according to seg20—HSFLT_seg20 (SEQ ID NO:363) amplicon and primers HSFLT_seg20F (SEQ ID NO:361) and HSFLT_seg20R (SEQ ID NO:362) on a normal panel;
  • FIG. 6 shows the structure of the HSI1RA mRNA and protein variants. Exons are represented by white boxes, while introns are represented by two headed arrows. Proteins are shown in boxes with upper right to lower left fill. The unique regions are represented by white boxes with dashed frame.
  • FIG. 7 is a histogram showing over expression of the Homo sapiens interleukin 1 receptor antagonist (IL1RN) HSI1RA transcripts which are detectable by amplicon as depicted in sequence name HSI1RA_junc15-17 (SEQ ID NO:376) in normal and cancerous Lung tissues;
  • FIG. 8 shows expression of Homo sapiens interleukin 1 receptor antagonist (IL1RN) HSI1RA transcripts which are detectable by amplicon as depicted in sequence name HSI1RA_junc15-17 (SEQ ID NO:376) in different normal tissues;
  • FIG. 9 is a histogram showing down regulation of the above-indicated Homo sapiens interleukin 1 receptor antagonist (IL1RN) transcripts in cancerous lung samples relative to the normal samples (junc23-30);
  • FIG. 10 shows expression of Homo sapiens interleukin 1 receptor antagonist (IL1RN) HSI1RA transcripts which are detectable by amplicon as depicted in sequence name HSI1RA_junc23-30 (SEQ ID NO: 383) in different normal tissues;
  • FIGS. 11A and 11B are histograms showing down regulation of the above-indicated Homo sapiens interleukin 1 receptor antagonist (IL1RN) transcripts in cancerous lung samples relative to the normal samples (seg 23-24);
  • FIG. 12 shows expression of Homo sapiens interleukin 1 receptor antagonist (IL1RN) HSI1RA transcripts which are detectable by amplicon as depicted in sequence name HSI1RA_seg23-24 (SEQ ID NO: 386) in different normal tissues;
  • FIGS. 13A and 13B are histograms showing down regulation of the above-indicated Homo sapiens interleukin 1 receptor antagonist (IL1RN) transcripts in cancerous lung samples relative to the normal samples (seg 36-37);
  • FIG. 14 shows expression of Homo sapiens interleukin 1 receptor antagonist (IL1RN) HSI1RA transcripts which are detectable by amplicon as depicted in sequence name HSI1RA_seg36-37WT (SEQ ID NO: 389) in different normal tissues;
  • FIG. 15 shows the structure of the HSPLGF mRNA and protein variants. Exons are represented by white boxes, while introns are represented by two headed arrows. Proteins are shown in boxes with upper right to lower left fill. The unique regions are represented by white boxes with dashed frame.
  • FIG. 16 is a histogram showing over expression of the above-indicated Homo sapiens placental growth factor, vascular endothelial growth factor-related protein (PLGF) transcripts in cancerous Lung samples relative to the normal samples;
  • FIG. 17 shows expression of Homo sapiens placental growth factor, vascular endothelial growth factor-related protein (PGF) HSPLGF transcripts which are detectable by amplicon as depicted in sequence name HSPLGF_seg7WT (SEQ ID NO: 393) in different normal tissues;
  • FIG. 18 shows expression of Homo sapiens placental growth factor, vascular endothelial growth factor-related protein (PlGF) HSPLGF transcripts which are detectable by amplicon as depicted in sequence name HSPLGF_seg15-16 (SEQ ID NO:396) in different normal tissues;
  • FIG. 19 is a histogram showing over expression of the above-indicated Homo sapiens placental growth factor, vascular endothelial growth factor-related protein (PGF) transcripts in cancerous Lung samples relative to the normal samples (seg16-21);
  • FIG. 20 is a histogram showing over expression of the above-indicated Homo sapiens placental growth factor, vascular endothelial growth factor-related protein (PGF) transcripts in cancerous colon samples relative to the normal samples (seg16-21);
  • FIG. 21 shows expression of Homo sapiens placental growth factor, vascular endothelial growth factor-related protein (PlGF) HSPLGF transcripts which are detectable by amplicon as depicted in sequence name HSPLGF_seg16-21 (SEQ ID NO:399) in different normal tissues;
  • FIG. 22 shows the structure of the mRNA and protein variants of cluster HUMSP18A;
  • FIG. 23 is a histogram showing down regulation of the above-indicated Homo sapiens surfactant, pulmonary-associated protein B (SFTPB), transcript variant 2 transcripts (seg32) in cancerous lung samples relative to the normal samples;
  • FIG. 24 shows expression of Homo sapiens surfactant, pulmonary-associated protein B (SFTPB) transcripts detectable by or according to seg32 in normal tissues;
  • FIG. 25 is a histogram showing down regulation of the above-indicated Homo sapiens surfactant, pulmonary-associated protein B (SFTPB), transcript variant 2 transcripts in cancerous lung samples relative to the normal samples (seg34-38WT);
  • FIG. 26 shows the results of expression of Homo sapiens surfactant, pulmonary-associated protein B (SFTPB) transcripts detectable by or according to seg34-38WT on a normal panel;
  • FIG. 27 shows cancer and cell-line vs. normal tissue expression for cluster F05068;
  • FIG. 28 shows the structure of the F05068 mRNA and protein variants. Exons are represented by white boxes, while introns are represented by two headed arrows. Proteins are shown in boxes with upper right to lower left fill. The unique regions are represented by white boxes with dashed frame.
  • FIG. 29 shows expression of Homo sapiens adrenomedullin (ADM) F05068 transcripts which are detectable by amplicon as depicted in sequence name F05068_seg3-5 (SEQ ID NO: 414) in different normal tissues;
  • FIG. 30 is a histogram showing down regulation of the above-indicated Homo sapiens adrenomedullin (ADM) transcripts in cancerous lung samples relative to the normal samples (seg9);
  • FIG. 31 shows expression of Homo sapiens adrenomedullin (ADM) F05068 transcripts which are detectable by amplicon as depicted in sequence name F05068_seg9 (SEQ ID NO: 417) in different normal tissues;
  • FIG. 32 shows expression of Homo sapiens adrenomedullin (ADM) F05068 transcripts which are detectable by amplicon as depicted in sequence name F05068_seg13_WT (SEQ ID NO:420) in different normal tissues;
  • FIG. 33 is a histogram showing the expression of Homo sapiens adrenomedullin (ADM) F05068 transcripts which are detectable by amplicon as depicted in sequence name F05068_seg3-5 (SEQ ID NO: 414) in normal and cancerous Lung tissues.
  • FIG. 34 shows the structure of the HUMIL10 mRNA and protein variants. Exons are represented by white boxes, while introns are represented by two headed arrows. Proteins are shown in boxes with upper right to lower left fill. The unique regions are represented by white boxes with dashed frame.
  • FIGS. 35A and 35B show expression of Homo sapiens interleukin 10 (IL10) transcripts detectable by or according to seg5, with the value of relative expression of each sample relative to median of the heart samples as shown in FIG. 35A, or with the value of relative expression of each sample relative to median of the blood samples as shown in FIG. 35B;
  • FIGS. 36A and 36B show expression of Homo sapiens interleukin 10 (IL10) transcripts detectable by or according to seg0WT, with the value of relative expression of each sample relative to median of the heart samples as shown in FIG. 36A, or with the value of relative expression of each sample relative to median of the blood samples as shown in FIG. 36B;
  • FIG. 37 shows that cluster AA336074 is overexpressed (at least at a minimum level) in the following pathological conditions: prostate cancer, a mixture of malignant tumors from different tissues and epithelial malignant tumors;
  • FIG. 38 shows the structure of the AA336074 mRNA and protein variants. Exons are represented by white boxes, while introns are represented by two headed arrows. Proteins are shown in boxes with upper right to lower left fill. The unique regions are represented by white boxes with dashed frame.
  • FIG. 39 is a histogram showing Expression of Homo sapiens kallikrein 4 ((KLK4) AA336074 transcripts which are detectable by amplicon as depicted in sequence name AA336074_junc9-28 (SEQ ID NO:431) in normal and cancerous Breast tissues.
  • FIG. 40 is a histogram showing the expression of Homo sapiens kallikrein 4 (KLK4) AA336074 transcripts which are detectable by amplicon as depicted in sequence name AA336074_junc9-28 (SEQ ID NO:431) in different normal tissues.
  • FIG. 41 is a histogram showing Expression of Homo sapiens kallikrein 4 ((KLK4) AA336074 transcripts which are detectable by amplicon as depicted in sequence name AA336074_seg13WT (SEQ ID NO:434) in normal and cancerous Breast tissues.
  • FIG. 42 is a histogram showing over expression of the Homo sapiens kallilrein 4 (KLK4) AA336074 transcripts which are detectable by amplicon as depicted in sequence name AA336074_seg13WT (SEQ ID NO:434) in different normal tissues.
  • FIG. 43 is a histogram showing over expression of the Homo sapiens kallikrein 4 (KLK4) AA336074 transcripts which are detectable by amplicon as depicted in sequence name AA336074 junc 9-28 (SEQ ID NO: 431) in normal and cancerous lung tissues.
  • FIG. 44 is a histogram showing over expression of the Homo sapiens kallikrein 4 (KLK4) AA336074 transcripts which are detectable by amplicon as depicted in sequence name AA336074 seg31 (SEQ ID NO: 437) in normal and cancerous breast tissues.
  • FIG. 45 is a histogram showing over expression of the Homo sapiens kallikrein 4 (prostase, enamel matrix, prostate) (KLK4) AA336074 transcripts which are detectable by amplicon as depicted in sequence name AA336074_seg31 (SEQ ID NO:437) in different normal tissues.
  • FIG. 46 shows that cluster HUMTREFAC is overexpressed (at least at a minimum level) in the following pathological conditions: a mixture of malignant tumors from different tissues, pancreas carcinoma, prostate cancer, breast malignant tumors and epithelial malignant tumors;
  • FIG. 47 shows mRNA and protein structure of HUMTREFAC variants. Exons are represented by white boxes, while introns are represented by two headed arrows. Proteins are shown in boxes with upper right to lower left fill. The unique regions are represented by white boxes with dashed frame.
  • FIG. 48 shows that cluster Z22012 is overexpressed (at least at a minimum level) in the following pathological conditions: brain malignant tumors, pancreas carcinoma, hepatocellular carcinoma, prostate cancer, a mixture of malignant tumors from different tissues and myosarcoma;
  • FIG. 49 shows that cluster Z39737 is overexpressed (at least at a minimum level) in the following pathological conditions: prostate cancer.
  • FIG. 50 shows mRNA and Protein Structure of Z39737 variants. Exons are represented by white boxes, while introns are represented by two headed arrows. Proteins are shown in boxes with upper right to lower left fill. The unique regions are represented by white boxes with dashed frame.
  • FIG. 51—shows a graph of cancer and cell-line vs. normal tissue expression for Z25299.
  • FIG. 52 shows mRNA and Protein Structure of Z25299 variants. Exons are represented by white boxes, while introns are represented by two headed arrows. Proteins are shown in boxes with upper right to lower left fill. The unique regions are represented by white boxes with dashed frame.
  • FIG. 53 is a histogram showing expression of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299_junc13-14-21 (SEQ ID NO:444) in different normal tissues.
  • FIG. 54 is a histogram showing expression of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299_seg12-13WT (SEQ ID NO: 447) in different normal tissues.
  • FIGS. 55-56 are histograms showing down regulation of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299_seg12-13WT (SEQ ID NO: 447) in cancerous lung samples relative to the normal samples.
  • FIG. 57 is a histogram showing over expression of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299_seg12-13WT (SEQ ID NO: 447) in cancerous Ovary samples relative to the normal samples.
  • FIGS. 58 and 59 are histograms showing down regulation of the Secretory leukocyte protease inhibitor Acid-stable proteinase inhibitor transcripts, which are detectable by amplicon as depicted in sequence name Z25299 seg20 (SEQ ID NO: 452) in cancerous lung samples relative to the normal samples.
  • FIG. 60 is a histogram showing expression of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299_seg20 (SEQ ID NO: 452) in different normal tissues.
  • FIG. 61 is a histogram showing over expression of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299_seg23 (SEQ ID NO: 455) in cancerous colon samples relative to the normal samples.
  • FIG. 62 is a histogram showing down regulation of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299_seg23 (SEQ ID NO: 455) in cancerous lung samples relative to the normal samples.
  • FIG. 63 is a histogram showing expression of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299_seg23 (SEQ ID NO: 455) in different normal samples.
  • FIG. 64 is a histogram showing over expression of Z25299_junc13-14-21 (SEQ ID NO: 444) transcripts in cancerous ovary samples relative to the normal samples.
  • FIG. 65 is a histogram showing over expression of Z25299 seg20 (SEQ ID NO: 452) transcripts in cancerous ovary samples relative to the normal samples.
  • FIG. 66 is a histogram showing over expression of Z25299 seg23 (SEQ ID NO: 455) transcripts in cancerous ovary samples relative to the normal samples.
  • FIG. 67 is a histogram showing down regulation of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299_junc13-14-21 (SEQ ID NO: 444) in cancerous lung tissues relative to the normal samples.
  • FIG. 68 is a histogram showing over expression of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299 seg20 (SEQ ID NO: 452) in cancerous Colon tissues relative to the normal samples.
  • FIG. 69 is a histogram showing down regulation of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299 seg20 (SEQ ID NO: 452) in cancerous breast tissues relative to the normal samples.
  • FIG. 70 is a histogram showing down regulation of the Homo sapiens secretory leukocyte protease inhibitor (antileukoproteinase) Z25299 transcripts which are detectable by amplicon as depicted in sequence name Z25299 seg23 (SEQ ID NO: 455) in cancerous breast tissues relative to the normal samples.
  • DESCRIPTION OF EMBODIMENTS
  • The present invention provides, in some embodiments, polynucleotides and polypeptides and uses thereof, as further described herein. polynucleotides and polypeptides described herein, in some embodiments, represent variants, which may optionally be used as diagnostic markers.
  • In some embodiments, these variants are useful as diagnostic markers for certain diseases, and as such the term “marker-detectable” or “variant-detectable” with regard to diseases is to be understood as encompassing use of the described polynucleotides and/or polypeptides.
  • In some embodiments, certain diseases are associated with differential expression, qualitatively or quantitatively, of the polynucleotides and polypeptides of this invention. Assessment of such expression, in turn, may in some embodiments, serve as a marker for a particular disease state, susceptibility, pathogenesis, etc., including any desired disease-specific event, whose analysis is useful, as will be appreciated by one skilled in the art. In one embodiment, such use as a marker is also referred to herein as the polynucleotides and polypeptides being “variant disease markers”.
  • The polynucleotides and polypeptides of the present invention, alone or in combination, in some embodiments, can be used for, and in some embodiments are a part of the methods of prognosis, prediction, screening, early diagnosis, staging, therapy selection and treatment monitoring of a marker-detectable disease. For example, in some embodiments, these markers may be used for the staging of disease in a patient (for example if the disease features cancer) and/or monitoring the progression of the disease. Furthermore, the markers of the present invention, alone or in combination, can be used for detection of the source of metastasis found in anatomical places other than the originating tissue, again in the example of cancer. Also, 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, in some embodiments, as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease.
  • In some embodiments, these markers are 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. In some embodiments, the measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate 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 provides, in some embodiments, diagnostic assays for a 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, for example in a sample taken from a subject (patient), which in some embodiments, is a blood sample.
  • Some embodiments of this invention have been exemplified herein wherein cellular localization was determined via the use of four different software programs: (i) tmhmm (from Center for Biological Sequence Analysis, Technical University of Denmark DTU, www.cbs.dtu.dk/services/TMHMM/TMHMM2.0b.guide.php) or (ii) tmpred (from EMBnet, maintained by the ISREC Bioinformatics group and the LICR Information Technology Office, Ludwig Institute for Cancer Research, Swiss Institute of Bioinformatics, www.ch.embnet.org/software/TMPRED_form.html) for transmembrane region prediction; (iii) signalp_hmm or (iv) signalp_nn (both from Center for Biological Sequence Analysis, Technical University of Denmark DTU, www.cbs.dtu.dk/services/SignalP/background/prediction.php) for signal peptide prediction. The terms “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. In some cases for the manual inspection of cellular localization prediction inventors used the ProLoc computational platform [Einat Hazkani-Covo, Erez Levanon, Galit Rotman, Dan Graur and Amit Novik; (2004) “Evolution of multicellularity in metazoa: comparative analysis of the subcellular localization of proteins in Saccharomyces, Drosophila and Caenorhabditis.” Cell Biology International 2004; 28(3):171-8.], which predicts protein localization based on various parameters including, protein domains (e.g., prediction of trans-membranous regions and localization thereof within the protein), pI, protein length, amino acid composition, homology to pre-annotated proteins, recognition of sequence patterns which direct the protein to a certain organelle (such as, nuclear localization signal, NLS, mitochondria localization signal), signal peptide and anchor modeling and using unique domains from Pfam that are specific to a single compartment.
  • Information is given in the text with regard to SNPs (single nucleotide polymorphisms). A description of the abbreviations is as follows. “T->C”, for example, means that the SNP results in a change at the position given in the table from T to C. Similarly, “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 (*). As part of the description of an SNP, 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 FTId is always the last component of a feature in the description field, as follows: FTId=XXX_number, in which XXX is the 3-letter code for the specific feature key, separated by an underscore from a 6-digit number. In the table of the amino acid mutations of the wild type proteins of the selected splice variants of the invention, 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. 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.
  • Some embodiments of this invention have been exemplified herein wherein homology to known proteins was determined by Smith-Waterman version 5.1.2 using special (non default) parameters as follows:
  • model=sw.model
  • GAPEXT=0
  • GAPOP=100.0
      • MATRIX=blosum100
  • Some embodiments of this invention have been exemplified herein wherein overexpression of a cluster in cancer was a determination based on ESTs. 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 (P1)
      • library based statistics: P-value including the level 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 clone 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.
  • Some embodiments of this invention have been exemplified herein wherein overexpression of a cluster in cancer was a determination based on microarray use. 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. For microarrays prepared according to a design by the present inventors, 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, Calif., USA (see for example data regarding the Human Genome U133 (HG-U133) Set at www.affymetrix.com/products/arrays/specific/hgu133.affx; GeneChip Human Genome U133A 2.0 Array at www.affymetrix.com/products/arrays/specific/hgu133av2.affx; and Human Genome U133 Plus 2.0 Array at www.affymetrix.com/products/arrays/specific/hgu133plus.affx). The probe names follow the Affymetrix naming convention. The data is available from NCBI Gene Expression Omnibus (see www.ncbi.nlm.nih.gov/projects/geo/ and Edgar et al, Nucleic Acids Research, 2002, Vol. 30, No. 1 207-210). The dataset (including results) is available from www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1133 for the Series GSE1133 database (published on March 2004); a reference to these results is as follows: Su et al (Proc Natl Acad Sci USA. 2004 Apr. 20; 101(16):6062-7. Epub 2004 Apr. 9).
  • Oligonucleotide microarray results taken from Affymetrix data were from chips available from Affymetrix Inc, Santa Clara, Calif., USA (see for example data regarding the Human Genome U133 (HG-U133) Set at www.affymetrix.com/products/arrays/specific/hgu133.affx; GeneChip Human Genome U133A 2.0 Array at www.affymetrix.com/products/arrays/specific/hgu133av2.affx; and Human Genome U133 Plus 2.0 Array at www.affymetrix.com/products/arrays/specific/hgu133plus.affx). The probe names follow the Affymetrix naming convention. The data is available from NCBI Gene Expression Omnibus (see www.ncbi.nlm.nih.gov/projects/geo/ and Edgar et al, Nucleic Acids Research, 2002, Vol. 30, No. 1 207-210). The dataset (including results) is available from www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1133 for the Series GSE1133 database (published on March 2004); a reference to these results is as follows: Su et al (Proc Natl Acad Sci USA. 2004 Apr. 20; 101(16):6062-7. Epub 2004 Apr. 9).
  • The following list of abbreviations for tissues was used in the TAA histograms. The term “TAA” stands for “Tumor Associated Antigen”, and the TAA histograms, given in the text, represent the cancerous tissue expression pattern as predicted by the biomarkers selection engine, as described in detail in examples 1-5 below (the first word is the abbreviation while the second word is the full name):
  • (“BONE”, “bone”);
    (“COL”, “colon”);
    (“EPI”, “epithelial”);
    (“GEN”, “general”);
    (“LIVER”, “liver”);
    (“LUN”, “lung”);
    (“LYMPH”, “lymph nodes”);
    (“MARROW”, “bone marrow”);
    (“OVA”, “ovary”);
    (“PANCREAS”, “pancreas”);
    (“PRO”, “prostate”);
    (“STOMACH”, “stomach”);
    (“TCELL”, “T cells”);
  • (“THYROID”, “Thyroid”);
  • (“MAM”, “breast”);
    (“BRAIN”, “brain”);
    (“UTERUS”, “uterus”);
    (“SKIN”, “skin”);
    (“KIDNEY”, “kidney”);
    (“MUSCLE”, “muscle”);
    (“ADREN”, “adrenal”);
    (“HEAD”, “head and neck”);
    (“BLADDER”, “bladder”);
  • It should be noted that the terms “segment”, “seg” and “node” (abbreviated as “N” in the names of nodes) are used interchangeably in reference to nucleic acid sequences of the present invention, they refer to portions of nucleic acid sequences that were shown to have one or more properties as described herein. They are also the building blocks that were used to construct complete nucleic acid sequences as described in greater detail elsewhere herein. Optionally and preferably, they are examples of 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.
  • In some embodiments, the phrase “disease” refers to its commonly understood meaning, and includes, inter alia, any type of pathology and/or damage, including both chronic and acute damage, as well as a progress from acute to chronic damage.
  • In some embodiments, the phrase “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.
  • In some embodiments, the phrase “differentially present” refers to differences in the quantity or quality of a marker present in a sample taken from patients having one of the herein-described diseases or conditions as compared to a comparable sample taken from patients who do not have one of the herein-described diseases or conditions. For example, a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT-based assays. A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample. It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present. Optionally, a relatively low amount of up-regulation may serve as the marker, as described herein. One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided in the description of each individual marker below.
  • In some embodiments, the phrase “diagnostic” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay are termed “true negatives.” The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the “false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • In some embodiments, the phrase “qualitative” when in reference to differences in expression levels of a polynucleotide, polypeptide or cluster as described herein, refers to the presence versus absence of expression, or in some embodiments, the temporal regulation of expression, or in some embodiments, the timing of expression, or in some embodiments, the variant expressed, or in some embodiments, any post-translational modifications to the expressed molecule, and others, as will be appreciated by one skilled in the art. In some embodiments, the phrase “quantitative” when in reference to differences in expression levels of a polynucleotide, polypeptide or cluster as described herein, refers to absolute differences in quantity of expression, as determined by any means, known in the art, or in other embodiments, relative differences, which may be statistically significant, or in some embodiments, when viewed as a whole or over a prolonged period of time, etc., indicate a trend in terms of differences in expression.
  • In some embodiments, the term “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, in some embodiments, be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease. It should be noted that 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.
  • In some embodiments, the term “level” refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention.
  • Typically 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. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.
  • Determining the level of the same variant in normal tissues of the same origin is preferably effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.
  • In some embodiments, the term “test amount” of a marker 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).
  • In some embodiments, the term “control amount” of a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, 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).
  • In some embodiments, the term “detect” refers to identifying the presence, absence or amount of the object to be detected.
  • In some embodiments, the term “label” includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, 35S, 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. For example, 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, optionally and preferably for use with immunoassays, 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. Alternatively, 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 phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” or “specifically interacts or binds” when referring to a protein or peptide (or other epitope), refers, in some embodiments, to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, 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. For example, polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein. This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, 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). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • In another embodiment, 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.
  • In some embodiments, the term “tail” refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (often 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.
  • In some embodiments, the term “head” refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein.
  • In some embodiments, the term “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.
  • In some embodiments, 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, or in some embodiments at least about 20 amino acids, or in some embodiments at least about 30 amino acids, or in some embodiments 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. In some embodiments, the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 11, 12, 13.37, 38, 39, 40 amino acids in length, or any number in between).
  • It should be noted that a bridge cannot be extended beyond the length of the sequence in either direction, and it should be assumed that every bridge description is to be read in such manner that the bridge length does not extend beyond the sequence itself.
  • Furthermore, bridges are described with regard to a sliding window in certain contexts below. For example, certain descriptions of the bridges feature the following format: a bridge between two edges (in which a portion of the known protein is not present in the variant) may optionally be described as follows: a bridge portion of CONTIG-NAME_P1 (representing the name of the protein), comprising a polypeptide having a length “n”, wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), having a structure as follows (numbering according to the sequence of CONTIG-NAME_P1): a sequence starting from any of amino acid numbers 49−x to 49 (for example); and ending at any of amino acid numbers 50+((n−2)−x) (for example), in which x varies from 0 to n−2. In this example, it should also be read as including bridges in which n is any number of amino acids between 10-50 amino acids in length. Furthermore, the bridge polypeptide cannot extend beyond the sequence, so it should be read such that 49−x (for example) is not less than 1, nor 50+((n−2)−x) (for example) greater than the total sequence length.
  • In another embodiment, this invention provides isolated nucleic acid molecules, which in some embodiments encode for splice variants, having a nucleotide sequence as set forth in any one of the sequences listed herein, being homologous to such sequences, at a percent as described herein, or a sequence complementary thereto. In another embodiment, this invention provides an oligonucleotide of at least about 12 nucleotides, which specifically hybridizes with the nucleic acid molecules of this invention. In another embodiment, this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids or polypeptides of this invention, as appropriate.
  • In another embodiment, this invention provides a method for detecting the polypeptides of this invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting said interaction; wherein the presence of an interaction correlates with the presence of a splice variant in the biological sample.
  • In another embodiment, this invention provides a method for detecting a polynucleotide of this invention in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a the polynucleotide in the biological sample.
  • In some embodiments of the present invention, the polypeptides/polynucleotides described herein are non-limiting examples of markers for diagnosing marker-detectable disease and/or an indicative condition. Each polypeptide/polynucleotide 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.
  • According to some embodiments of the present invention, 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. Furthermore, 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. With regard to such a ratio between any marker described herein (or a combination thereof) and a known marker, more preferably the known marker comprises the “known protein” as described in greater detail below with regard to each cluster or gene.
  • In some embodiments of the present invention, there are provided of methods, uses, devices and assays for the diagnosis of a disease or condition. Optionally a plurality of biomarkers (or markers) may be used with the present invention. The plurality of markers may optionally include a plurality of markers described herein, and/or one or more known markers. The plurality of markers is preferably then correlated with the disease or condition. For example, such correlating may optionally comprise determining the concentration of each of the plurality of markers, and individually comparing each marker concentration to a threshold level. Optionally, if the marker concentration is above or below the threshold level (depending upon the marker and/or the diagnostic test being performed), the marker concentration correlates with the disease or condition. Optionally and preferably, a plurality of marker concentrations correlate with the disease or condition.
  • Alternatively, such correlating may optionally comprise determining the concentration of each of the plurality of markers, calculating a single index value based on the concentration of each of the plurality of markers, and comparing the index value to a threshold level.
  • Also alternatively, such correlating may optionally comprise determining a temporal change in at least one of the markers, and wherein the temporal change is used in the correlating step.
  • Also alternatively, such correlating may optionally comprise determining whether at least “X” number of the plurality of markers has a concentration outside of a predetermined range and/or above or below a threshold (as described above). The value of “X” may optionally be one marker, a plurality of markers or all of the markers; alternatively or additionally, rather than including any marker in the count for “X”, one or more specific markers of the plurality of markers may optionally be required to correlate with the disease or condition (according to a range and/or threshold).
  • Also alternatively, such correlating may optionally comprise determining whether a ratio of marker concentrations for two markers is outside a range and/or above or below a threshold. Optionally, if the ratio is above or below the threshold level and/or outside a range, the ratio correlates with the disease or condition.
  • Optionally, a combination of two or more these correlations may be used with a single panel and/or for correlating between a plurality of panels.
  • Optionally, the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to normal subjects. As used herein, sensitivity relates to the number of positive (diseased) samples detected out of the total number of positive samples present; specificity relates to the number of true negative (non-diseased) samples detected out of the total number of negative samples present. Preferably, the method distinguishes a disease or condition with a sensitivity of at least 80% at a specificity of at least 90% when compared to normal subjects. More preferably, the method distinguishes a disease or condition with a sensitivity of at least 90% at a specificity of at least 90% when compared to normal subjects. Also more preferably, the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to subjects exhibiting symptoms that mimic disease or condition symptoms.
  • A marker panel may be analyzed in a number of fashions well known to those of skill in the art. For example, each member of a panel may be compared to a “normal” value, or a value indicating a particular outcome. A particular diagnosis/prognosis may depend upon the comparison of each marker to this value; alternatively, if only a subset of markers are outside of a normal range, this subset may be indicative of a particular diagnosis/prognosis. The skilled artisan will also understand that diagnostic markers, differential diagnostic markers, prognostic markers, time of onset markers, disease or condition differentiating markers, etc., may be combined in a single assay or device. Markers may also be commonly used for multiple purposes by, for example, applying a different threshold or a different weighting factor to the marker for the different purpose(s).
  • In one embodiment, the panels comprise markers for the following purposes: diagnosis of a disease; diagnosis of disease and indication if the disease is in an acute phase and/or if an acute attack of the disease has occurred; diagnosis of disease and indication if the disease is in a non-acute phase and/or if a non-acute attack of the disease has occurred; indication whether a combination of acute and non-acute phases or attacks has occurred; diagnosis of a disease and prognosis of a subsequent adverse outcome; diagnosis of a disease and prognosis of a subsequent acute or non-acute phase or attack; disease progression (for example for cancer, such progression may include for example occurrence or recurrence of metastasis).
  • The above diagnoses may also optionally include differential diagnosis of the disease to distinguish it from other diseases, including those diseases that may feature one or more similar or identical symptoms.
  • In certain embodiments, one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicator(s). In other embodiments, threshold level(s) of a diagnostic or prognostic indicator(s) can be established, and the level of the indicator(s) in a patient sample can simply be compared to the threshold level(s). The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical “quality” of the test—they also depend on the definition of what constitutes an abnormal result. In practice, Receiver Operating Characteristic curves, or “ROC” curves, are typically calculated by plotting the value of a variable versus its relative frequency in “normal” and “disease” populations, and/or by comparison of results from a subject before, during and/or after treatment. For any particular marker, a distribution of marker levels for subjects with and without a disease will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition.
  • The horizontal axis of the ROC curve represents (1-specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cutoff selected, the value of (1-specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.
  • ROC curves can be used even when test results don't necessarily give an accurate number. As long as one can rank results, one can create an ROC curve. For example, results of a test on “disease” samples might be ranked according to degree (say 1=low, 2=normal, and 3=high). This ranking can be correlated to results in the “normal” population, and a ROC curve created. These methods are well known in the art (see for example Hanley et al., Radiology 143: 29-36 (1982), incorporated by reference as if fully set forth herein).
  • One or more markers may lack diagnostic or prognostic value when considered alone, but when used as part of a panel, such markers may be of great value in determining a particular diagnosis/prognosis. In some embodiments, particular thresholds for one or more markers in a panel are not relied upon to determine if a profile of marker levels obtained from a subject are indicative of a particular diagnosis/prognosis. Rather, the present invention may utilize an evaluation of the entire marker profile by plotting ROC curves for the sensitivity of a particular panel of markers versus 1-(specificity) for the panel at various cutoffs. In these methods, a profile of marker measurements from a subject is considered together to provide a global probability (expressed either as a numeric score or as a percentage risk) that an individual has had a disease, is at risk for developing such a disease, optionally the type of disease which the individual has had or is at risk for, and so forth etc. In such embodiments, an increase in a certain subset of markers may be sufficient to indicate a particular diagnosis/prognosis in one patient, while an increase in a different subset of markers may be sufficient to indicate the same or a different diagnosis/prognosis in another patient. Weighting factors may also be applied to one or more markers in a panel, for example, when a marker is of particularly high utility in identifying a particular diagnosis/prognosis, it may be weighted so that at a given level it alone is sufficient to signal a positive result. Likewise, a weighting factor may provide that no given level of a particular marker is sufficient to signal a positive result, but only signals a result when another marker also contributes to the analysis.
  • In some embodiments, markers and/or marker panels are selected to exhibit at least 70% sensitivity, more preferably at least 80% sensitivity, even more preferably at least 85% sensitivity, still more preferably at least 90% sensitivity, and most preferably at least 95% sensitivity, combined with at least 70% specificity, more preferably at least 80% specificity, even more preferably at least 85% specificity, still more preferably at least 90% specificity, and most preferably at least 95% specificity. In some embodiments, both the sensitivity and specificity are at least 75%, more preferably at least 80%, even more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95%. Sensitivity and/or specificity may optionally be determined as described above, with regard to the construction of ROC graphs and so forth, for example.
  • According to some embodiments of the present invention, individual markers and/or combinations (panels) of markers may optionally be used for diagnosis of time of onset of a disease or condition. Such diagnosis may optionally be useful for a wide variety of conditions, preferably including those conditions with an abrupt onset.
  • The phrase “determining the prognosis” as used herein refers to methods by which the skilled artisan can predict the course or outcome of a condition in a patient. The term “prognosis” does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, the skilled artisan will understand that the term “prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition. For example, in individuals not exhibiting the condition, the chance of a given outcome may be about 3%. In some embodiments, a prognosis is about a 5% chance of a given outcome, about a 7% chance, about a 10% chance, about a 12% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, and about a 95% chance. The term “about” in this context refers to +/−1%.
  • The skilled artisan will understand that associating a prognostic indicator with a predisposition to an adverse outcome is a statistical analysis. For example, a marker level of greater than 80 pg/mL may signal that a patient is more likely to suffer from an adverse outcome than patients with a level less than or equal to 80 pg/mL, as determined by a level of statistical significance. Additionally, a change in marker concentration from baseline levels may be reflective of patient prognosis, and the degree of change in marker level may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. In one embodiment the confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001. Exemplary statistical tests for associating a prognostic indicator with a predisposition to an adverse outcome are described hereinafter.
  • In other embodiments, a threshold degree of change in the level of a prognostic or diagnostic indicator can be established, and the degree of change in the level of the indicator in a patient sample can simply be compared to the threshold degree of change in the level. A preferred threshold change in the level for markers of the invention is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. The term “about” in this context refers to +/−10%. In yet other embodiments, a “nomogram” can be established, by which a level of a prognostic or diagnostic indicator can be directly related to an associated disposition towards a given outcome. The skilled artisan is acquainted with the use of such nomograms to relate two numeric values with the understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration because individual sample measurements are referenced, not population averages.
  • Exemplary, non-limiting methods and systems for identification of suitable biomarkers for marker panels are now described. Methods and systems for the identification of one or more markers for the diagnosis, and in particular for the differential diagnosis, of disease have been described previously. Suitable methods for identifying markers useful for the diagnosis of disease states are described in detail in U.S. patent application no. 2004-0126767, entitled METHOD AND SYSTEM FOR DISEASE DETECTION USING MARKER COMBINATIONS, filed Dec. 27, 2002, hereby incorporated by reference in its entirety as if fully set forth herein. One skilled in the art will also recognize that univariate analysis of markers can be performed and the data from the univariate analyses of multiple markers can be combined to form panels of markers to differentiate different disease conditions.
  • In developing a panel of markers useful in diagnosis, data for a number of potential markers may be obtained from a group of subjects by testing for the presence or level of certain markers. The group of subjects is divided into two sets, and preferably the first set and the second set each have an approximately equal number of subjects. The first set includes subjects who have been confirmed as having a disease or, more generally, being in a first condition state. For example, this first set of patients may be those that have recently had a disease and/or a particular type of the disease. The confirmation of this condition state may be made through more rigorous and/or expensive testing, preferably according to a previously defined diagnostic standard. Hereinafter, subjects in this first set will be referred to as “diseased”.
  • The second set of subjects are simply those who do not fall within the first set. Subjects in this second set may be “non-diseased;” that is, normal subjects. Alternatively, subjects in this second set may be selected to exhibit one symptom or a constellation of symptoms that mimic those symptoms exhibited by the “diseased” subjects.
  • The data obtained from subjects in these sets includes levels of a plurality of markers. Preferably, data for the same set of markers is available for each patient. This set of markers may include all candidate markers which may be suspected as being relevant to the detection of a particular disease or condition. Actual known relevance is not required. Embodiments of the methods and systems described herein may be used to determine which of the candidate markers are most relevant to the diagnosis of the disease or condition. The levels of each marker in the two sets of subjects may be distributed across a broad range, e.g., as a Gaussian distribution. However, no distribution fit is required.
  • As noted above, a marker often is incapable of definitively identifying a patient as either diseased or non-diseased. For example, if a patient is measured as having a marker level that falls within the overlapping region, the results of the test will be useless in diagnosing the patient. An artificial cutoff may be used to distinguish between a positive and a negative test result for the detection of the disease or condition. Regardless of where the cutoff is selected, the effectiveness of the single marker as a diagnosis tool is unaffected. Changing the cutoff merely trades off between the number of false positives and the number of false negatives resulting from the use of the single marker. The effectiveness of a test having such an overlap is often expressed using a ROC (Receiver Operating Characteristic) curve as described above.
  • As discussed above, the measurement of the level of a single marker may have limited usefulness. The measurement of additional markers provides additional information, but the difficulty lies in properly combining the levels of two potentially unrelated measurements. In the methods and systems according to embodiments of the present invention, data relating to levels of various markers for the sets of diseased and non-diseased patients may be used to develop a panel of markers to provide a useful panel response. The data may be provided in a database such as Microsoft Access, Oracle, other SQL databases or simply in a data file. The database or data file may contain, for example, a patient identifier such as a name or number, the levels of the various markers present, and whether the patient is diseased or non-diseased.
  • Next, an artificial cutoff region may be initially selected for each marker. The location of the cutoff region may initially be selected at any point, but the selection may affect the optimization process described below. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer. In an embodiment method, the cutoff region is initially centered about the center of the overlap region of the two sets of patients. In one embodiment, the cutoff region may simply be a cutoff point. In other embodiments, the cutoff region may have a length of greater than zero. In this regard, the cutoff region may be defined by a center value and a magnitude of length. In practice, the initial selection of the limits of the cutoff region may be determined according to a pre-selected percentile of each set of subjects. For example, a point above which a pre-selected percentile of diseased patients are measured may be used as the right (upper) end of the cutoff range.
  • Each marker value for each patient may then be mapped to an indicator. The indicator is assigned one value below the cutoff region and another value above the cutoff region. For example, if a marker generally has a lower value for non-diseased patients and a higher value for diseased patients, a zero indicator will be assigned to a low value for a particular marker, indicating a potentially low likelihood of a positive diagnosis. In other embodiments, the indicator may be calculated based on a polynomial. The coefficients of the polynomial may be determined based on the distributions of the marker values among the diseased and non-diseased subjects.
  • The relative importance of the various markers may be indicated by a weighting factor. The weighting factor may initially be assigned as a coefficient for each marker. As with the cutoff region, the initial selection of the weighting factor may be selected at any acceptable value, but the selection may affect the optimization process. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer. In an embodiment method, acceptable weighting coefficients may range between zero and one, and an initial weighting coefficient for each marker may be assigned as 0.5. In one embodiment, the initial weighting coefficient for each marker may be associated with the effectiveness of that marker by itself. For example, a ROC curve may be generated for the single marker, and the area under the ROC curve may be used as the initial weighting coefficient for that marker.
  • Next, a panel response may be calculated for each subject in each of the two sets. The panel response is a function of the indicators to which each marker level is mapped and the weighting coefficients for each marker. One advantage of using an indicator value rather than the marker value is that an extraordinarily high or low marker levels do not change the probability of a diagnosis of diseased or non-diseased for that particular marker. Typically, a marker value above a certain level generally indicates a certain condition state. Marker values above that level indicate the condition state with the same certainty. Thus, an extraordinarily high marker value may not indicate an extraordinarily high probability of that condition state. The use of an indicator which is constant on one side of the cutoff region eliminates this concern.
  • The panel response may also be a general function of several parameters including the marker levels and other factors including, for example, race and gender of the patient. Other factors contributing to the panel response may include the slope of the value of a particular marker over time. For example, a patient may be measured when first arriving at the hospital for a particular marker. The same marker may be measured again an hour later, and the level of change may be reflected in the panel response. Further, additional markers may be derived from other markers and may contribute to the value of the panel response. For example, the ratio of values of two markers may be a factor in calculating the panel response.
  • Having obtained panel responses for each subject in each set of subjects, the distribution of the panel responses for each set may now be analyzed. An objective function may be defined to facilitate the selection of an effective panel. The objective function should generally be indicative of the effectiveness of the panel, as may be expressed by, for example, overlap of the panel responses of the diseased set of subjects and the panel responses of the non-diseased set of subjects. In this manner, the objective function may be optimized to maximize the effectiveness of the panel by, for example, minimizing the overlap.
  • In a some embodiment, the ROC curve representing the panel responses of the two sets of subjects may be used to define the objective function. For example, the objective function may reflect the area under the ROC curve. By maximizing the area under the curve, one may maximize the effectiveness of the panel of markers. In other embodiments, other features of the ROC curve may be used to define the objective function. For example, the point at which the slope of the ROC curve is equal to one may be a useful feature. In other embodiments, the point at which the product of sensitivity and specificity is a maximum, sometimes referred to as the “knee,” may be used. In an embodiment, the sensitivity at the knee may be maximized. In further embodiments, the sensitivity at a predetermined specificity level may be used to define the objective function. Other embodiments may use the specificity at a predetermined sensitivity level may be used. In still other embodiments, combinations of two or more of these ROC-curve features may be used.
  • It is possible that one of the markers in the panel is specific to the disease or condition being diagnosed. When such markers are present at above or below a certain threshold, the panel response may be set to return a “positive” test result. When the threshold is not satisfied, however, the levels of the marker may nevertheless be used as possible contributors to the objective function.
  • An optimization algorithm may be used to maximize or minimize the objective function. Optimization algorithms are well-known to those skilled in the art and include several commonly available minimizing or maximizing functions including the Simplex method and other constrained optimization techniques. It is understood by those skilled in the art that some minimization functions are better than others at searching for global minimums, rather than local minimums. In the optimization process, the location and size of the cutoff region for each marker may be allowed to vary to provide at least two degrees of freedom per marker. Such variable parameters are referred to herein as independent variables. In one embodiment, the weighting coefficient for each marker is also allowed to vary across iterations of the optimization algorithm. In various embodiments, any permutation of these parameters may be used as independent variables.
  • In addition to the above-described parameters, the sense of each marker may also be used as an independent variable. For example, in many cases, it may not be known whether a higher level for a certain marker is generally indicative of a diseased state or a non-diseased state. In such a case, it may be useful to allow the optimization process to search on both sides. In practice, this may be implemented in several ways. For example, in one embodiment, the sense may be a truly separate independent variable which may be flipped between positive and negative by the optimization process. Alternatively, the sense may be implemented by allowing the weighting coefficient to be negative.
  • The optimization algorithm may be provided with certain constraints as well. For example, the resulting ROC curve may be constrained to provide an area-under-curve of greater than a particular value. ROC curves having an area under the curve of 0.5 indicate complete randomness, while an area under the curve of 1.0 reflects perfect separation of the two sets. Thus, a minimum acceptable value, such as 0.75, may be used as a constraint, particularly if the objective function does not incorporate the area under the curve. Other constraints may include limitations on the weighting coefficients of particular markers. Additional constraints may limit the sum of all the weighting coefficients to a particular value, such as 1.0.
  • The iterations of the optimization algorithm generally vary the independent parameters to satisfy the constraints while minimizing or maximizing the objective function. The number of iterations may be limited in the optimization process. Further, the optimization process may be terminated when the difference in the objective function between two consecutive iterations is below a predetermined threshold, thereby indicating that the optimization algorithm has reached a region of a local minimum or a maximum.
  • Thus, the optimization process may provide a panel of markers including weighting coefficients for each marker and cutoff regions for the mapping of marker values to indicators. In order to develop lower-cost panels which require the measurement of fewer marker levels, certain markers may be eliminated from the panel. In this regard, the effective contribution of each marker in the panel may be determined to identify the relative importance of the markers. In one embodiment, the weighting coefficients resulting from the optimization process may be used to determine the relative importance of each marker. The markers with the lowest coefficients may be eliminated.
  • Individual panel response values may also be used as markers in the methods described herein. For example, a panel may be constructed from a plurality of markers, and each marker of the panel may be described by a function and a weighting factor to be applied to that marker (as determined by the methods described above). Each individual marker level is determined for a sample to be tested, and that level is applied to the predetermined function and weighting factor for that particular marker to arrive at a sample value for that marker. The sample values for each marker are added together to arrive at the panel response for that particular sample to be tested. For a “diseased” and “non-diseased” group of patients, the resulting panel responses may be treated as if they were just levels of another disease marker.
  • Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003 (hereby incorporated by reference as if fully set forth herein), and used to determine the effectiveness of a given marker or panel of markers. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. As discussed above, suitable tests may exhibit one or more of the following results on these various measures: at least 75% sensitivity, combined with at least 75% specificity; ROC curve area of at least 0.7, more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; and/or a positive likelihood ratio (calculated as sensitivity/(1−specificity)) of at least 5, more preferably at least 10, and most preferably at least 20, and a negative likelihood ratio (calculated as (1−sensitivity)/specificity) of less than or equal to 0.3, more preferably less than or equal to 0.2, and most preferably less than or equal to 0.1.
  • According to embodiments of the present invention, a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof, may be featured as a biomarker for detecting marker-detectable disease and/or an indicative condition, such that a biomarker may optionally comprise any of the above.
  • According to still other embodiments, the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein. Any oligopeptide or peptide relating to such an amino acid sequence or fragment thereof may optionally also (additionally or alternatively) be used as a biomarker, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges. The present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.
  • The present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.
  • Non-limiting examples of methods or assays are described below.
  • The present invention also relates to kits based upon such diagnostic methods or assays.
  • 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.
  • In cases where the polynucleotide sequences of the present invention encode previously unidentified polypeptides, 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).
  • As used herein 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.
  • As used herein the phrase “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
  • As used herein the phrase “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.
  • Some 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).
  • Alternatively, an oligonucleotide probe of the present invention can be designed to hybridize with a nucleic acid sequence encompassed by any of the above nucleic acid sequences, particularly the portions specified above, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
  • Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988) and “Oligonucleotide Synthesis” Gait, M. J., ed. (1984) utilizing solid phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting and purification by for example, an automated trityl-on method or HPLC.
  • 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. Preferably, 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.
  • Preferably used oligonucleotides are those modified at one or more of the backbone, internucleoside linkages or bases, as is broadly described hereinunder.
  • Specific examples of oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat. Nos. 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.
  • 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′. Various salts, mixed salts and free acid forms can also be used.
  • Alternatively, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having 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; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.
  • Other oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside 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). United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No. 6,303,374.
  • Oligonucleotides of the present invention may also include base modifications or substitutions. As used herein, “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 cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. 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 O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
  • Another modification of the 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. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-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 palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, as disclosed in U.S. Pat. No. 6,303,374.
  • It is not necessary for all positions in a given oligonucleotide molecule to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.
  • It will be appreciated that 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.
  • To enable cellular expression of the polynucleotides of the present invention, a nucleic acid construct according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and further includes at least one cis acting regulatory element. As used herein, the phrase “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.
  • In some embodiments, the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed. Examples of 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. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). 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 further includes, in some embodiments, an appropriate selectable marker and/or an origin of replication. In some embodiments, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
  • In some embodiments, in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, retrovirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. Such vector constructs may comprise a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used. In addition, such a construct may include a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs may 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.
  • Variant Recombinant Expression Vectors and Host Cells
  • Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a variant protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., variant proteins, mutant forms of variant proteins, fusion proteins, etc.).
  • The recombinant expression vectors of the invention can be designed for production of variant proteins in prokaryotic or eukaryotic cells. For example, variant proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, to the amino or carboxyl terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, PreScission, TEV and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) and pTrcHis (Invitrogen Life Technologies) that fuse glutathione S-transferase (GST), maltose E binding protein, protein A or 6×His, respectively, to the target recombinant protein.
  • Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques. Another optional strategy to solve codon bias is by using BL21-codon plus bacterial strains (Invitrogen) or Rosetta bacterial strain (Novagen), as these strains contain extra copies of rare E. coli tRNA genes.
  • In another embodiment, the expression vector encoding for the variant protein is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
  • Alternatively, variant protein can be produced in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195), pIRESpuro (Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen) pREP4 (Invitrogen), pcDNA3 (Invitrogen). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, Rous Sarcoma Virus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the O-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
  • The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to mRNA encoding for variant protein. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., “Antisense RNA as a molecular tool for genetic analysis,” Reviews-Trends in Genetics, Vol. 1(1) 1986.
  • Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • A host cell can be any prokaryotic or eukaryotic cell. For example, variant protein can be produced in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS or 293 cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
  • For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin, puromycin, blasticidin and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding variant protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) variant protein. Accordingly, the invention further provides methods for producing variant protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the present invention (into which a recombinant expression vector encoding variant protein has been introduced) in a suitable medium such that variant protein is produced. In another embodiment, the method further comprises isolating variant protein from the medium or the host cell.
  • For efficient production of the protein, it is preferable to place the nucleotide sequences encoding the variant protein under the control of expression control sequences optimized for expression in a desired host. For example, the sequences may include optimized transcriptional and/or translational regulatory sequences (such as altered Kozak sequences).
  • 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). 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.
  • Thus, the isolated polynucleotides (oligonucleotides) of the present invention are preferably hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions.
  • Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10% dextrane sulfate, 1 M NaCl, 1% SDS and 5×106 cpm 32P labeled probe, at 65° C., with a final wash solution of 0.2×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 5×106 cpm 32P labeled probe, at 65° C., with a final wash solution of 1×SSC and 0.1% SDS and final wash at 50° C.
  • More generally, hybridization of short nucleic acids (below 200 bp in length, e.g. 17-40 bp in length) can be effected using the following exemplary hybridization protocols which can be modified according to the desired stringency; (i) hybridization solution of 6×SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 1-1.5° C. below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm; (ii) hybridization solution of 6×SSC and 0.1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 2-2.5° C. below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm, final wash solution of 6×SSC, and final wash at 22° C.; (iii) hybridization solution of 6×SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature.
  • The detection of hybrid duplexes can be carried out by a number of methods. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Such labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. A label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.
  • Probes can be labeled according to numerous well known methods. Non-limiting examples of radioactive labels include 3H, 14C, 32P, and 35S, Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radio-nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
  • For example, 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, Alternatively, when fluorescently-labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluor X (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.
  • It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays. For instance, samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.
  • Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods.
  • As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples of radioactive labels include 3H, 14C, 32P, and 35S.
  • 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.
  • It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays.
  • 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.
  • NAT 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).
  • As used herein, a “primer” defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the q3 replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. 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).
  • The terminology “amplification pair” (or “primer 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. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions.
  • In one particular embodiment, 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. In one embodiment, RT-PCR is carried out on an mRNA sample from a patient under conditions which favor the amplification of the most abundant mRNA. In another embodiment, 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. Optionally, 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. As commonly known in the art, the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, 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.
  • Polymerase Chain Reaction (PCR): The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al., 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. Following hybridization, the primers are extended with polymerase so as to form complementary strands. The steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.
  • The length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and, therefore, this length is a controllable parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be “PCR-amplified.”
  • Ligase Chain Reaction (LCR or LAR): The ligase chain reaction [LCR; sometimes referred to as “Ligase Amplification Reaction” (LAR)] has developed into a well-recognized alternative method of amplifying nucleic acids. In 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. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, 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. WO9001069 A1 (1990). However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
  • 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. In a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo- and deoxyribonucleoside triphosphates, 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 (Qβ) Replicase: In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Qβ replicase. A previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step. However, available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 degrees C.). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere.
  • A successful diagnostic method must be very specific. A straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA, and Qβ systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature (i.e., >55 degrees C.). Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons, PCR and LCR currently dominate the research field in detection technologies.
  • The basis of the amplification procedure in the PCR and LCR is the fact that the products of one cycle become usable templates in all subsequent cycles, consequently doubling the population with each cycle. The final yield of any such doubling system can be expressed as: (1+X)n=y, where “X” is the mean efficiency (percent copied in each cycle), “n” is the number of cycles, and “y” is the overall efficiency, or yield of the reaction. If every copy of a target DNA is utilized as a template in every cycle of a polymerase chain reaction, then the mean efficiency is 100%. If 20 cycles of PCR are performed, then the yield will be 220, or 1,048,576 copies of the starting material. If the reaction conditions reduce the mean efficiency to 85%, then the yield in those 20 cycles will be only 1.8520, or 220,513 copies of the starting material. In other words, a PCR running at 85% efficiency will yield only 21% as much final product, compared to a reaction running at 100% efficiency. A reaction that is reduced to 50% mean efficiency will yield less than 1% of the possible product.
  • In practice, routine polymerase chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate for the lower yield. At 50% mean efficiency, it would take 34 cycles to achieve the million-fold amplification theoretically possible in 20, and at lower efficiencies, the number of cycles required becomes prohibitive. In addition, any background products that amplify with a better mean efficiency than the intended target will become the dominant products.
  • Also, many variables can influence the mean efficiency of PCR, including target DNA length and secondary structure, primer length and design, primer and dNTP concentrations, and buffer composition, to name but a few. Contamination of the reaction with exogenous DNA (e.g., DNA spilled onto lab surfaces) or cross-contamination is also a major consideration. Reaction conditions must be carefully optimized for each different primer pair and target sequence, and the process can take days, even for an experienced investigator. The laboriousness of this process, including numerous technical considerations and other factors, presents a significant drawback to using PCR in the clinical setting. Indeed, PCR has yet to penetrate the clinical market in a significant way. The same concerns arise with LCR, as LCR must also be optimized to use different oligonucleotide sequences for each target sequence. In addition, both methods require expensive equipment, capable of precise temperature cycling.
  • Many applications of nucleic acid detection technologies, such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few, or single, nucleotide differences. One method of the detection of allele-specific variants by PCR is based upon the fact that it is difficult for Taq polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3′ end of the primer. An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence. This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect.
  • A similar 3′-mismatch strategy is used with greater effect to prevent ligation in the LCR. Any mismatch effectively blocks the action of the thermostable ligase, but LCR still has the drawback of target-independent background ligation products initiating the amplification. Moreover, the combination of PCR with subsequent LCR to identify the nucleotides at individual positions is also a clearly cumbersome proposition for the clinical laboratory.
  • The direct detection method according to various embodiments of the present invention may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.
  • When a sufficient amount of a nucleic acid to be detected is available, there are advantages to detecting that sequence directly, instead of making more copies of that target, (e.g., as in PCR and LCR). Most notably, a method that does not amplify the signal exponentially is more amenable to quantitative analysis. Even if the signal is enhanced by attaching multiple dyes to a single oligonucleotide, the correlation between the final signal intensity and amount of target is direct. Such a system has an additional advantage that the products of the reaction will not themselves promote further reaction, so contamination of lab surfaces by the products is not as much of a concern. Recently devised techniques have sought to eliminate the use of radioactivity and/or improve the sensitivity in automatable formats. Two examples are the “Cycling Probe Reaction” (CPR), and “Branched DNA” (bDNA).
  • Cycling probe reaction (CPR): The cycling probe reaction (CPR), uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
  • Branched DNA: Branched DNA (bDNA), 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 according to various embodiments of the present invention 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).
  • The demand for tests which allow the detection of specific nucleic acid sequences and sequence changes is growing rapidly in clinical diagnostics. As nucleic acid sequence data for genes from humans and pathogenic organisms accumulates, the demand for fast, cost-effective, and easy-to-use tests for as yet mutations within specific sequences is rapidly increasing.
  • A handful of methods have been devised to scan nucleic acid segments for mutations. One option is to determine the entire gene sequence of each test sample (e.g., a bacterial isolate). For sequences under approximately 600 nucleotides, this may be accomplished using amplified material (e.g., PCR reaction products). This avoids the time and expense associated with cloning the segment of interest. However, 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.
  • In view of the difficulties associated with sequencing, 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 an ordered map. The presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs.
  • Restriction fragment length polymorphism (RFLP): For detection of single-base differences between like sequences, the requirements of the analysis are often at the highest level of resolution. For cases in which the position of the nucleotide in question is known in advance, several methods have been developed for examining single base changes without direct sequencing. For example, if a mutation of interest happens to fall within a restriction recognition sequence, a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymorphism [RFLP] analysis).
  • 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). However, this method requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory.
  • RFLP analysis suffers from low sensitivity and requires a large amount of sample. When 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. Moreover, the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA manipulations. Thus, it is applicable only in a small fraction of cases, as most mutations do not fall within such sites.
  • A handful of rare-cutting restriction enzymes with 8 base-pair specificities have been isolated and these are widely used in genetic mapping, but these enzymes are few in number, are limited to the recognition of G+C-rich sequences, and cleave at sites that tend to be highly clustered. Recently, endonucleases encoded by group I introns have been discovered that might have greater than 12 base-pair specificity, but again, these are few in number.
  • Allele specific oligonucleotide (ASO): If the change is not in a recognition sequence, then allele-specific oligonucleotides (ASOs), 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 (ASO) 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.
  • Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE): Two other methods rely on detecting changes in electrophoretic mobility in response to minor sequence changes. One of these methods, termed “Denaturing Gradient Gel Electrophoresis” (DGGE) is based on the observation that slightly different sequences will display different patterns of local melting when electrophoretically resolved on a gradient gel. In this manner, variants can be distinguished, as differences in melting properties of homoduplexes versus heteroduplexes differing in a single nucleotide can detect the presence of mutations in the target sequences because of the corresponding changes in their electrophoretic mobilities. The fragments to be analyzed, usually PCR products, are “clamped” at one end by a long stretch of G-C 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. Furthermore, the method requires specialized equipment to prepare the gels and maintain the needed high temperatures during electrophoresis. The expense associated with the synthesis of the clamping tail on one oligonucleotide for each sequence to be tested is also a major consideration. In addition, long running times are required for DGGE. The long running time of DGGE was shortened in a modification of DGGE called constant denaturant gel electrophoresis (CDGE). CDGE requires that gels be performed under different denaturant conditions in order to reach high efficiency for the detection of mutations.
  • A technique analogous to DGGE, termed temperature gradient gel electrophoresis (TGGE), uses a thermal gradient rather than a chemical denaturant gradient. TGGE requires the use of specialized equipment which can generate a temperature gradient perpendicularly oriented relative to the electrical field. TGGE can detect mutations in relatively small fragments of DNA therefore scanning of large gene segments requires the use of multiple PCR products prior to running the gel.
  • Single-Strand Conformation Polymorphism (SSCP): Another common method, called “Single-Strand Conformation Polymorphism” (SSCP) was developed by Hayashi, Sekya and colleagues and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations.
  • The SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run. This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
  • Dideoxy fingerprinting (ddF): The dideoxy fingerprinting (ddF) is another technique developed to scan genes for the presence of mutations. The ddF technique combines components of Sanger dideoxy sequencing with SSCP. A dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis. While ddF is an improvement over SSCP in terms of increased sensitivity, ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).
  • In addition to the above limitations, all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed. For the direct sequencing approach, 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. Although SSCP is reportedly able to detect 90% of single-base substitutions 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.
  • According to a one embodiment of the present invention 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, Qβ-Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing gradient gel electrophoresis, constant denaturant gel electrophoresis, temperature gradient gel electrophoresis and dideoxy fingerprinting.
  • 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 incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.
  • 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.
  • Amino Acid Sequences and Peptides
  • The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms “polypeptide,” “peptide” and “protein” include glycoproteins, as well as non-glycoproteins.
  • Polypeptide products can be biochemically synthesized such as by employing standard solid phase techniques. Such methods include but are not limited to exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
  • Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
  • Synthetic polypeptides can optionally be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.], after which their composition can be confirmed via amino acid sequencing.
  • In cases where large amounts of a polypeptide are desired, it can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.
  • 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. Preferably, 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 also preferably include having an E value of 10, filtering low complexity sequences and a word size of 11. Finally, 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.
  • It will be appreciated that 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.
  • In addition to the above, 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).
  • As used herein in the specification and in the claims section below the term “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids. Non-conventional or modified amino acids can be incorporated in the polypeptides of this invention as well, as will be known to one skilled in the art.
  • Since the peptides of the present invention are utilized, in some embodiments, in diagnostics which require the peptides to be in soluble form, the peptides of the present invention may 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 may be utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • The peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis well known in the art, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
  • Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing.
  • In cases where large amounts of the peptides of the present invention are desired, the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 and also as described above.
  • Antibodies:
  • “Antibody” refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad-immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab′ and F(ab)′2 fragments. The term “antibody,” as used herein, 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, CH1, CH2 and CH3, but does not include the heavy chain variable region.
  • The functional fragments of antibodies, such as Fab, F(ab′)2, and Fv that are capable of binding to macrophages, are described as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) 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; (3) (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; (4) 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; and (5) Single chain antibody (“SCA”), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
  • Monoclonal antibody development may optionally be performed according to any method that is known in the art. The method described below is provided for the purposes of description only and is not meant to be limiting in any way.
  • Antibody Engineering in Phage Display Libraries:
  • Antibodies of this invention may be prepared through the use of phage display libraries, as is known in the art, for example, as described in PCT Application No. WO 94/18219, U.S. Pat. No. 6,096,551, both of which are hereby fully incorporated by reference, The method involves inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene for the purpose of producing light chain gene libraries for use in combination with heavy chain genes and gene libraries to produce antibody libraries of diverse and novel immuno-specificities. The method comprises amplifying a CDR portion of an immunoglobulin light chain gene by polymerase chain reaction (PCR) using a PCR primer oligonucleotide. The resultant gene portions are inserted into phagemids for production of a phage display library, wherein the engineered light chains are displayed by the phages, for example for testing their binding specificity.
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using Papain produces two monovalent Fab′ fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • 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 (1972)]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) 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. A scFv antibody fragment is an engineered antibody derivative that includes heavy- and light chain variable regions joined by a peptide linker. The minimal size of antibody molecules are those that still comprise the complete antigen binding site. ScFv antibody fragments are potentially more effective than unmodified IgG antibodies. The reduced size of 27-30 kDa permits them to penetrate tissues and solid tumors more readily. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)]. Optionally, there may be 1, 2 or 3 CDRs of different chains, but preferably there are 3 CDRs of 1 chain. The chain could be the heavy or the light chain.
  • 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, or fragments thereof may comprise the antibodies of this invention. Humanized antibodies are well known in the art. Methods for humanizing non-human antibodies are well known in the art, for example, as described in 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)], U.S. Pat. No. 4,816,567, Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991), Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985), Boerner et al., J. Immunol., 147(1):86-95 (1991), U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995), all of which are incorporated herein by reference.
  • Preferably, the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention. As used herein, the term “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.
  • Optionally, 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.
  • Immunoassays
  • In another embodiment of the present invention, an immunoassay can be used to qualitatively or quantitatively detect and analyze markers in a sample. This method comprises: providing an antibody that specifically binds to a marker; contacting a sample with the antibody; and detecting the presence of a complex of the antibody bound to the marker in the sample.
  • To prepare an antibody that specifically binds to a marker, 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). Generally, a sample obtained from a subject can be contacted with the antibody that specifically binds the marker.
  • Optionally, 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. Examples of 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 incubating the washed mixture with a detection reagent. Alternatively, 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.
  • Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, marker, volume of solution, concentrations and the like. Usually the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10° C. to 40° C.
  • The immunoassay can be used to determine a test amount of a marker in a sample from a subject. First, a test amount of a marker in a sample can be detected using the immunoassay methods described above. If a marker is present in the sample, it will form an antibody-marker complex with an antibody that specifically binds the marker under suitable incubation conditions described above. The amount of an antibody-marker complex can optionally be determined by comparing to a standard. As noted above, 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.
  • In some embodiments, antibodies which specifically interact with the polypeptides of the present invention and not with wild type proteins or other isoforms thereof, are used. Such antibodies are directed, for example, to the unique sequence portions of the polypeptide variants of the present invention, including but not limited to bridges, heads, tails and insertions described in greater detail below. Some embodiments of antibodies according to the present invention are described in greater detail with regard to the section entitled “Antibodies”.
  • Radio-immunoassay (RIA): In one version, this method involves precipitation of the desired substrate and in the methods detailed hereinbelow, with a specific antibody and radiolabelled antibody binding protein (e.g., protein A labeled with I125) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
  • In an alternate version of the RIA, a labeled substrate and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of substrate is added in varying amounts. The decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
  • Enzyme linked immunosorbent assay (ELISA): 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
  • These 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, U.S. Pat. No. 6,696,686 describes the use of SPECT for detection of breast cancer, and is hereby incorporated by reference as if fully set forth herein.
  • Display Libraries
  • According to 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.
  • Methods of constructing such display libraries are well known in the art. Such methods are described in, for example, Young A C, et al., “The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes” J Mol Biol 1997 Dec. 12; 274(4):622-34; Giebel L B et al. “Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities” Biochemistry 1995 Nov. 28; 34(47):15430-5; Davies E L et al., “Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes” J Immunol Methods 1995 Oct. 12; 186(1):125-35; Jones C R T al. “Current trends in molecular recognition and bioseparation” J Chromatogr A 1995 Jul. 14; 707(1):3-22; Deng S J et al. “Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries” Proc Natl Acad Sci USA 1995 May 23; 92(11):4992-6; and Deng S J et al. “Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display” J Biol Chem 1994 Apr. 1; 269(13):9533-8, which are incorporated herein by reference.
  • Theranostics:
  • The term theranostics describes the use of diagnostic testing to diagnose the disease, choose the correct treatment regime according to the results of diagnostic testing and/or monitor the patient response to therapy according to the results of diagnostic testing. Theranostic tests can be used to select patients for treatments that are particularly likely to benefit them and unlikely to produce side-effects. They can also provide an early and objective indication of treatment efficacy in individual patients, so that (if necessary) the treatment can be altered with a minimum of delay. For example: DAKO and Genentech together created HercepTest and Herceptin (trastuzumab) for the treatment of breast cancer, the first theranostic test approved simultaneously with a new therapeutic drug. In addition to HercepTest (which is an immunohistochemical test), other theranostic tests are in development which use traditional clinical chemistry, immunoassay, cell-based technologies and nucleic acid tests. PPGx's recently launched TPMT (thiopurine S-methyltransferase) test, which is enabling doctors to identify patients at risk for potentially fatal adverse reactions to 6-mercaptopurine, an agent used in the treatment of leukemia. Also, Nova Molecular pioneered SNP genotyping of the apolipoprotein E gene to predict Alzheimer's disease patients' responses to cholinomimetic therapies and it is now widely used in clinical trials of new drugs for this indication. Thus, the field of theranostics represents the intersection of diagnostic testing information that predicts the response of a patient to a treatment with the selection of the appropriate treatment for that particular patient.
  • Surrogate Markers:
  • A surrogate marker is a marker, that is detectable in a laboratory and/or according to a physical sign or symptom on the patient, and that is used in therapeutic trials as a substitute for a clinically meaningful endpoint. The surrogate marker is a direct measure of how a patient feels, functions, or survives which is expected to predict the effect of the therapy. The need for surrogate markers mainly arises when such markers can be measured earlier, more conveniently, or more frequently than the endpoints of interest in terms of the effect of a treatment on a patient, which are referred to as the clinical endpoints. Ideally, a surrogate marker should be biologically plausible, predictive of disease progression and measurable by standardized assays (including but not limited to traditional clinical chemistry, immunoassay, cell-based technologies, nucleic acid tests and imaging modalities).
  • Surrogate endpoints were used first mainly in the cardiovascular area. For example, antihypertensive drugs have been approved based on their effectiveness in lowering blood pressure. Similarly, in the past, cholesterol-lowering agents have been approved based on their ability to decrease serum cholesterol, not on the direct evidence that they decrease mortality from atherosclerotic heart disease. The measurement of cholesterol levels is now an accepted surrogate marker of atherosclerosis. In addition, currently two commonly used surrogate markers in HIV studies are CD4+ T cell counts and quantitative plasma I-V RNA (viral load). In some embodiments of this invention, the polypeptide/polynucleotide expression pattern may serve as a surrogate marker for a particular disease, as will be appreciated by one skilled in the art.
  • Monoclonal Antibody Therapy:
  • In some embodiments, monoclonal antibodies are useful for the identification of cancer cells. In some embodiments, monoclonal antibody therapy is a form of passive immunotherapy useful in cancer treatment. Such antibodies may comprise naked monoclonal antibodies or conjugated monoclonal antibodies—joined to a chemotherapy drug, radioactive particle, or a toxin (a substance that poisons cells). In some embodiments, the former is directly cytotoxic to the target (cancer) cell, or in another embodiment, stimulates or otherwise participates in an immune response ultimately resulting in the lysis of the target cell.
  • In some embodiments, the conjugated monoclonal antibodies are joined to drugs, toxins, or radioactive atoms. They are used as delivery vehicles to take those substances directly to the cancer cells. The MAb acts as a homing device, circulating in the body until it finds a cancer cell with a matching antigen. It delivers the toxic substance to where it is needed most, minimizing damage to normal cells in other parts of the body. Conjugated MAbs are also sometimes referred to as “tagged,” “labeled,” or “loaded” antibodies. MAbs with chemotherapy drugs attached are generally referred to as chemolabeled. MAbs with radioactive particles attached are referred to as radiolabeled, and this type of therapy is known as radioimmunotherapy (RIT). MAbs attached to toxins are called immunotoxins.
  • An illustrative, non-limiting example is provided herein of a method of treatment of a patient with an antibody to a variant as described herein, such that the variant is a target of the antibody. A patient with breast cancer is treated with a radiolabeled humanized antibody against an appropriate breast cancer target as described herein. The patient is optionally treated with a dosage of labeled antibody ranging from 10 to 30 mCi. Of course any type of therapeutic label may optionally be used.
  • The following sections relate to Candidate Marker Examples. It should be noted that Table numbering is restarted within each Example, which starts with the words “Description for Cluster”.
  • Candidate Marker Examples Section
  • This Section relates to Examples of sequences according to the present invention, including illustrative methods of selection thereof with regard to cancer; other markers were selected as described below for the individual markers.
  • Description of the methodology undertaken to uncover the biomolecular sequences of the present invention
  • Human ESTs and cDNAs were obtained from GenBank versions 136 (Jun. 15, 2003 ftp.ncbi.nih.gov/genbank/release.notes/gb136.release.notes); NCBI genome assembly of April 2003; RefSeq sequences from June 2003; Genbank version 139 (December 2003); Human Genome from NCBI (Build 34) (from October 2003); and RefSeq sequences from December 2003. With regard to GenBank sequences, the human EST sequences from the EST (GBEST) section and the human mRNA sequences from the primate (GBPR1) 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. 1993 August; 4(4):332-3; all of which are hereby incorporated by reference as if fully set forth herein).
  • Novel splice variants were predicted using the LEADS clustering and assembly system as described in Sorek, R., Ast, G. & Graur, D. Alu-containing exons are alternatively spliced. Genome Res 12, 1060-7 (2002); U.S. Pat. No. 6,625,545; and U.S. patent application Ser. No. 10/426,002, published as US20040101876 on May 27, 2004; all of which are hereby incorporated by reference as if fully set forth herein. Briefly, the software cleans the expressed sequences from repeats, vectors and immunoglobulins. It then aligns the expressed sequences to the genome taking alternatively splicing into account and clusters overlapping expressed sequences into “clusters” that represent genes or partial genes.
  • These were annotated using the GeneCarta (Compugen, Tel-Aviv, Israel) platform. 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.
  • A brief explanation is provided with regard to the method of selecting the candidates. However, it should be noted that this explanation is provided for descriptive purposes only, and is not intended to be limiting in any way. 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. Various parameters related to the information in the EST libraries, determined according to classification by library annotation, were used to assist in locating genes and/or splice variants thereof that are specifically and/or differentially expressed in heart tissues. The detailed description of the selection method and of these parameters is presented in Example 1 below.
  • Selecting Candidates with Regard to Cancer
  • A brief explanation is provided with regard to a non-limiting method of selecting the candidates for cancer diagnostics. However, it should noted that this explanation is provided for descriptive purposes only, and is not intended to be limiting in any way. The potential markers were identified by a computational process that was designed to find genes and/or their splice variants that are over-expressed in tumor tissues, by using databases of expressed sequences. Various parameters related to the information in the EST libraries, determined according to a manual classification process, were used to assist in locating genes and/or splice variants thereof that are over-expressed in cancerous tissues. The detailed description of the selection method is presented in Example 1 below. The cancer biomarkers selection engine and the following wet validation stages are schematically summarized in FIG. 1.
  • Example 1 Identification of Differentially Expressed Gene Products—Algorithm
  • In order to distinguish between differentially expressed gene products and constitutively expressed genes (i.e., house keeping genes), 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 (Jun. 15, 2003 ftp.ncbi.nih.gov/genbank/release.notes/gb136.release.notes) and Genbank version 139 (December 2003); and from the LifeSeq library of Incyte Corporation (ESTs only; Wilmington, Del., 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.
      • 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 (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. If the percentage of such sequences (as compared to all other sequences) was at least 4 standard deviations above the average for all libraries being analyzed, this library was tagged as being contaminated and was eliminated from further consideration in the below analysis (see also Sorek, R. & Safer, H. M. A novel algorithm for computational identification of contaminated EST libraries. Nucleic Acids Res 31, 1067-74 (2003) for further details).
  • Clusters (genes) having at least five sequences including at least two sequences from the tissue of interest were analyzed. Splice variants were identified by using the LEADS software package as described above
  • Example 2 Identification of Genes Over Expressed in Cancer
  • Two different scoring algorithms were developed.
  • Libraries score—candidate sequences which are supported by a number of cancer libraries, are more likely to serve as specific and effective diagnostic markers.
  • 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.
  • The algorithm—
  • Clone counting: For counting EST clones each library protocol class was given a weight based on an assessment of how much the protocol reflects actual expression levels:
  • (i) non-normalized: 1
  • (ii) normalized: 0.2
  • (iii) all other classes: 0.1
  • Clones number score—The total weighted number of EST clones from cancer libraries was compared to the EST clones from normal libraries. To avoid cases where one library contributes to the majority of the score, the contribution of the library that gives most clones for a given cluster was limited to 2 clones.
  • The score was computed as
  • c + 1 C / n + 1 N
  • where:
  • c—weighted number of “cancer” clones in the cluster.
  • C—weighted number of clones in all “cancer” libraries.
  • n—weighted number of “normal” clones in the cluster.
  • N—weighted number of clones in all “normal” libraries.
  • Clones number score significance—Fisher exact test was used to check if EST clones from cancer libraries are significantly over-represented in the cluster as compared to the total number of EST clones from cancer and normal libraries.
  • Two search approaches were used to find either general cancer-specific candidates or tumor specific candidates.
      • Libraries/sequences originating from tumor tissues are counted as well as libraries originating from cancer cell-lines (“normal” cell-lines were ignored).
      • Only libraries/sequences originating from tumor tissues are counted
    Example 3 Identification of Tissue Specific Genes
  • For detection of tissue specific clusters, 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 referred 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 (S1).
  • (ii) Supporting transcripts of group TB (S2).
  • (iii) Supporting transcripts from both groups (S3).
  • Library and clones number scores described above were given to S1 group.
  • Fisher Exact Test P-values were used to check if:
  • S1 is significantly enriched by cancer EST clones compared to S2; and
  • S1 is significantly enriched by cancer EST clones compared to cluster background (S1+S2+S3).
  • Identification of unique sequence regions and division of the group of transcripts accordingly is illustrated in FIG. 2. Each of these unique sequence regions corresponds 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: T1 unique regions (2+6) against T2+3 unique regions (3+4); Region 3: specific to Transcripts 2+3: T2+3 unique regions (3+4) against T1 unique regions (2+6); Region 4: specific to Transcript 3: T3 unique regions (4) against T1+2 unique regions (2+5+6); Region 5: specific to Transcript 1+2: T1+2 unique regions (2+5+6) against T3 unique regions (4); Region 6: specific to Transcript 1: same as region 2.
  • Example 5 Diseases and Conditions that May be Diagnosed with One or More Variant(s) According to the Present Invention Cardiovascular and Cerebrovascular Conditions
  • Various examples are listed below for conditions that affect the vascular system, including various cardiovascular and cerebrovascular conditions, for which one or more variants according to the present invention may have a diagnostic utility. Based on these diseases mechanisms and the correlation between the known proteins and the cardiovascular and cerebrovascular conditions, such correlation was predicted also for one or more variants according to the present invention, as described below. Each variant marker of the present invention described herein as potential marker for cardiovascular conditions, might optionally be used alone or in combination with one or more other variant markers described herein, and or in combination with known markers for cardiovascular conditions, including but not limited to Heart-type fatty acid binding protein (H-FABP), Angiotensin, C-reactive protein (CRP), myeloperoxidase (MPO), and/or in combination with the known protein(s) for the variant marker as described herein. Each variant marker of the present invention described herein as potential marker for cerebrovascular conditions, might optionally be used alone or in combination with one or more other variant markers described herein, and or in combination with known markers for cerebrovascular conditions, including but not limited to CRP, S100b, BNGF, CD40, MCP1, N-Acetyl-Aspartate (NAA), N-methyl-d-aspartate (NMDA) receptor antibodies (NR2Ab), and/or in combination with the known protein(s) for the variant marker as described herein.
  • Myocardial Infarction
  • HSFLT variants, HSI1Ra variants, HSPLGF variants, HUMSP18A variants, F05068 variants and/or HUMIL10 variants are potential markers for myocardial infarction. Other conditions that may be diagnosed by these markers or variants of them include but are not limited to the presence, risk and/or extent of the following:
    • 1. Myocarditis—in myocarditis cardiac muscle cells can go through cell lysis and leakage with the release of intracellular content to the extracellular space and blood, a similar process as happens in myocardial infarction (see also extended description below).
    • 2. Angina—stable or unstable, as the reduction of oxygen delivery to part of the heart often leads to local ischemic conditions that facilitate leakage of intracellular content.
    • 3. Traumatic injury to myocardial tissue—blunt or penetrating, may also result in myocardial cell leakage.
    • 4. Opening an occluded coronary artery following thrombolytic therapy—If such treatment is successful, proteins and other products of the local tissue are washed into the blood and can be detected there.
    • 5. Cardiomyopathy—which is characterized by slow degeneration of the heart muscle (see also extended description below).
    • 6. Myocardial injury after rejection of heart transplant.
    • 7. Congestive heart failure where heart myocytes slowly degenerate (as had been shown for Troponin-I; see also extended description below).
    • 8. Future cardiovascular disease (as a risk factor).
    • 9. Conditions which have similar clinical symptoms as myocardial infarction and where the differential diagnosis between them and myocardial infarction is of clinical importance including but not limited to:
      • a. Clinical symptoms resulting from lung related tissue (e.g. Pleuritis, pulmonary embolism)
      • b. Musculoskeletal origin of pain
      • c. Clinical symptoms resulting from heart related tissue which are not due to myocardial infarction, e.g. acute pericarditis
      • d. Upper abdominal pain from abdominal organs including but nor limited to esophagitis, gastro-esophageal reflux, gastritis, gastric ulcer, duodenitis, duodenal ulcer, enteritis, gastroenteritis, cholecystitis, cholelithiasis, cholangiolithiasis, pancreatitis, splenic infarction, splenic trauma, Aortic dissection.
  • One or more of these markers (variants according to the present invention) may optionally be used a tool to decide on treatment options e.g. anti platelet inhibitors (as has been shown for Troponin-I); as a tool in the assessment of pericardial effusion; and/or as a tool in the assessment of endocarditis and/or rheumatic fever, where progressive damage to the heart muscle may occur.
  • Acute and Chronic Inflammation and Risk Factors for CVS Diseases
  • HSFLT variants, HSI1Ra variants, HSPLGF variants, HUMSP18A variants, F05068 variants and/or HUMIL10 variants are potential markers for inflammation, including a spectrum of diseases where an inflammatory process plays a substantial role. In addition CRP levels and in particular baseline levels serve as a risk factor for various diseases, particularly cardiovascular diseases where inflammation is thought to participate in the pathogenesis. Conditions that may be diagnosed by these markers or variants of them include but are not limited to the presence, risk and/or extent of the following:
    • 1. Conditions that entail an inflammatory process that involves blood vessels including but not limited to hypercholesterolemia, diabetes, atherosclerosis, inflammation that involves blood vessels—whether acute or chronic including but not limited to the coronary arteries and blood vessels of the brain, myocardial infarction, cerebral stroke, peripheral vascular disease, vasculitis, polyarteritis nodosa, ANCA associated small vessel vasculitis, Churg-Strauss syndrome, Henoch-Schonlein purpura, scleroderma, thromboangiitis obliterans, temporal arteritis, Takayasu's arteritis, hypersensitivity vasculitis, Kawasaki disease, Behçet syndrome, and their complications including but not limited to coronary disease, angina pectoris, deep vein thrombosis, renal disease, diabetic nephropathy, lupus nephritis, renal artery thrombosis, renal artery stenosis, atheroembolic disease of the renal arteries, renal vein thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, arteriolar nephrosclerosis, preeclampsia, eclampsia, albuminuria, microalbuminuria, glomerulonephritis, renal failure, hypertension, uremia, cerebrovascular disease, peripheral vascular disease, intermittent claudication, abdominal angina.
    • 2. Rheumatic/autoimmune diseases that involve systemic immune reaction including but not limited to rheumatoid arthritis, scleroderma, mixed connective tissue disease, Sjogren syndrome, ankylosing spondylitis, spondyloarthropathy, psoriasis, psoriatic arthritis, myositis and systemic lupus erythematosus.
    • 3. Acute and/or chronic infective processes that involve systemic immune reaction including but not limited to pneumonia, bacteremia, sepsis, pyelonephritis, cellulitis, osteomyelitis, meningitis and viral hepatitis.
    • 4. Malignant and idiopathic processes that involve systemic immune reaction and/or proliferation of immune cells including but not limited to granulomatous disorders, Wegener's granulomatosis, lymphomatoid granulomatosis/polymorphic reticulosis, idiopathic midline granuloma, multiple myeloma, Waldenstrom's macroglobulinemia, Castleman's disease, amyloidosis, lymphoma, histiocytosis, renal cell carcinoma and paraneoplastic syndromes.
    • 5. Conditions where CRP was shown to have a positive correlation with the presence of the condition including but not limited to weight loss, anorexia-cachexia syndrome, extent of disease, recurrence in advanced cancer, diabetes (types 1 & 2), obesity, hypertension, preterm delivery.
    • 6. Conditions which have similar symptoms, signs and complications as the conditions above and where the differential diagnosis between them and the conditions above is of clinical importance including but not limited to:
      • a. Other (non vascular) causes of heart disease, renal disease and cerebral disease.
      • b. Other (non rheumatic) causes of arthropathy and musculoskeletal pain.
      • c. Other causes of non-specific symptoms and signs such as fever of unknown origin, loss of appetite, weight loss, nonspecific pains, breathing difficulties and anxiety.
    Stroke
  • Stroke is a manifestation of vascular injury to the brain which is commonly secondary to atherosclerosis or hypertension, and is the third leading cause of death (and the second most common cause of neurologic disability) in the United States. Embodiments of marker(s) for diagnosis of stroke and related conditions as described herein may optionally be selected from the group consisting of HSFLT variants, HSI1Ra variants, HSPLGF variants, HUMSP18A variants, F05068 variants and/or HUMIL10 variants or markers related thereto.
  • Specific markers of neural tissue injury are found in the blood or in blood components such as serum and plasma, as well as the CSF of a patient experiencing stroke or TIAs. Furthermore, clearance of the obstructing object in ischemic stroke can cause injury from oxidative insult during reperfusion, and patients with ischemic stroke can sometimes experience hemorrhagic transformation as a result of reperfusion or thrombolytic therapy.
  • Fibrinolysis is the process of proteolytic clot dissolution. In a manner analogous to coagulation, fibrinolysis is mediated by serine proteinases that are activated from their zymogen form. The serine proteinase plasmin is responsible for the degradation of fibrin into smaller degradation products that are liberated from the clot, resulting in clot dissolution. Fibrinolysis is activated soon after coagulation in order to regulate clot formation. Endogenous serine proteinase inhibitors also function as regulators of fibrinolysis.
  • The presence of a coagulation or fibrinolysis marker in cerebrospinal fluid would indicate that activation of coagulation or fibrinolysis, depending upon the marker used, coupled with increased permeability of the blood-brain barrier has occurred. In this regard, more definitive conclusions regarding the presence of coagulation or fibrinolysis markers associated with acute stroke may be obtained using cerebrospinal fluid.
  • Stroke can be categorized into two broad types, “ischemic stroke” and “hemorrhagic stroke.” Additionally, a patient may experience transient ischemic attacks, which are in turn a high risk factor for the future development of a more severe episode.
  • Ischemic stroke encompasses thrombotic, embolic, lacunar and hypoperfusion types of strokes. Thrombi are occlusions of arteries created in situ within the brain, while emboli are occlusions caused by material from a distant source, such as the heart and major vessels, often dislodged due to myocardial infarct or atrial fibrillation. Less frequently, thrombi may also result from vascular inflammation due to disorders such as meningitis. Thrombi or emboli can result from atherosclerosis or other disorders, for example, arteritis, and lead to physical obstruction of arterial blood supply to the brain. Lacunar stroke refers to an infarct within non-cortical regions of the brain. Hypoperfusion embodies diffuse injury caused by non-localized cerebral ischemia, typically caused by myocardial infarction and arrhythmia.
  • The onset of ischemic stroke is often abrupt, and can become an “evolving stroke” manifested by neurologic deficits that worsen over a 24-48 hour period. In evolving stroke, “stroke-associated symptom(s)” commonly include unilateral neurologic dysfunction which extends progressively, without producing headache or fever. Evolving stroke may also become a “completed stroke,” in which symptoms develop rapidly and are maximal within a few minutes.
  • Hemorrhagic stroke is caused by intracerebral or subarachnoid hemorrhage, i.e., bleeding into brain tissue, following blood vessel rupture within the brain. Intracerebral and subarachnoid hemorrhage are subsets of a broader category of hemorrhage referred to as intracranial hemorrhage. Intracerebral hemorrhage is typically due to chronic hypertension, and a resulting rupture of an arteriosclerotic vessel. Stroke-associated symptom(s) of intracerebral hemorrhage are abrupt, with the onset of headache and steadily increasing neurological deficits. Nausea, vomiting, delirium, seizures and loss of consciousness are additional common stroke-associated symptoms.
  • In contrast, most subarachnoid hemorrhage is caused by head trauma or aneurysm rupture which is accompanied by high pressure blood release which also causes direct cellular trauma. Prior to rupture, aneurysms may be asymptomatic, or occasionally associated with tension or migraine headaches. However, headache typically becomes acute and severe upon rupture, and may be accompanied by varying degrees of neurological deficit, vomiting, dizziness, and altered pulse and respiratory rates.
  • Transient ischemic attacks (TIAs) have a sudden onset and brief duration, typically 2-30 minutes. Most TIAs are due to emboli from atherosclerotic plaques, often originating in the arteries of the neck, and can result from brief interruptions of blood flow. The symptoms of TIAs are identical to those of stroke, but are only transient. Concomitant with underlying risk factors, patients experiencing TIAs are at a markedly increased risk for stroke.
  • Current diagnostic methods for stroke include costly and time-consuming procedures such as noncontrast computed tomography (CT) scan, electrocardiogram, magnetic resonance imaging (MRI), and angiography. Determining the immediate cause of stroke and differentiating ischemic from hemorrhagic stroke is difficult. CT scans can detect parenchymal bleeding greater than 1 cm and 95% of all subarachnoid hemorrhages. CT scan often cannot detect ischemic strokes until 6 hours from onset, depending on the infarct size. MRI may be more effective than CT scan in early detection of ischemic stroke, but it is less accurate at differentiating ischemic from hemorrhagic stroke, and is not widely available. An electrocardiogram (ECG) can be used in certain circumstances to identify a cardiac cause of stroke. Angiography is a definitive test to identify stenosis or occlusion of large and small cranial blood vessels, and can locate the cause of subarachnoid hemorrhages, define aneurysms, and detect cerebral vasospasm. It is, however, an invasive procedure that is also limited by cost and availability. Coagulation studies can also be used to rule out a coagulation disorder (coagulopathy) as a cause of hemorrhagic stroke.
  • Immediate diagnosis and care of a patient experiencing stroke can be critical. For example, tissue plasminogen activator (TPA) given within three hours of symptom onset in ischemic stroke is beneficial for selected acute stroke patients. Alternatively, patients may benefit from anticoagulants (e.g., heparin) if they are not candidates for TPA therapy. In contrast, thrombolytics and anticoagulants are strongly contraindicated in hemorrhagic strokes. Thus, early differentiation of ischemic events from hemorrhagic events is imperative. Moreover, delays in the confirmation of stroke diagnosis and the identification of stroke type limit the number of patients that may benefit from early intervention therapy. Finally, there are currently no diagnostic methods that can identify a TIA, or predict delayed neurological deficits which are often detected at a time after onset concurrent with the presentation of symptoms.
  • Accordingly, there is a present need in the art for a rapid, sensitive and specific diagnostic assay for stroke and TIA that can also differentiate the stroke type and identify those individuals at risk for delayed neurological deficits. Such a diagnostic assay would greatly increase the number of patients that can receive beneficial stroke treatment and therapy, and reduce the costs associated with incorrect stroke diagnosis.
  • The present invention relates to the identification and use of diagnostic markers for stroke and neural tissue injury. The methods and compositions described herein can meet the need in the art for rapid, sensitive and specific diagnostic assay to be used in the diagnosis and differentiation of various forms of stroke and TIAs. Moreover, the methods and compositions of the present invention can also be used to facilitate the treatment of stroke patients and the development of additional diagnostic and/or prognostic indicators.
  • In various aspects, the invention relates to materials and procedures for identifying markers that are associated with the diagnosis, prognosis, or differentiation of stroke and/or TIA in a patient; to using such markers in diagnosing and treating a patient and/or to monitor the course of a treatment regimen; to using such markers to identify subjects at risk for one or more adverse outcomes related to stroke and/or TIA; and for screening compounds and pharmaceutical compositions that might provide a benefit in treating or preventing such conditions.
  • In a first aspect, the invention discloses methods for determining a diagnosis or prognosis related to stroke, or for differentiating between types of strokes and/or TIA. These methods comprise analyzing a test sample obtained from a subject for the presence or amount of one or more markers for neural tissue injury. These methods can comprise identifying one or more markers, the presence or amount of which is associated with the diagnosis, prognosis, or differentiation of stroke and/or TIA. Once such marker(s) are identified, the level of such marker(s) in a sample obtained from a subject of interest can be measured. In certain embodiments, these markers can be compared to a level that is associated with the diagnosis, prognosis, or differentiation of stroke and/or TIA. By correlating the subject's marker level(s) to the diagnostic marker level(s), the presence or absence of stroke, the probability of future adverse outcomes, etc., in a patient may be rapidly and accurately determined.
  • In a related aspect, the invention discloses methods for determining the presence or absence of a disease in a subject that is exhibiting a perceptible change in one or more physical characteristics (that is, one or more “symptoms”) that are indicative of a plurality of possible etiologies underlying the observed symptom(s), one of which is stroke. These methods comprise analyzing a test sample obtained from the subject for the presence or amount of one or more markers selected to rule in or out stroke, or one or more types of stroke, as a possible etiology of the observed symptom(s). Etiologies other than stroke that are within the differential diagnosis of the symptom(s) observed are referred to herein as “stroke mimics”, and marker(s) able to differentiate one or more types of stroke from stroke mimics are referred to herein as “stroke differential diagnostic markers”. The presence or amount of such marker(s) in a sample obtained from the subject can be used to rule in or rule out one or more of the following: stroke, thrombotic stroke, embolic stroke, lacunar stroke, hypoperfusion, intracerebral hemorrhage, and subarachnoid hemorrhage, thereby either providing a diagnosis (rule-in) and/or excluding a diagnosis (rule-out).
  • Obtaining information on the true time of onset can be critical, as early treatments have been reported to be critical for proper treatment. Obtaining this time-of-onset information may be difficult, and is often based upon interviews with companions of the stroke victim. Thus, in various embodiments, markers and marker panels are selected to distinguish the approximate time since stroke onset. For purposes of the present invention, the term “acute stroke” refers to a stroke that has occurred within the prior 12 hours, more preferably within the prior 6 hours, and most preferably within the prior 3 hours; while the term “non-acute stroke” refers to a stroke that has occurred more than 12 hours ago, preferably between 12 and 48 hours ago, and most preferably between 12 and 24 hours ago. Embodiments of markers for differentiating between acute and non-acute strokes, referred to herein as stroke “time of onset markers” are described hereinafter.
  • For markers appearing in the patent which are already linked to stroke, either ischemic or hemorrhagic, variants could also help to diagnose, directly or by elimination of other conditions including but not limited to:
  • 1. Transient ischemic attack
    2. Brain trauma, in case it is unclear whether accompanied by stroke or not
  • 3. Migraine
  • 4. Bleeding in any part of the brain or inside the skull that cause or didn't cause damage to brain tissue
  • 5. Tumor
  • In addition, such markers may help determine:
    1. The time of stroke
    2. The type of stroke
    3. The extent of tissue damage as a result of the stroke
    4. Response to immediate treatments that are meant to alleviate the extent of stroke and brain damage, when available.
  • With regard to stroke, according to embodiments of the present invention, the panel may optionally and preferably provide diagnosis of stroke and indication if an ischemic stroke has occurred; diagnosis of stroke and indication if a hemorrhagic stroke has occurred; diagnosis of stroke, indication if an ischemic stroke has occurred, and indication if a hemorrhagic stroke has occurred; diagnosis of stroke and prognosis of a subsequent cerebral vasospasm; and diagnosis of stroke, indication if a hemorrhagic stroke has occurred, and prognosis of a subsequent cerebral vasospasm.
  • According to other optional embodiments of the present invention, there are provided methods of identifying a patient at risk for cerebral vasospasm. Such methods preferably comprise comparing an amount of one or more marker(s) predictive of a subsequent cerebral vasospasm in a test sample from a patient diagnosed with a subarachnoid hemorrhage. Such markers may be one or more markers related to blood pressure regulation, markers related to inflammation, markers related to apoptosis, and/or specific markers of neural tissue injury. As discussed herein, such marker may be used in panels comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual markers. Embodiments of marker(s) may be selected from the group consisting of HSFLT variants, HSI1Ra variants, HSPLGF variants, HUMSP18A variants, F05068 variants and/or HUMIL10 variants or markers related thereto. The levels of one or more markers may be compared to a predictive level of said marker(s), wherein said patient is identified as being at risk for cerebral vasospasm by a level of said marker(s) equal to or greater than said predictive level. In the alternative, a panel response value for a plurality of such markers may be determined, optionally considering a change in the level of one or more such markers as an additional independent marker.
  • According to yet other embodiments of the present invention, there are provided methods of differentiating ischemic stroke from hemorrhagic stroke using such marker panels.
  • Cardiomyopathy and Myocarditis
  • Cardiomyopathy may be treated with the polynucleotides/polypeptides and/or methods of this invention. Cardiomyopathy is a general diagnostic term designating primary myocardial disease which may progress to heart failure. The disease comprises inflammatory cardiomyopathies, cardiomyopathies resulting from a metabolic disorder such as a nutritional deficiency or by altered endocrine function, exposure to toxic substances, for example from alcohol or exposure to cobalt or lead, infiltration and deposition of abnormal. In some embodiments, the marker(s) for diagnosis of cardiomyopathy and myocarditis, and related conditions as described herein, may optionally be selected from the group consisting of HSFLT variants, HSI1Ra variants, HSPLGF variants, HUMSP18A variants, F05068 variants and/or HUMIL10 variants
  • Congestive Heart Failure (CHF)
  • HSFLT variants, HSI1Ra variants, HSPLGF variants, HUMSP18A variants, F05068 variants and/or HUMIL10 variants are potential markers for, and may be used to treat, etc., CHF.
  • The invention provides a means for the identification/prognostication, etc., of a number of conditions including the assessment of the presence, risk and/or extent of the following:
    • 1. A risk factor for sudden cardiac death, from arrhythmia or any other heart related reason.
    • 2. Rejection of a transplanted heart.
    • 3. Conditions that lead to heart failure including but not limited to myocardial infarction, angina, arrhythmias, valvular diseases, atrial and/or ventricular septal defects.
    • 4. Conditions that cause atrial and or ventricular wall volume overload. Wall stretch results in enhanced secretion of cardiac extracellular regulators. Such conditions include but are not limited to systemic arterial hypertension, pulmonary hypertension and pulmonary embolism.
    • 5. Conditions which have similar clinical symptoms as heart failure and as states that cause atrial and or ventricular pressure-overload, where the differential diagnosis between these conditions to the latter is of clinical importance including but not limited to breathing difficulty and/or hypoxia due to pulmonary disease, anemia or anxiety.
    Cancerous Conditions
  • Various non-limiting examples are given below of cancerous conditions for which one or more variants according to the present invention may have a diagnostic, or therapeutic utility.
  • Ovarian Cancer
  • Ovarian cancer causes more deaths than any other cancer of the female reproductive system, however, only 25% of ovarian cancers are detected in stage I. No single marker has been shown to be sufficiently sensitive or specific to contribute to the diagnosis of ovarian cancer.
  • In one embodiment, the markers of this invention are utilized alone, or in combination with other markers, for the diagnosis, treatment or assessment of prognosis of ovarian cancer. Such other markers may comprise CA-125 or mucin 16, CA-50, CA 54-61, CA-195 and CA 19-9, STN and TAG-72, kallikreins, cathepsin L, urine gonadotropin, inhibins, cytokeratins, such as TPA and TPS, members of the Transforming Growth Factors (TGF) beta superfamily, Epidermal Growth Factor, p53 and HER-2 or any combination thereof.
  • Immunohistochemistry may be used to assess the origin of the tumor and staging as part of the methods of this invention, and as protected uses for the polypeptides of this invention.
  • In some embodiments, this invention provides polypeptides/polynucleotides which serves as markers for ovarian cancer. In some embodiments, the marker is any polypeptide/polynucleotide as described herein. In some embodiments, the marker is an HSFLT, Z25299 or variants as described herein or markers related thereto. Each variant marker of the present invention described herein may be used alone or in combination with one or more other variant ovarian cancer described herein, and/or in combination with known markers for ovarian cancer, as described herein. Diagnosis of ovarian cancer and or of other conditions that may be diagnosed by these markers or variants of them include but are not limited to the presence, risk and/or extent of the following:
      • 1. The identification of a metastasis of unknown origin which originated from a primary ovarian cancer, for example gastric carcinoma (such as Krukenberg tumor), breast cancer, colorectal carcinoma and pancreatic carcinoma.
      • 2. As a marker to distinguish between different types of ovarian cancer, therefore potentially affect treatment choice (e.g. discrimination between epithelial tumors and germ cell tumors).
      • 3. As a tool in the assessment of abdominal mass and in particular in the differential diagnosis between a benign and malignant ovarian cysts.
      • 4. As a tool for the assessment of infertility.
      • 5. Other conditions that may elevate serum levels of ovary related markers. These include but are not limited to: cancers of the endometrium, cervix, fallopian tubes, pancreas, breast, lung and colon; nonmalignant conditions such as pregnancy, endometriosis, pelvic inflammatory disease and uterine fibroids.
      • 6. Conditions which have similar symptoms, signs and complications as ovarian cancer and where the differential diagnosis between them and ovarian cancer is of clinical importance including but not limited to:
        • a. Non-malignant causes of pelvic mass. Including, but not limited to: benign (functional) ovarian cyst, uterine fibroids, endometriosis, benign ovarian neoplasms and inflammatory bowel lesions
        • b. Any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, skeletal or abdominal pain, paraneoplastic syndrome.
        • c. Ascites.
      • 7. Prediction of patient's drug response
      • 8. As surrogate markers for clinical outcome of a treated cancer.
    Breast Cancer
  • Breast cancer is the most commonly occurring cancer in women, comprising almost a third of all malignancies in females. In one embodiment, the polypeptides and/or polynucleotides of this invention are utilized alone, or in combination with other markers, for the diagnosis, treatment or assessment of prognosis of breast cancer. In one embodiment, the polypeptides and/or polynucleotides serve as markers of disease.
  • Such markers may be used alone, or in combination with other known markers for breast cancer, including, inter alia, Mucin1 (measured as CA 15-3), CEA (CarcinoEmbryonic Antigen), HER-2, CA125, CA 19-9, PCNA, Ki-67, E-Cadherin, Cathepsin D, TFF1, epidermal growth factor receptor (EGFR), cyclin E, p53, bcl-2, vascular endothelial growth factor, urokinase-type plasminogen activator-1, survivin, or any combination thereof, and includes use of any compound which detects or quantifies the same. ESR (Erythrocyte Sedimentation Rate) values may be obtained, and comprise the marker panel for breast cancer.
  • In some embodiments, the polypeptides/polynucleotides of this invention serve as prognosticators, in identifying, inter alia, patients at minimal risk of relapse, patients with a worse prognosis, or patients likely to benefit from specific treatments.
  • There are some non-cancerous pathological conditions which represent an increased risk factor for development breast cancer, and as such, patients with these conditions may be evaluated using the polypeptides/polynucleotides and according to the methods of this invention, for example, as part of the screening methods of this invention, Some of these conditions include, but are not limited to ductal hyperplasia without atypia, atypical hyperplasia, and others.
  • In some embodiments, the polypeptides/polynucleotides of this invention serve as markers for breast cancer, including, but not limited to: HSFLT, AA336074, Z25299 or homologues thereof. In some embodiments, the HSFLT, AA336074, Z25299 or polynucleotides encoding the same, can be used alone or in combination with any other desired marker, including, inter alia, Calcitonin, CA15-3 (Mucin1), CA27-29, TPA, a combination of CA 15-3 and CEA, CA 27.29 (monoclonal antibody directed against MUC1), Estrogen 2 (beta), HER-2 (c-erbB2), or any combinations thereof.
  • In some embodiments, the polypeptides/polynucleotides of this invention may be useful in, inter alia, assessing the presence, risk and/or extent of the following:
    • 1. The identification of a metastasis of unknown origin which originated from a primary breast cancer tumor.
    • 2. In the assessment of lymphadenopathy, and in particular axillary lymphadenopathy.
    • 3. As a marker to distinguish between different types of breast cancer, therefore potentially affect treatment choice (e.g. as HER-2)
    • 4. As a tool in the assessment of palpable breast mass and in particular in the differential diagnosis between a benign and malignant breast mass.
    • 5. As a tool in the assessment of conditions affecting breast skin (e.g. Paget's disease) and their differentiation from breast cancer.
    • 6. As a tool in the assessment of breast pain or discomfort resulting from either breast cancer or other possible conditions (e.g. Mastitis, Mondors syndrome).
    • 7. Other conditions not mentioned above which have similar symptoms, signs and complications as breast cancer and where the differential diagnosis between them and breast cancer is of clinical importance including but not limited to:
      • a. Abnormal mammogram and/or nipple retraction and/or nipple discharge due to causes other than breast cancer. Such causes include but are not limited to benign breast masses, melanoma, trauma and technical and/or anatomical variations.
      • b. Any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, paraneoplastic syndrome.
  • Lymphadenopathy, weight loss and other signs and symptoms associated with breast cancer but originate from diseases different from breast cancer including but not limited to other malignancies, infections and autoimmune diseases.
    • 8. Prediction of patient's drug response
    • 9. As surrogate markers for clinical outcome of a treated cancer.
    Lung Cancer
  • Lung cancer is the primary cause of cancer death among both men and women in the U.S. In one embodiment, the polypeptides and/or polynucleotides of this invention are utilized alone, or in combination with other markers, for the diagnosis, treatment or assessment of prognosis of lung cancer. In one embodiment, the term “lung cancer” is to be understood as encompassing small cell or non-small cell lung cancers, including adenocarcinomas, bronchoalveolar-alveolar, squamous cell and large cell carcinomas.
  • In some embodiments, the polypeptides/polynucleotides of this invention are utilized in conjunction with other screening procedures, as well as the use of other markers, for the diagnosis, or assessment of prognosis of lung cancer in a subject. In some embodiments, such screening procedures may comprise the use of chest x-rays, analysis of the type of cells contained in sputum, fiberoptic examination of the bronchial passages, or any combination thereof. Such evaluation in turn may impact the type of treatment regimen pursued, which in turn may reflect the type and stage of the cancer, and include surgery, radiation therapy and/or chemotherapy.
  • Current radiotherapeutic agents, chemotherapeutic agents and biological toxins are potent cytotoxins, yet do not discriminate between normal and malignant cells, producing adverse effects and dose-limiting toxicities. In some embodiments of this invention, the polypeptides/polynucleotides provide a means for more specific targeting to neoplastic versus normal cells.
  • In some embodiments, the polypeptides for use in the diagnosis, treatment and/or assessment of progression of lung cancer may comprise: HSFLT, HSI1RA, HSPLGF, HUMSP18A, F05068, Z25299, AA336074 or homologous thereof, or polynucleotides encoding the same. In some embodiments, these polypeptides/polynucleotides may be used alone or in combination with one or more other appropriate markers, including, inter alia, other polypeptides/polynucleotides of this invention. In some embodiments, such use may be in combination with other known markers for lung cancer, including but not limited to CEA, CA15-3, Beta-2-microglobulin, CA19-9, TPA, and/or in combination with native sequences associated with the polypeptides/polynucleotides of this invention, as herein described.
  • In some embodiments, the polypeptides/polynucleotides of this invention may be useful in, inter alia, assessing the presence, risk and/or extent of the following:
    • 1. The identification of a metastasis of unknown origin which originated from a primary lung cancer.
    • 2. The assessment of a malignant tissue residing in the lung that is from a non-lung origin, including but not limited to: osteogenic and soft tissue sarcomas; colorectal, uterine, cervix and corpus tumors; head and neck, breast, testis and salivary gland cancers; melanoma; and bladder and kidney tumors.
    • 3. Distinguishing between different types of lung cancer, therefore potentially affect treatment choice (e.g. small cell vs. non small cell tumors).
    • 4. Unexplained dyspnea and/or chronic cough and/or hemoptysis, and analysis thereof.
    • 5. Differential diagnosis of the origin of a pleural effusion.
    • 6. Conditions which have similar symptoms, signs and complications as lung cancer and where the differential diagnosis between them and lung cancer is of clinical importance including but not limited to:
      • a. Non-malignant causes of lung symptoms and signs. Symptoms and signs include, but are not limited to: lung lesions and infiltrates, wheeze, stridor.
      • b. Other symptoms, signs and complications suggestive of lung cancer, such as tracheal obstruction, esophageal compression, dysphagia, recurrent laryngeal nerve paralysis, hoarseness, phrenic nerve paralysis with elevation of the hemidiaphragm and Horner syndrome.
      • c. Any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, hypophosphatemia, hyponatremia, syndrome of inappropriate secretion of antidiuretic hormone, elevated ANP, elevated ACTH, hypokalemia, clubbing, neurologic-myopathic syndromes and thrombophlebitis.
    • 7. Prediction of patient's drug response
    • 8. As surrogate markers for clinical outcome of a treated cancer.
    Colorectal Cancer:
  • Colon and rectal cancers are malignant conditions which occur in the corresponding segments of the large intestine. In one embodiment, the polypeptides and/or polynucleotides of this invention are utilized alone, or in combination with other markers, for the diagnosis, treatment or assessment of prognosis of colorectal cancer. In some embodiments, the term “colorectal cancers” is to be understood as encompassing adenocarcinomas, carcinoid tumors, for example, found in the appendix and rectum; gastrointestinal stromal tumors for example, found in connective tissue in the wall of the colon and rectum; and lymphomas, which are malignancies of immune cells in the colon, rectum and lymph nodes. In some embodiments, the polypeptides/polynucleotides are useful in diagnosing, treating and/or assessing progression of colorectal pathogenesis, including the maturation of adenomatous polyps, to larger polyps, and all relevant stages in the neoplastic transformation of the tissue.
  • In some embodiments, the polypeptides/polynucleotides of this invention are utilized in conjunction with other screening procedures, as well as the use of other markers, for the diagnosis, or assessment of prognosis of colorectal cancer in a subject. In some embodiments, such screening procedures may comprise fecal occult blood tests, sigmoidoscopy, barium enema X-ray, digital rectal exam, colonoscopy, detection of carcinoembryonic antigen (CEA) or combinations thereof.
  • In some embodiments, the polypeptides/polynucleotides are useful in assessing progression of colorectal pathogenesis. Such assessment may reflect the staging of the colorectal cancer. In some embodiments, the polypeptides/polynucleotides are useful in assessing or altering stage progression in a subject with colorectal cancer. When in reference to cancer staging, it is to be understood that any known means or classification system will apply, for any embodiment as described herein. In some embodiments, staging in reference to colorectal cancer may be via the Dukes' system and/or the International Union against Cancer-American Joint Committee on Cancer TNM staging system. Staging will reflect, in some embodiments, the extent of tumor penetration into the colon wall, with greater penetration generally correlating with a more dangerous tumor; the extent of invasion of the tumor through the colon wall and into other neighboring tissues, with greater invasion generally correlating with a more dangerous tumor; the extent of invasion of the tumor into the regional lymph nodes, and the extent of metastatic invasion into more distant tissues, such as the liver. It is to be understood that the polypeptides/polynucleotides of this invention may be useful both in the identification/assessment of colorectal cancer pathogenesis as a function of stage designation, as well as
  • In some embodiments, the polypeptides/polynucleotides of this invention may be useful in the diagnosis, treatment and/or assessment of prognosis of colon cancer. According to this aspect and in one embodiment, the polypeptides useful in this context are: HSFLT, HSPLGF, Z25299 or homologues thereof, or polynucleotides encoding the same. In some embodiments, these polypeptides/polynucleotides are used alone or in combination with one or more other polypeptides/polynucleotides of this invention, and/or in combination with other markers for colorectal cancer, including but not limited to CEA, CA19-9, CA50, and/or in combination with a native protein associated with the polypeptides of this invention, for example, native proteins of which the polypeptides are variants thereof. In some embodiments, the polypeptides/polynucleotides of this invention may be useful in, inter alia, assessing the presence, risk and/or extent of the following:
    • 1. Early diagnosis, staging, grading, prognosis, monitoring, and treatment of diseases associated with colon cancer, or to indicate a predisposition to such for preventative measures.
    • 2. The identification of a metastasis of unknown origin which originated from a primary colorectal cancer tumor, in particular when the metastasis is located in the liver, lung, bones, supraclavicluar lymph nodes or brain.
    • 3. In the assessment of lymphadenopathy, in particular supraclavicluar or internal abdominal lymphadenopathy.
    • 4. As a marker to distinguish between different types of colorectal tumors including but not limited to nonhereditary carcinoma, Familial Polyposis Coli, Hereditary nonpolyposis colon cancer (Lynch syndrome) and Carcinoid; therefore potentially affect treatment choice.
    • 5. In the assessment of cancer staging, in addition and as a complementary measure to the Dukes system for staging colorectal cancer.
    • 6. As a risk factor to the development of colorectal tumor, and in particular in diseases known to have high incidence of colorectal tumor, including but not limited to Crohn's disease and Ulcerative Colitis.
    • 7. As a tool in the assessment of fecal occult blood or imaging findings suspected for colorectal tumor or abnormal blood tests associated with colorectal cancer including but not limited to elevated CEA level.
    • 8. In the differential diagnosis between malignant and benign colorectal tumors, in particular adenomas and polyps.
    • 9. Other conditions not mentioned above which have similar symptoms, signs and complications as colorectal cancer and where the differential diagnosis between them and colorectal cancer is of clinical importance including but not limited to:
      • a. Any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, paraneoplastic syndrome.
      • b. Lymphadenopathy, weight loss and other signs and symptoms associated with colorectal cancer but originate from diseases different from colorectal cancer including but not limited to other malignancies, infections and autoimmune diseases.
    • 10. Prediction of patient's drug response
    • 11. As surrogate markers for clinical outcome of a treated cancer.
    Prostate Cancer
  • Prostate cancer is the most commonly diagnosed malignancy and the second most frequent cause of cancer-related deaths in the western male population. In one embodiment, the polypeptides and/or polynucleotides of this invention are utilized alone, or in combination with other markers, for the diagnosis, treatment or assessment of prognosis of prostate cancer.
  • In some embodiments, the polypeptides/polynucleotides of this invention are utilized in conjunction with other screening procedures, as well as the use of other markers, for the diagnosis, or assessment of prognosis of colorectal cancer in a subject. In some embodiments, such markers may comprise prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), prostate-specific membrane antigen (PSM), PCA3 DD3 or combinations thereof.
  • In some embodiments, the polypeptides/polynucleotides of this invention may be useful in the diagnosis, treatment and/or assessment of prognosis of prostate cancer. According to this aspect and in one embodiment, the polypeptides useful in this context are: AA336074, Z22012, HUMTREFAC, homologues thereof, or polynucleotides encoding the same. In some embodiments, these polypeptides/polynucleotides are used alone or in combination with one or more other polypeptides/polynucleotides of this invention, and/or in combination with other markers, including, inter alia, PSA, PAP (prostatic acid phosphatase), CPK-BB, PSMA, PCA3, DD3, and/or a native protein associated with the polypeptides of this invention, for example, native proteins of which the polypeptides are variants thereof. In some embodiments the polypeptides/polynucleotides of this invention are useful in the diagnosis of prostate cancer, which includes, inter alia, the differential diagnosis between prostate cancer and BPH, prostatitis and/or prostatism.
  • Candidate Markers
  • The markers of the present invention were tested with regard to their expression in various tissue samples. Unless otherwise noted, all experimental data relates to variants of the present invention, named according to the segment being tested (as expression was tested through RT-PCR as described). A description of the samples used in the ovarian cancer testing panel is provided in Table 1 below. A description of the samples used in the colon cancer testing panel is provided in Table 2 below. A description of the samples used in the lung cancer testing panel is provided in Table 3 below. A description of the samples used in the breast cancer testing panel is provided in Table 4 and Table 41 below. Table 42 provides a key for various terms listed in table 41. A description of the samples used in the normal tissue panel is provided in Table 5 and table 51 below.
  • TABLE 1
    Tissue samples in ovarian cancer testing panel:
    Sample name Lot number Source Pathology Grade age
    33-B-Pap Sero CystAde G1 A503175 BioChain Serous papillary cystadenocarcinoma 1 41
    41-G-Mix Sero/Muc/Endo G2 98-03-G803 GOG Mixed epithelial cystadenocarcinoma with mucinous, 2 38
    endometrioid, squamous and papillary serous (Stage 2)
    35-G-Endo Adeno G2 94-08-7604 GOG Endometrioid adenocarcinoma 2 39
    14-B-Adeno G2 A501111 BioChain Adenocarcinoma 2 41
    12-B-Adeno G3 A406023 Biochain Adenocarcinoma 3 45
    40-G-Mix Sero/Endo G2 95-11-G006 GOG Papillary serous and endometrioid cystadenocarcinoma 2 49
    (Stage 3C)
    4-A-Pap CystAdeno G2 ILS-7286 ABS Papillary cystadenocarcinoma 2 50
    3-A-Pap Adeno G2 ILS-1431 ABS Papillary adenocarcinoma 2 52
    2-A-Pap Adeno G2 ILS-1408 ABS Papillary adenocarcinoma 2 53
    5-G-Adeno G3 99-12-G432 GOG Adenocarcinoma (Stage 3C) 3 46
    11-B-Adeno G3 A407068 Biochain Adenocarcinoma 3 49
    39--G-Mix Sero/Endo G3 2001-12-G037 GOG Mixed serous and endometrioid adenocarcinoma 3 49
    29-G-Sero Adeno G3 2001-12-G035 GOG Serous adenocarcinoma (Stage 3A) 3 50
    70-G-Pap Sero Adeno G3 95-08-G069 GOG Papillary serous adenocarcinoma 3 50
    6-A-Adeno G3 A0106 ABS adenocarcinoma 3 51
    31-B-Pap Sero CystAde G3 A503176 BioChain Serous papillary cystadenocarcinoma 3 52
    25-A-Pap Sero Adeno G3 N0021 ABS Papillary serous adenocarcinoma (Stage T3CN1MX) 3 55
    37-G-Mix Sero/Endo G3 2002-05-G513 GOG Mixed serous and endometrioid adenocarcinoma 3 56
    7-A-Adeno G3 IND-00375 ABS adenocarcinoma 3 59
    8-B-Adeno G3 A501113 BioChain adenocarcinoma 3 60
    10-B-Adeno G3 A407069 Biochain Adenocarcinoma 3 60
    38-G-Mix Sero/Endo G3 2002-05-G509 GOG Mixed serous and endometrioid adenocarcinoma of mullerian 3 64
    (Stage 3C)
    13-G-Adeno G3 94-05-7603 GOG Poorly differentiated adenocarcinoma from primary peritoneal 3 67
    24-G-Pap Sero Adeno G3 2001-07-G801 GOG Papillary serous adenocarcinoma 3 68
    34-G-Pap Endo Adeno G3 95-04-2002 GOG Papillary endometrioid adenocarcinoma (Stage 3C) 3 68
    30-G-Pap Sero Adeno G3 2001-08-G011 GOG Papillary serous carcinoma (Stage 1C) 3 72
    1-A-Pap Adeno G3 ILS-1406 ABS Papillary adenocarcinoma 3 73
    9-G-Adeno G3 99-06-G901 GOG Adenocarcinoma (maybe serous) 3 84
    32-G-Pap Sero CystAde G3 93-09-4901 GOG Serous papillary cystadenocarcinoma 3 67
    66-G-Pap Sero Adeno G3 SIV 2000-01-G413 GOG Papillary serous carcinoma (metastais of primary peritoneum) 3 67
    (Stage 4)
    19-B-Muc Adeno G3 A504085 BioChain Mucinous adenocarcinoma 3 34
    21-G-Muc CystAde G2-3 95-10-G020 GOG Mucinous cystadenocarcinoma (Stage 2) 2-3 44
    18-B-Muc Adeno G3 A504083 BioChain Mucinous adenocarcinoma 3 45
    20-A-Pap Muc CystAde USA-00273 ABS Papillary mucinous cystadenocarcinoma 46
    17-B-Muc Adeno G3 A504084 BioChain Mucinous adenocarcinoma 3 51
    22-A-Muc CystAde G2 A0139 ABS Mucinous cystadenocarcinoma (Stage 1C) 2 72
    43-G-Clear cell Adeno G3 2001-10-G002 GOG Clear cell adenocarcinoma 3 74
    44-G-Clear cell Adeno 2001-07-G084 GOG Clear cell adenocarcinoma (Stage 3A) 73
    15-B-Adeno G3 A407065 BioChain Carcinoma 3 27
    16-Ct-Adeno 1090387 Clontech Carcinoma NOS NA 58
    23-A-Muc CystAde G3 VNM-00187 ABS Mucinous cystadenocarcinoma with low malignant 3 45
    42-G-Adeno borderline 98-08-G001 GOG Epithelial adenocarcinoma of borderline malignancy 46
    63-G-Sero CysAdenoFibroma 2000-10-G620 GOG Serous CysAdenoFibroma of borderline malignancy 71
    62-G-Ben Muc CysAdenoma 99-10-G442 GOG Benbin mucinus cysadenoma 32
    60-G-Muc CysAdenoma 99-01-G043 GOG Mucinous Cysadenoma 40
    56-G-Ben Muc CysAdeno 99-01-G407 GOG Bengin mucinus cysadenoma 46
    64-G-Ben Sero CysAdenoma 99-06-G039 GOG Bengin Serous CysAdenoma 57
    61-G-Muc CysAdenoma 99-07-G011 GOG Mucinous Cysadenoma 63
    59-G-Sero CysAdenoFibroma 98-12-G401 GOG Serous CysAdenoFibroma 77
    51-G-N M41 98-03-G803N GOG Normal (matched tumor 98-03-G803) 38
    75-G-N M60 99-01-G043N GOG Normal (matched tumor 99-01-G043) 40
    49-B-N M14 A501112 BioChain Normal (matched tumor A501111) 41
    52-G-N M42 98-08-G001N GOG Normal (matched tumor 98-08-G001) 46
    68-G-N M56 99-01-G407N GOG Normal (matched bengin 99-01-G407) 46
    50-B-N M8 A501114 BioChain Normal (matched tumor A501113) 60
    67-G-N M38 2002-05-509N GOG Normal (matched tumor 2002-05-G509) 64
    69-G-N M24 2001-07-G801N GOG Normal (matched tumor 2001-07-G801) 68
    73-G-N M59 98-12-G401N GOG Normal (matched tumor 98-12-G401) 77
    72-G-N M66 2000-01-G413N GOG Normal (matched tumor 2000-01-G413)
    45-B-N A503274 BioChain Normal PM 41
    46-B-N A504086 BioChain Normal PM 41
    71-CG-N CG-188-7 Ichilov Normal PM 49
    48-B-N A504087 BioChain Normal PM 51
  • TABLE 2
    Tissue samples in colon cancer testing panel
    sample name Lot No. tissue source pathology Grade gender/age
    58-B-Adeno G1 A609152 Colon biochain Adenocarcinoma 1 M/73
    59-B-Adeno G1 A609059 Colon biochain Adenocarcinoma, Ulcer 1 M/58
    14-CG-Polypoid Adeno G1 D-C CG-222 (2) Rectum Ichilov Well polypoid adeocarcinoma Duke's C F/49
    17-CG-Adeno G1-2 CG-163 Rectum Ichilov Adenocarcinoma 2 M/73
    10-CG-Adeno G1-2 D-B2 CG-311 Sigmod colon Ichilov Adenocarcinoma Astler-Coller B2. 1-2 M/88
    11-CG-Adeno G1-2 D-C2 CG-337 Colon Ichilov Adenocarcinoma Astler-Coller C2. 1-2 NA
    6-CG-Adeno G1-2 D-C2 CG-303 (3) Colon Ichilov Adenocarcinoma Astler-Coller C2. 1-2 F/77
    5-CG-Adeno G2 CG-308 Colon Sigma Ichilov Adenocarcinoma. 2 F/80
    16-CG-Adeno G2 CG-278C colon Ichilov Adenocarcinoma 2 F/60
    56-B-Adeno G2 A609148 Colon biochain Adenocarcinoma 2 F48
    61-B-Adeno G2 A606258 Colon biochain Adenocarcinoma, Ulcer 2 M/41
    60-B-Adeno G2 A609058 Colon biochain Adenocarcinoma, Ulcer 2 M/67
    22-CG-Adeno G2 D-B CG-229C Colon Ichilov Adenocarcinoma Duke's B 2 F/55
    1-CG-Adeno G2 D-B2 CG-335 Cecum Ichilov Adenocarcinoma Dukes B2. 2 F/66
    12-CG-Adeno G2 D-B2 CG-340 Colon Sigma Ichilov Adenocarcinoma Astler-Coller B2. 2 M/66
    28-CG-Adeno G2 D-B2 CG-284 sigma Ichilov Adenocarcinoma Duke's B2 2 F/72
    2-CG-Adeno G2 D-C2 CG-307 X2 Cecum Ichilov Adenocarcinoma Astler-Coller C2. 2 F/89
    9-CG-Adeno G2 D-D CG-297 X2 Rectum Ichilov Adenocarcinoma Dukes D. 2 M/62
    13-CG-Adeno G2 D-D CG-290 X2 Rectosigmoidal colon Ichilov Adenocarcinoma Dukes D. 2 M/47
    26-CG-Adeno G2 D-D CG-283 sigma Ichilov Colonic adenocarcinoma Duke's D 2 F/63
    4-CG-Adeno G3 CG-276 Colon Ichilov Carcinoma. 3 M/64
    53-B-Adeno G3 A609161 Colon biochain Adenocarcinoma 3 F/53
    54-B-Adeno G3 A609142 Colon biochain Adenocarcinoma 3 M/53
    55-B-Adeno G3 A609144 Colon biochain Adenocarcinoma 3 M/68
    57-B-Adeno G3 A609150 Colon biochain Adenocarcinoma 3 F/45
    72-CG-Adeno G3 CG-309 colon Ichilov Adenocarcinoma 3 F/88
    20-CG-Adeno G3 D-B2 CG-249 Colon Ichilov Ulcerated adenocarcinoma Duke's B2 3 M/36
    7-CG-Adeno D-A CG-235 Rectum Ichilov Adenocarcinoma intramucosal Duke's F/66
    A.
    23-CG-Adeno D-C CG-282 sigma Ichilov Mucinus adenocarcinoma Astler Coller C M/51
    3-CG-Muc adeno D-D CG-224 Colon Ichilov Mucinois adenocarcinoma Duke's D M/48
    18-CG-Adeno CG-22C Colon Ichilov Adenocarcinoma NA
    19-CG-Adeno CG-19C (1) Colon Ichilov Adenocarcinoma NA
    21-CG-Adeno CG-18C Colon Ichilov Adenocarcinoma NA
    24-CG-Adeno CG-12 (2) Colon Ichilov Adenocarcinoma NA
    25-CG-Adeno CG-2 Colon Ichilov Adenocarcinoma NA
    27-CG-Adeno CG-4 Colon Ichilov Adenocarcinoma NA
    8-CG-diverticolosis, diverticulitis CG-291 Wall of sigma Ichilov Diverticolosis and F/65
    diverticulitis of the Colon
    46-CG-Crohn's disease CG-338C Cecum Ichilov Crohn's disease M/22
    47-CG-Crohn's disease CG-338AC Colon Ichilov Crohn's disease. M/22
    42-CG-N M20 CG-249N Colon Ichilov Normal M/36
    43-CG-N M8 CG-291N Wall of sigma Ichilov Normal F/65
    44-CG-N M21 CG-18N Colon Ichilov Normal NA
    45-CG-N M11 CG-337N Colon Ichilov Normal M/75
    49-CG-N M14 CG-222N Rectum Ichilov Normal F/49
    50-CG-N M5 CG-308N Sigma Ichilov Within normal limits F/80
    51-CG-N M26 CG-283N Sigma Ichilov Normal F/63
    41-B-N A501156 Colon biochain Normal PM M/78
    52-CG-N CG-309TR Colon Ichilov Within normal limits F/88
    62-B-N A608273 Colon biochain Normal PM M/66
    63-B-N A609260 Colon biochain Normal PM M/61
    64-B-N A609261 Colon biochain Normal PM F/68
    65-B-N A607115 Colon biochain Normal PM M/24
    66-B-N A609262 Colon biochain Normal PM M/58
    67-B-N A406029 Colon biochain Normal PM (Pool of 10)
    69-B-N A411078 Colon biochain Normal PM (Pool of 10) F&M
    70-Cl-N 1110101 Colon clontech Normal PM (Pool of 3)
    71-Am-N 071P10B Colon Ambion Normal (IC BLEED) F/34
  • TABLE 3
    Tissue samples in lung cancer testing panel
    sample name Lot No. source pathology Grade gender/age
    1-B-Adeno G1 A504117 Biochain Adenocarcinoma 1 F/29
    2-B-Adeno G1 A504118 Biochain Adenocarcinoma 1 M/64
    95-B-Adeno G1 A610063 Biochain Adenocarcinoma 1 F/54
    12-B-Adeno G2 A504119 Biochain Adenocarcinoma 2 F/74
    75-B-Adeno G2 A609217 Biochain Adenocarcinoma 2 M/65
    77-B-Adeno G2 A608301 Biochain Adenocarcinoma 2 M/44
    13-B-Adeno G2-3 A504116 Biochain Adenocarcinoma 2-3 M/64
    89-B-Adeno G2-3 A609077 Biochain Adenocarcinoma 2-3 M/62
    76-B-Adeno G3 A609218 Biochain Adenocarcinoma 3 M/57
    94-B-Adeno G3 A610118 Biochain Adenocarcinoma 3 M/68
    3-CG-Adeno CG-200 Ichilov Adenocarcinoma NA
    14-CG-Adeno CG-111 Ichilov Adenocarcinoma M/68
    15-CG-Bronch adeno CG-244 Ichilov Bronchioloalveolar adenocarcinoma M/74
    45-B-Alvelous Adeno A501221 Biochain Alveolus carcinoma F/50
    44-B-Alvelous Adeno G2 A501123 Biochain Alveolus carcinoma 2 F/61
    19-B-Squamous G1 A408175 Biochain Squamous carcinoma 1 M/78
    16-B-Squamous G2 A409091 Biochain Squamous carcinoma 2 F/68
    17-B-Squamous G2 A503183 Biochain Squamous carcinoma 2 M/57
    21-B-Squamous G2 A503187 Biochain Squamous carcinoma 2 M/52
    78-B-Squamous G2 A607125 Biochain Squamous Cell Carcinoma 2 M/62
    80-B-Squamous G2 A609163 Biochain Squamous Cell Carcinoma 2 M/74
    18-B-Squamous G2-3 A503387 Biochain Squamous Cell Carcinoma 2-3 M/63
    81-B-Squamous G3 A609076 Biochain Squamous Carcinoma 3 m/53
    79-B-Squamous G3 A609018 Biochain Squamous Cell Carcinoma 3 M/67
    20-B-Squamous A501121 Biochain Squamous Carcinoma M/64
    22-B-Squamous A503386 Biochain Squamous Carcinoma M/48
    88-B-Squamous A609219 Biochain Squamous Cell Carcinoma M/64
    100-B-Squamous A409017 Biochain Squamous Carcinoma M/64
    23-CG-Squamous CG-109 (1) Ichilov Squamous Carcinoma M/65
    24-CG-Squamous CG-123 Ichilov Squamous Carcinoma M/76
    25-CG-Squamous CG-204 Ichilov Squamous Carcinoma M/72
    87-B-Large cell G3 A609165 Biochain Large Cell Carcinoma 3 F/47
    38-B-Large cell A504113 Biochain Large cell M/58
    39-B-Large cell A504114 Biochain Large cell F/35
    82-B-Large cell A609170 Biochain Large Cell Neuroendocrine Carcinoma M/68
    30-B-Small cell carci G3 A501389 Biochain small cell 3 M/34
    31-B-Small cell carci G3 A501390 Biochain small cell 3 F/59
    32-B-Small cell carci G3 A501391 Biochain small cell 3 M/30
    33-B-Small cell carci G3 A504115 Biochain small cell 3 M
    86-B-Small cell carci G3 A608032 Biochain Small Cell Carcinoma 3 F/52
    83-B-Small cell carci A609162 Biochain Small Cell Carcinoma F/47
    84-B-Small cell carci A609167 Biochain Small Cell Carcinoma F/59
    85-B-Small cell carci A609169 Biochain Small Cell Carcinoma M/66
    46-B-N M44 A501124 Biochain Normal M44 F/61
    47-B-N A503205 Biochain Normal PM M/26
    48-B-N A503206 Biochain Normal PM M/44
    49-B-N A503384 Biochain Normal PM M/27
    50-B-N A503385 Biochain Normal PM M/28
    90-B-N A608152 Biochain Normal (Pool 2) PM pool 2
    91-B-N A607257 Biochain Normal (Pool 2) PM pool 2
    92-B-N A503204 Biochain Normal PM m/28
    93-Am-N 111P0103A Ambion Normal PM F/61
    96-Am-N 36853 Ambion Normal PM F/43
    97-Am-N 36854 Ambion Normal PM M/46
    98-Am-N 36855 Ambion Normal PM F/72
    99-Am-N 36856 Ambion Normal PM M/31
  • TABLE 4
    Tissue samples in breast cancer testing panel
    sample name Lot no source pathology grade age TNM stage
    14-A-IDC G2 A0135T ABS IDC 2 37 T2N2Mx
    43-B-IDC G2 A609183 Biochain IDC 2 40
    54-B-IDC G2 A605353 Biochain IDC 2 41
    55-B-IDC G2 A609179 Biochain IDC 2 42
    47-B-IDC G2 A609221 Biochain IDC 2 42
    17-A-IDC G2 4904020036T ABS IDC 2-3 42 T3N1Mx
    42-A-IDC G3 6005020031T ABS IDC 3 42 T1cN0Mx
    7-A-IDC G2 7263T ABS IDC 2 43 T1N0M0 stage 1
    48-B-IDC G2 A609222 Biochain IDC 2 44
    53-B-IDC G2 A605151 Biochain IDC 2 44
    12-A-IDC G2 1432T ABS IDC 2 46 T2N0M0 stage 2A
    61-B-IDC G2 A610029 Biochain IDC 2 46
    46-B-Carci G2 A609177 Biochain Carcinoma 2 48
    16-A-IDC G2 4904020032T ABS IDC 2 49 T3N1Mx
    62-B-IDC G2 A609194 Biochain IDC 2 51
    49-B-IDC G2 A609223 Biochain IDC 2 54
    32-A-Muc Carci 7116T ABS Mucinous carcinoma 54 T2N0M0 stage 2A
    45-B-IDC G2 A609181 Biochain IDC 2 58
    15-A-IDC G2 7259T ABS IDC 2 59 T3N1M0 stage 3A
    52-B-ILC G1 A605360 Biochain Invasive Lobular Carcinoma 1 60
    6-A-IDC G1 7238T ABS IDC 1 60 T2N0M0 stage 2A
    26-A-IDC G3 7249T ABS IDC 3 60 T2N0M0 stage 2A
    13-A-IDC G2 A0133T ABS IDC 2 63 T2N1aMx
    50-B-IDC G2 A609224 Biochain IDC 2 69
    44-B-IDC G2 A609198 Biochain IDC 2 77
    51-B-IDC G1 A605361 Biochain IDC 1 79
    31-CG-IDC CG-154 Ichilov IDC 83
    27-A-IDC G3 4907020072T ABS IDC 3 91 T2N0Mx
    36-A-N M7 7263N ABS Normal matched to 7T 43
    40-A-N M12 1432N ABS Normal matched to 12T 46
    39-A-N M15 7259N ABS Normal matched to 15T 59
    35-A-N M6 7238N ABS Normal matched to 6T 60
    41-A-N M26 7249N ABS Normal matched to 26T 60
    57-B-N A609233 Biochain Normal PM 34
    59-B-N A607155 Biochain Normal PM 35
    60-B-N A609234 Biochain Normal PM 36
    63-Am-N 26486 Ambion Normal PS 43
    66-Am-N 36678 Ambion Normal PM 45
    64-Am-N 23036 Ambion Normal PM 57
    56-B-N A609235 Biochain Normal PM 59
    65-Am-N 31410 Ambion Normal PM 63
    67-Am-N 073P01060286A Ambion Normal PM 64
    58-B-N A609232 Biochain Normal PM 65
  • TABLE 4_1
    Tissue samples in Breast cancer testing panel
    sample_id
    (GCI)/
    case id
    (Asterand)/ Age Re-
    Source/ lot no. TISSUE_ID Sample # of cov- Year
    De- sample (old (GCI)/specimen ID SampD C Tum WT HT Ethnic # of Live first Br ery of
    Tissue livery name samples) ID(Asterand) (Asterand) IAG Grade TNM Stage % age MS (KG) (CM) BMI B Preg. Bir child child Type birth
    BC_in- Aster 1-As-DCIS 19723 42509 42509A1 DCIS High T1aN0M0 0 100 39 Pre-M 102 157 41.4 CAU 2 1 Surg
    situ S0 Grade
    BC GCI 2-GC-IDC 5IRTK 5IRTKAXT IDC I 75 39 Pre-M 48.1 147 22.2 WCAU 1 0 Surg 1962
    SI
    BC ABS 3-(42)-AB- 6005020031T IDC 3 T1cN0Mx I 42
    IDC SI
    BC ABS 4-(7)-AB- 7263T IDC 2 T1N0M0 I 43
    IDC SI
    BC GCI 5-GC-IDC DSI52 DSI52AH3 IDC I 50 50 Post-M 113.4 175 36.9 WCAU 2 2 17 0 Surg 1951
    SI
    BC GCI 6-GC-IDC S2GBY S2GBYAGC IDC I 55 56 Post-M 57.6 168 20.5 WCAU 0 Surg 1945
    SI
    BC GCI 7-GC-IDC POPHP POPHPAZ4 IDC I 65 57 Post-M 76.2 165 28.0 WCAU 2 2 17 0 Surg 1944
    SI
    BC GCI 8-GC-IDC I2YLE I2YLEACP IDC I 65 60 Post-M 68 140 34.8 WCAU 1 1 23 0 Surg 1942
    SI
    BC Aster 9-As-IDC SI 17959 31225 31225A1 IDC 2 T1NXM0 I 90 65 Post-M 70 168 24.8 CAU 3 2 Aut
    BC ABS 10-(12)-AB- 1432T IDC 2 T2N0M0 IIA 46
    IDC SIIA
    BC Aster 11-As-IDC 17138 30697 30697A1 IDC 3 T2NXM0 IIA 90 46 Pre-M 69 174 22.8 CAU 1 1 Surg
    SIIA
    BC GCI 12-GC-IDC YSZ67 YSZ67A48 IDC IIA 70 46 Pre-M 76.2 165 28.0 WCAU 1 0  0 0 Surg 1956
    SIIA
    BC ABS 13-(6)-AB- 7238T IDC 1 T2N0M0 IIA 60
    IDC SIIA
    BC ABS 14-(26)-AB- 7249T IDC 3 T2N0M0 IIA 60
    IDC SIIA
    BC GCI 15-GC-IDC UT3SE UT3SEAQY IDC IIA 80 67 Post-M 113.4 168 40.4 WCAU 3 1 34 0 Surg 1938
    SIIA
    BC GCI 16-GC-IDC PVSYX PVSYXA72 IDC IIA 65 70 Pre-M 79.4 163 30.0 WCAU 2 2 20 0 Surg 1932
    SIIA
    BC GCI 17-GC-IDC GETCV GETCVAY2 IDC IIA 55 70 Post-M 72.6 163 27.5 WCAU 2 2 21 0 Surg 1931
    SIIA
    BC ABS 18-(27)-AB- 4907020072T IDC 3 T2N0Mx IIA 91
    IDC SIIA
    BC GCI 19-GC-IDC SE5BK SE5BKAEQ IDC IIB 55 41 Pre-M 61.2 165 22.5 WCAU 0 Surg 1960
    SIIB
    BC GCI 20-GC-IDC OLKL4 OLKL4AO6 IDC IIB 60 46 Pre-M 111.1 168 39.5 WCAU 2 2 24 2 Surg 1955
    SIIB
    BC GCI 21-GC-IDC VK1EJ VK1EJAQE IDC IIB 60 54 Post-M 72.6 168 25.8 WCAU 0 Surg 1947
    SIIB
    BC GCI 22-GC-IDC 3Z5Z4 3Z5Z4ANH IDC IIB 85 60 Post-M 80 163 30.3 WCAU 1 1 22 0 Surg 1942
    SIIB
    BC ABS 23-(13)-AB- A0133T IDC 2 T2N1aMx IIB 63
    IDC SIIB
    BC GCI 24-GC-IDC J5MPN J5MPNA9Q IDC IIB 55 64 Post-M 68.5 157 27.6 WCAU 4 3 25 3 Surg 1938
    SIIB
    BC GCI 25-GC-IDC 54NTA 54NTAAKT IDC IIB 70 67 Post-M 56.7 160 22.1 WCAU 5 5 20 0 Surg 1934
    SIIB
    BC GCI 27-GC-IDC RD3F9 RD3F9AFQ IDC IIIA 90 41 ? 63.5 157 25.6 WCAU 1 1 25 0 Surg 1962
    SIIIA
    BC ABS 28-(17)-AB- 4904020036T IDC 2-3 T3N1Mx IIIA 42
    IDC SIIIA
    BC ABS 29-(16)-AB- 4904020032T IDC 2 T3N1Mx IIIA 49
    IDC IIIA
    BC ABS 30-(15)-AB- 7259T IDC 2 T3N1M0 IIIA 59
    IDC SIIIA
    BC GCI 31-GC-IDC YOLOF YOLOFARG IDC IIIA 85 62 Post-M 82.6 160 32.2 WCAU 3 3 20 0 Surg 1943
    SIIIA
    BC GCI 32-GC-IDC 4W2NY 4W2NYAC1 IDC IIIB 50 39 Pre-M 54.4 163 20.6 WCAU 2 2 26 0 Surg 1962
    SIIIB
    BC GCI 33-GC-IDC YQ1WW YQ1WWAUV IDC IIIB 60 62 Pre-M 78 163 29.5 WCAU 0 Surg 1940
    SIIIB
    BC GCI 34-GC-IDC KIOE7 KIOE7AI9 IDC IIIB 55 65 Post-M 73.5 157 29.6 WCAU 2 2 18 2 Surg 1939
    SIIIB
    BC Bioch 70-(43)-Bc- A609183 IDC 2 40
    IDC
    BC Bioch 71-(54)-Bc- A605353 IDC 2 41
    IDC
    BC ABS 72-(55)-Bc- A609179 IDC 2 42
    IDC
    BC Bioch 73-(47)-Bc- A609221 IDC 2 42
    IDC
    BC Bioch 74-(48)-Bc- A609222 IDC 2 44
    IDC
    BC Bioch 75-(53)-Bc- A605151 IDC 2 44
    IDC
    BC Bioch 76-(61)-Bc- A610029 IDC 2 46
    IDC
    BC Bioch 77-(46)-Bc- A609177 Carc 2 48
    Carci
    BC Ichilov 78-(62)-Bc- A609194 IDC 2 51
    IDC
    BC Amb 79-(32)-AB- 7116T MC T2N0M0 IIA 54
    Muc Carci
    SIIA
    BC GCI 80-(49)-Bc- A609223 IDC 2 54
    IDC
    BC GCI 81-(45)-Bc- A609181 IDC 2 58
    IDC
    BC GCI 82-(50)-Bc- A609224 IDC 2 69
    IDC
    BC Bioch 83-(44)-Bc- A609198 IDC 2 77
    IDC
    BC Bioch 84-(51)-Bc- A605361 IDC 1 79
    IDC
    BC Amb 85-(31)-Ic- CG-154 IDC 83
    IDC
    BC Aster 35-As-ILC 17090 30738 30738A1 ILC T1cNXM0 I 100 50 94 170 32.5 W 2 2 Surg
    SI
    BC GCI 36-GC-ILC I35US I35USA9G ILC IIA 60 70 77.1 178 24.4 WCAU Surg 1932
    SIIA
    BC GCI 37-GC-ILC IS84Y IS84YAAY ILC IIB 65 67 Post-M 62.6 163 23.7 WCAU 2 2 16 0 Surg 1934
    SIIB
    BC Bioch 38-(52)-Bc- A605360 ILC 1 60
    ILC
    BB Aster 39-As-Ben 11975 15478 15478B1 FIBR 100 24 Pre-M 80 164 29.7 CAU 2 0 Surg
    BB GCI 40-GC-Ben ZT15M ZT15MAMR FIBR 100 34 57.6 165 21.1 WCAU Surg 1967
    BB GCI 41-GC-Ben NNP3Q NNP3QA4V FIBR 95 54 56.7 157 22.9 WCAU Surg 1948
    BB GCI 42-GC-Ben QK8IY QK8IYALU FIBR 100 41 59 173 19.7 WCAU Surg 1960
    BN-PS GCI 43-GC-N 83LO7 83LO7NEH NB-PS 32 78.5 155 32.7 WCAU Surg 1969
    PS
    BN-PS GCI 45-GC-N O6JBJ O6JBJNT1 NB-PS 38 67.1 173 22.5 WCAU Surg 1963
    PS
    BN-PS GCI 46-GC-N E6UDD E6UDDNCF NB-PS 40 99.8 170 34.5 WCAU Surg 1961
    PS
    BN-PS GCI 47-GC-N DHLR1 DHLR1NIQ NB-PS 40 90.7 168 32.3 WCAU Surg 1961
    PS
    BN-PS GCI 48-GC-N JHQEH JHQEHN4D NB-PS 41 65.3 157 26.3 WCAU Surg 1960
    PS
    BN-PS Amb 49-(63)-Am- 26486 NB-PS 43
    N PS
    BN-PS GCI 50-GC-N ONBFK ONBFKNO2 NB-PS 45 81.6 165 30.0 WCAU Surg 1956
    PS
    BN-PS GCI 51-GC-N TG6J6 TG6J6NNA NB-PS 46 90.7 173 30.4 WCAU Surg 1955
    PS
    BN-PS Aster 52-GC-N 14398 20021 20021D1 NB-PS 49 Pre-M 68 165 25.0 CAU 3 3 Surg
    PS
    BN-PS GCI 54-GC-N AJGXV AJGXVNFC NB-PS 52 70 168 24.8 WCAU Surg 1949
    PS
    BN-PS GCI 56-GC-N HLCZX HLCZXNLS NB-PS 54 67 163 25.2 WCAU Surg 1947
    PS
    BN-PS GCI 58-GC-N FGV8P FGV8PNQ6 NB-PS 61 106.6 168 37.9 WCAU Surg 1940
    PS
    BN-? Aster 59-As-N PS 9264 9486 9486A1 NB-PS 0 0 0 0.0
    BN-PM Bioch 60-(57)-Bc- A609233 A609233 NB-PM 34 Aut
    N PM
    BN-PM Bioch 61-(59)-Bc- A607155 A607155 NB-PM 35 Aut
    N PM
    BN-PM Bioch 62-(60)-Bc- A609234 A609234 NB-PM 36 Aut
    N PM
    BN-PM Amb 63-(66)-Am- 36678 36678 NB-PM 45 Aut
    N PM
    BN-PM Amb 64-(64)-Am- 23036 23036 NB-PM 57 Aut
    N PM
    BN-PM Amb 65-(65)-Am- 31410 31410 NB-PM 63 Aut
    N PM
    BN-PM Amb 66-(67)-Am- 073P010602086A 073P010602086A NB-PM 64 Aut
    N PM
    BN-PM Bioch 67-(58)-Bc- A609232 A609232 NB-PM 65 Aut
    N PM
    BN-PM Aster 68-As-N 8862 8766 8766B1 NB-PM 74 64 157 26.0 CAU Aut
    PM
    BN-PM Aster 69-As-N 8457 7928 7928M1 NB-PM 87 59 165 21.7 CAU Aut
    PM
  • TABLE 4_2
    Key Full Name
    # Live Bir # Live Births
    # of Preg Number of Pregnancies
    Amb Ambion
    Aster Asterand
    Aut Autopsy
    BB Breast Benign
    BC Breast Cancer
    Bioch Biochain
    BN Breast Normal
    BN-PM Breast_Normal-PM
    BN-PS Breast_Normal-PS
    Breastfeed child Br Child
    C Stage Cancer Stage
    Carc Carcinoma
    CAU Caucasian
    DCIS Ductal Carcinoma In Situ
    Ethnic B Ethnic Background
    FIBR FIBROADENOMA
    IDC INFILTRATING DUCTAL CARCINOMA
    ILC INFILTRATING LOBULAR CARCINOMA
    M Menopausal
    MC Mucinous carcinoma
    MS Menopausal Status
    NB-PM NORMAL BREAST-PM
    NB-PS NORMAL BREAST-PS
    Post-M Post-Menopausal
    Pre-M Pre-Menopausal
    Samp DIAG Sample Diagnosis
    Surg Surgical
    Tum % Percentage of Tumor
    W White
    WCAU White Caucasian
  • TABLE 5
    Tissue samples in normal panel:
    Lot no. Source Tissue Pathology Sex/Age comments
    1-Am-Colon (C71) 071P10B Ambion Colon PM IC bleed F/43 IC-intracarnial
    bleed
    2-B-Colon (C69) A411078 Biochain Colon PM-Pool of 10 M (26-78)&F (53-77)
    3-Cl-Colon (C70) 1110101 Clontech Colon PM-Pool of 3 sudden death M&F (20-50)
    4-Am-Small Intestine 091P0201A Ambion Small Intestine PM ICH M/85
    5-B-Small Intestine A501158 Biochain Small Intestine PM M/63
    6-B-Rectum A605138 Biochain Rectum PM M/25
    7-B-Rectum A610297 Biochain Rectum PM M/24
    8-B-Rectum A610298 Biochain Rectum PM M/27
    9-Am-Stomach 110P04A Ambion Stomach PM GSW M/16
    10-B-Stomach A501159 Biochain Stomach PM M/24
    11-B-Esophagus A603814 Biochain Esophagus PM M/26
    12-B-Esophagus A603813 Biochain Esophagus PM M/41
    13-Am-Pancreas 071P25C Ambion Pancreas PM MVA F/25
    14-CG-Pancreas CG-255-2 Ichilov Pancreas PM M/75
    15-B-Lung A409363 Biochain Lung PM-Pool of 5 M (24-28)&F62
    16-Am-Lung (L93) 111P0103A Ambion Lung PM ICH F/61
    17-B-Lung (L92) A503204 Biochain Lung PM M/28
    19-B-Ovary (O48) A504087 Biochain Ovary PM F/51
    20-B-Ovary (O46) A504086 Biochain Ovary PM F/41
    75-G-Ovary L629FRV1 GCI Ovary PS DIGESTIVE HEMORRHAGE F/47
    (ALCOHOLISM)
    76-G-Ovary DWHTZRQX GCI Ovary PS LEIOMYOMAS F/42
    77-G-Ovary FDPL9NJ6 GCI Ovary PS VAGINAL BLEEDING F/56
    78-G-Ovary GWXUZN5M GCI Ovary PS ABNORMAL PAP SMEARS F/53
    21-Am-Cervix 101P0101A Ambion Cervix PM Surgery F/40
    23-B-Cervix A504089 Biochain Cervix PM-Pool of 5 F (36-55)
    24-B-Uterus A411074 Biochain Uterus PM-Pool of 10 F (32-53)
    25-B-Uterus A409248 Biochain Uterus PM F/35
    26-B-Uterus A504090 Biochain Uterus PM-Pool of 5 F (40-53)
    28-Am-Bladder 071P02C Ambion Bladder PM GSW M/28
    29-B-Bladder A504088 Biochain Bladder PM-Pool of 5 M (26-44)&F30
    30-Am-Placenta 021P33A Ambion Placenta PB F/33 PB - post
    birth
    31-B-Placenta A410165 Biochain Placenta PB F/26
    32-B-Placenta A411073 Biochain Placenta PB-Pool of 5 F (24-30)
    33-B-Breast (B59) A607155 Biochain Breast PM F/36
    34-Am-Breast (B63) 26486 Ambion Breast PS bilateral breast reduction F/43
    35-Am-Breast (B64) 23036 Ambion Breast PM lung cancer F/57
    36-Cl-Prostate (P53) 1070317 Clontech Prostate PM-Pool of 47 sudden death M (14-57)
    37-Am-Prostate (P42) 061P04A Ambion Prostate PM IC bleed M/47
    38-Am-Prostate (P59) 25955 Ambion Prostate PM head trauma M/62
    39-Am-Testis 111P0104A Ambion Testis PM GSW M/25
    40-B-Testis A411147 Biochain Testis PM M/74
    41-Cl-Testis 1110320 Clontech Testis PM-Pool of 45 sudden death M (14-64)
    42-CG-Adrenal CG-184-10 Ichilov Adrenal PM F/81
    43-B-Adrenal A610374 Biochain Adrenal PM F/83
    44-B-Heart A411077 Biochain Heart PM-Pool of 5 M (23-70)
    45-CG-Heart CG-255-9 Ichilov Heart focal PM M/75
    fibrosis
    46-CG-Heart CG-227-1 Ichilov Heart PM F/36
    47-Am-Liver 081P0101A Ambion Liver PM ICH M/64
    48-CG-Liver CG-93-3 Ichilov Liver PM F/19
    49-CG-Liver CG-124-4 Ichilov Liver of fetus PM fetus
    50-Cl-BM 1110932 Clontech Bone Marrow PM-Pool of 8 sudden death M&F (22-65)
    51-CGEN-Blood WBC#5 CGEN Blood M
    52-CGEN-Blood WBC#4 CGEN Blood M
    53-CGEN-Blood WBC#3 CGEN Blood M
    54-CG-Spleen CG-267 Ichilov Spleen PM F/25
    55-CG-Spleen 111P0106B Ambion Spleen PM GSW M/25
    56-CG-Spleen A409246 Biochain Spleen PM F/12
    57-CG-Thymus CG-98-7 Ichilov Thymus PM F/28
    58-Am-Thymus 101P0101A Ambion Thymus PM head injury M/14
    59-B-Thymus A409278 Biochain Thymus PM M/28
    60-B-Thyroid A610287 Biochain Thyroid PM M/27
    61-B-Thyroid A610286 Biochain Thyroid PM M/24
    62-CG-Thyroid CG-119-2 Ichilov Thyroid PM F/66
    63-Cl-Salivary Gland 1070319 Clontech Salivary Gland PM-Pool of 24 sudden death M&F 15-60
    64-Am-Kidney 111P0101B Ambion Kidney PM ICH M 60
    65-Cl-Kidney 1110970 Clontech Kidney PM-Pool of 14 sudden death M&F 18-59
    66-B-Kidney A411080 Biochain Kidney PM-Pool of 5 M 24-46
    67-CG-Cerebellum CG-183-5 Ichilov Cerebellum PM M/74
    68-CG-Cerebellum CG-212-5 Ichilov Cerebellum PM M/54
    69-B-Brain A411322 Biochain Brain PM M/28
    70-Cl-Brain 1120022 Clontech Brain PM
    71-B-Brain A411079 Biochain Brain PM-Pool of 2 M 27-28
    72-CG-Brain CG-151-1 Ichilov Brain PM F/86
    73-Am-Skeletal Muscle 101P013A Ambion Skeletal Muscle PM head injury F/28
    74-Cl-Skeletal Muscle 1061038 Clontech Skeletal Muscle PM-Pool of 2 sudden death M&F 43-46
  • TABLE 5_1
    Normal panel
    7-B-Rectum 1-(7)-Bc-Rectum Biochain A610297
    8-B-Rectum 2-(8)-Bc-Rectum Biochain A610298
    new colon 3-GC-Colon GCI CDSUV CDSUVNR3
    new colon 4-As-Colon Asterand 16364 31802 31802B1
    new colon 5-As-Colon Asterand 22900 74446 74446B1
    new small bowl 6-GC-Small bowl GCI V9L7D V9L7DN6Z
    new small bowl 7-GC-Small bowl GCI M3GVT M3GVTN5R
    new small bowl 8-GC-Small bowl GCI 196S2 196S2AJN
    9-Am-Stomach 9-(9)-Am-Stomach Ambion 110P04A
    10-B-Stomach 10-(10)-Bc-Stomach Biochain A501159
    11-B-Esophagus 11-(11)-Bc-Esoph Biochain A603814
    12-B-Esophagus 12-(12)-Bc-Esoph Biochain A603813
    new pancreas 13-As-Panc Asterand 8918 9442 9442C1
    new pancreas 14-As-Panc Asterand 10082 11134 11134B1
    48-CG-Liver 15-(48)-Ic-Liver Ichilov CG-93-3
    new liver 16-As-Liver Asterand 7916 7203 7203B1
    28-Am-Bladder 17-(28)-Am-Bladder Ambion 071P02C
    29-B-Bladder 18-(29)-Bc-Bladder Biochain A504088
    64-Am-Kidney 19-(64)-Am-Kidney Ambion 111P0101B
    65-Cl-Kidney 20-(65)-Cl-Kidney Clontech 1110970
    66-B-Kidney 21-(66)-Bc-Kidney Biochain A411080
    new kidney 22-GC-Kidney GCI N1EVZ N1EVZN91
    new kidney 23-GC-Kidney GCI BMI6W BMI6WN9F
    42-CG-Adrenal 24-(42)-Ic-Adrenal Ichilov CG-184-10
    43-B-Adrenal 25-(43)-Bc-Adrenal Biochain A610374
    16-Am-Lung (L93) 26-(16)-Am-Lung Ambion 111P0103A
    17-B-Lung (L92) 27-(17)-Bc-Lung Biochain A503204
    new lung 28-As-Lung Asterand 9078 9275 9275B1
    new lung 29-As-Lung Asterand 6692 6161 6161A1
    new lung 30-As-Lung Asterand 7900 7180 7180F1
    75-G-Ovary 31-(75)-GC-Ovary GCI L629FRV1
    76-G-Ovary 32-(76)-GC-Ovary GCI DWHTZRQX
    77-G-Ovary 33-(77)-GC-Ovary GCI FDPL9NJ6
    78-G-Ovary 34-(78)-GC-Ovary GCI GWXUZN5M
    21-Am-Cervix 35-(21)-Am-Cerix Ambion 101P0101A
    new cervix 36-GC-cervix GCI E2P2N E2P2NAP4
    24-B-Uterus 37-(24)-Bc-Uterus Biochain A411074
    26-B-Uterus 38-(26)-Bc-Uterus Biochain A504090
    30-Am-Placenta 39-(30)-Am-Placen Ambion 021P33A
    32-B-Placenta 40-(32)-Bc-Placen Biochain A411073
    new breast 41-GC-Breast GCI DHLR1
    new breast 42-GC-Breast GCI TG6J6
    new breast 43-GC-Breast GCI E6UDD E6UDDNCF
    38-Am-Prostate (P59) 44-(38)-Am-Prostate Ambion 25955
    add prostate from 45-Bc-Prostate Biochain A609258
    prostate panel
    new testis 46-As-Testis Asterand 13071 19567 19567B1
    new testis 47-As-Testis Asterand 19671 42120 42120A1
    ARTERY 48-GC-Artery GCI 7FUUP 7FUUPAMP
    ARTERY 49-GC-Artery GCI YGTVY YGTVYAIN
    blood cells? 50-Th-Blood-MONO Tel-Hashomer 52497
    blood cells? 51-Th-Blood-MONO Tel-Hashomer 31055
    blood cells? 52-Th-Blood-MONO Tel-Hashomer 31058
    54-CG-Spleen 53-(54)-Ic-Spleen Ichilov CG-267
    55-CG-Spleen 54-(55)-Ic-Spleen Ichilov 111P0106B
    57-CG-Thymus 55-(57)-Ic-Thymus Ichilov CG-98-7
    58-Am-Thymus 56-(58)-Am-Thymus Ambion 101P0101A
    60-B-Thyroid 57-(60)-Bc-Thyroid Biochain A610287
    62-CG-Thyroid 58-(62)-Ic-Thyroid Ichilov CG-119-2
    new salivary gland 59-Gc-Sali gland GCI NNSMV NNSMVNJC
    67-CG-Cerebellum 60-(67)-Ic-Cerebellum Ichilov CG-183-5
    68-CG-Cerebellum 61-(68)-Ic-Cerebellum Ichilov CG-212-5
    69-B-Brain 62-(69)-Bc-Brain Biochain A411322
    71-B-Brain 63-(71)-Bc-Brain Biochain A411079
    72-CG-Brain 64-(72)-Ic-Brain Ichilov CG-151-1
    44-B-Heart 65-(44)-Bc-Heart Biochain A411077
    46-CG-Heart 66-(46)-Ic-Heart Ichilov CG-227-1
    45-CG-Heart (Fibrotic) 67-(45)-Ic-Heart (Fibrotic) Ichilov CG-255-9
    new skeletal muscle 68-GC-Skel Mus GCI T8YZS T8YZSN7O
    new skeletal muscle 69-GC-Skel Mus GCI Q3WKA Q3WKANCJ
    new skeletal muscle 70-As-Skel Mus Asterand 8774 8235 8235G1
    new skeletal muscle 71-As-Skel Mus Asterand 8775 8244 8244A1
    new skeletal muscle 72-As-Skel Mus Asterand 10937 12648 12648C1
    new skeletal muscle 73-As-Skel Mus Asterand 6692 6166 6166A1
  • Materials and Experimental Procedures
  • RNA preparation—RNA was obtained from ABS (Wilmington, Del. 19801, USA, http://www.absbioreagents.com), BioChain Inst. Inc. (Hayward, Calif. 94545 USA www.biochain.com), GOG for ovary samples—Pediatic Cooperative Human Tissue Network, Gynecologic Oncology Group Tissue Bank, Children Hospital of Columbus (Columbus Ohio 43205 USA), Clontech (Franklin Lakes, N.J. USA 07417, www.clontech.com), Ambion (Austin, Tex. 78744 USA, http://www.ambion.com), Asternad (Detroit, Mich. 48202-3420, USA, www.asterand.com), and from Genomics Collaborative Inc. a Division of Seracare (Cambridge, Mass. 02139, USA, www.genomicsinc.com). Alternatively, RNA was generated from tissue samples using TRI-Reagent (Molecular Research Center), according to Manufacturer's instructions. Tissue and RNA samples were obtained from patients or from postmortem. Total RNA samples were treated with DNaseI (Ambion).
  • RT PCR—Purified RNA (1 μg) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 μM dNTP in a total volume of 15.6 μl. The mixture was incubated for 5 min at 65° C. and then quickly chilled on ice. Thereafter, 5 μl of 5× SuperscriptII first strand buffer (Invitrogen), 2.4 μl 0.1M DTT and 40 units RNasin (Promega) were added, and the mixture was incubated for 10 min at 25° C., followed by further incubation at 42° C. for 2 min. Then, 1 μl (200 units) of SuperscriptII (Invitrogen) was added and the reaction (final volume of 25 μl) was incubated for 50 min at 42° C. and then inactivated at 70° C. for 15 min. The resulting cDNA was diluted 1:20 in TE buffer (10 mM Tris pH=8, 1 mM EDTA pH=8).
  • Real-Time RT-PCR analysis—cDNA (5 μl), prepared as described above, was used as a template in Real-Time PCR reactions using the SYBR Green I assay (PE Applied Biosystem) with specific primers and UNG Enzyme (Eurogentech or ABI or Roche). The amplification was effected as follows: 50° C. for 2 min, 95° C. for 10 min, and then 40 cycles of 95° C. for 15 sec, followed by 60° C. for 1 min. Detection was performed by using the PE Applied Biosystem SDS 7000. The cycle in which the reactions achieved a threshold level (Ct) of fluorescence was registered and was used to calculate the relative transcript quantity in the RT reactions. The relative quantity was calculated using the equation Q=efficiencŷ−Ct. The efficiency of the PCR reaction was calculated from a standard curve, created by using serial dilutions of several reverse transcription (RT) reactions. prepared from RNA purified from 5 cell lines (HCT116, H1299, DU145, MCF7, ES-2). To minimize inherent differences in the RT reaction, the resulting relative quantities were normalized to normalization factor calculated in one of the following methods as indicated in the text:
  • Method 1—the geometric mean of the relative quantities of the selected housekeeping (HSKP) genes was used as normalization factor.
  • Method 2—The expression of several housekeeping (HSKP) genes was checked on every panel. The relative quantity (O) of each housekeeping gene in each sample, calculated as described above, was divided by the median quantity of this gene in all panel samples to obtain the “relative Q rel to MED”. Then, for each sample the median of the “relative Q rel to MED” of the selected housekeeping genes was calculated and served as normalization factor of this sample for further calculations. Schematic summary of quantitative real-time PCR analysis is presented in FIG. 3. As shown, the x-axis shows the cycle number. The CT=Threshold Cycle point, which is the cycle that the amplification curve crosses the fluorescence threshold that was set in the experiment. This point is a calculated cycle number in which PCR products signal is above the background level (passive dye ROX) and still in the Geometric/Exponential phase (as shown, once the level of fluorescence crosses the measurement threshold, it has a geometrically increasing phase, during which measurements are most accurate, followed by a linear phase and a plateau phase; for quantitative measurements, the latter two phases do not provide accurate measurements). The y-axis shows the normalized reporter fluorescence. It should be noted that this type of analysis provides relative quantification.
  • The sequences of the housekeeping genes measured in all the examples on the ovary cancer tissue testing panel were as follows:
  • SDHA (GenBank Accession No. NM_004168 (SEQ ID NO:
    364),)
    SDHA Forward primer (SEQ ID NO: 556):
    TGGGAACAAGAGGGCATCTG
    SDHA Reverse primer (SEQ ID NO: 557):
    CCACCACTGCATCAAATTCATG
    SDHA-amplicon (SEQ ID NO: 365):
    TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGT
    ATCCAGTAGTGGATCATGAATTTGATGCAGTGGTGG
    PBGD (GenBank Accession No. BC019323 (SEQ ID NO:
    381),),
    PBGD Forward primer (SEQ ID NO: 558):
    TGAGAGTGATTCGCGTGGG
    PBGD Reverse primer (SEQ ID NO: 559):
    CCAGGGTACGAGGCTTTCAAT
    PBGD-amplicon (SEQ ID NO: 382):
    TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAG
    ACGGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG
    HPRT1 (GenBank Accession No. NM_000194 (SEQ ID NO:
    379),
    HPRT1 Forward primer (SEQ ID NO: 560):
    TGACACTGGCAAAACAATGCA
    HPRT1 Reverse primer (SEQ ID NO: 561):
    GGTCCTTTTCACCAGCAAGCT
    HPRT1-amplicon (SEQ ID NO: 380):
    TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATA
    ATCCAAAGATGGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
    GAPDH (GenBank Accession No. BC026907 (SEQ ID NO:
    451))
    GAPDH Forward primer (SEQ ID NO: 562):
    TGCACCACCAACTGCTTAGC
    GAPDH Reverse primer (SEQ ID NO: 563):
    CCATCACGCCACAGTTTCC
    GAPDH-amplicon (SEQ ID NO: 450):
    TGCACCACCAACTGCTTAGCACCCCTGGCCAAGGTCATCCATGACAACTT
    TGGTATCGTGGAAGGACTCATGACCACAGTCCATGCCATCACTGCCACCC
    AGAAGACTGTGGATGG
  • The sequences of the housekeeping genes measured in all the examples on colon cancer tissue testing panel were as follows:
  • PBGD (GenBank Accession No. BC019323 (SEQ ID NO:
    381)),
    PBGD Forward primer (SEQ ID NO: 558):
    TGAGATGATTCGCGTGGG
    PBGD Reverse primer (SEQ ID NO: 559):
    CCAGGGTACGAGGCTTTCAAT
    PBGD-amplicon (SEQ ID NO: 382):
    TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAG
    ACGGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG
    HPRT1 (GenBank Accession No. NM_000194 (SEQ ID NO:
    379),
    HPRT1 Forward primer (SEQ ID NO: 560):
    TGACACTGGCAAAACAATGCA
    HPRT1 Reverse primer (SEQ ID NO: 561):
    GGTCCTTTTCACCAGCAAGCT
    HPRT1-amplicon (SEQ ID NO: 380):
    TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATA
    ATCCAAAGATGGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
    G6PD (GenBank Accession No. NM_000402 (SEQ ID NO:
    405))
    G6PDForward primer (SEQ ID NO: 564):
    gaggccgtcaccaagaacat
    G6PD Reverse primer (SEQ ID NO: 565):
    ggacagccggtcagagctc
    G6PD-amplicon (SEQ ID NO: 404):
    gaggccgtcaccaagaacattcacgagtcctgcatgagccagataggctg
    gaaccgcatcatcgtggagaagcccttcgggagggacctgcagagctctg
    accggctgtcc
    RPS27A (GenBank Accession No. NM_002954 (SEQ ID
    NO:403),)
    RPS27A Forward primer (SEQ ID NO: 566):
    CTGGCAAGCAGCTGGAAGAT
    RPS27A Reverse primer (SEQ ID NO: 567):
    TTCTTAGCACCACCACGAAGTC
    RPS27A-amplicon (SEQ ID NO: 402):
    CTGGCAAGCAGCTGGAAGATGGACGTACTTTGTCTGACTACAATATTCAA
    AAGGAGTCTACTCTTCATCTTGTGTTGAGACTTCGTGGTGGTGCTAAGAA
    A
  • The sequences of the housekeeping genes measured in all the examples in the lung panel were as follows:
  • Ubiquitin (GenBank Accession No. BC000449 (SEQ ID
    NO:366),)
    Ubiquitin Forward primer (SEQ ID NO: 568):
    ATTTGGGTCGCGGTTCTTG
    Ubiquitin Reverse primer (SEQ ID NO: 569):
    TGCCTTGACATTCTCGATGGT
    Ubiquitin-amplicon (SEQ ID NO: 367):
    ATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGACAA
    TGCAGATCTTCGTGAAGACTCTGACTGGTAAGACCATCACCCTCGAGGTT
    GAGCCCAGTGACACCATCGAGAATGTCAAGGCA
    SDHA (GenBank Accession No. NM_004168 (SEQ ID NO:
    364),)
    SDHA Forward primer (SEQ ID NO: 556):
    TGGGAACAAGAGGGCATCTG
    SDHA Reverse primer (SEQ ID NO: 557):
    CCACCACTGCATCAAATTCATG
    SDHA-amplicon (SEQ ID NO: 365):
    TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGT
    ATCCAGTAGTGGATCATGAATTTGATGCAGTGGTGG
    PBGD (GenBank Accession No. BC019323 (SEQ ID NO:
    381)),
    PBGD Forward primer (SEQ ID NO: 558):
    TGAGAGTGATTCGCGTGGG
    PBGD Reverse primer (SEQ ID NO: 559):
    CCAGGGTACGAGGCTTTCAAT
    PBGD-amplicon (SEQ ID NO: 382):
    TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAG
    ACGGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG
    HPRT1 (GenBank Accession No. NM_000194 (SEQ ID NO:
    379)),
    HPRT1 Forward primer (SEQ ID NO: 560):
    TGACACTGGCAAAACAATGCA
    HPRT1 Reverse primer (SEQ ID NO: 561):
    GGTCCTTTTCACCAGCAAGCT
    HPRT1-amplicon (SEQ ID NO: 380):
    TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATA
    ATCCAAAGATGGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
  • The sequences of the housekeeping genes measured in all the examples on breast cancer panel were as follows:
  • G6PD (GenBank Accession No. NM_000402 (SEQ ID NO:
    405))
    G6PD Forward primer (SEQ ID NO: 564):
    gaggccgtcaccaagaacat
    G6PD Reverse primer (SEQ ID NO: 565):
    ggacagccggtcagagctc
    G6PD-amplicon (SEQ ID NO: 404):
    gaggccgtcaccaagaacattcacgagtcctgcatgagccagataggctg
    gaaccgcatcatcgtggagaagcccttcgggagggacctgcagagctctg
    accggctgtcc
    SDHA (GenBank Accession No. NM_004168 (SEQ ID NO:
    364),)
    SDHA Forward primer (SEQ ID NO: 556):
    TGGGAACAAGAGGGCATCTG
    SDHA Reverse primer (SEQ ID NO: 557):
    CCACCACTGCATCAAATTCATG
    SDHA-amplicon (SEQ ID NO: 365):
    TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGT
    ATCCAGTAGTGGATCATGAATTTGATGCAGTGGTGG
    PBGD (GenBank Accession No. BC019323 (SEQ ID NO:
    381)),
    PBGD Forward primer (SEQ ID NO: 558):
    TGAGAGTGATTCGCGTGGG
    PBGD Reverse primer (SEQ ID NO: 559):
    CCAGGGTACGAGGCTTTCAAT
    PBGD-amplicon (SEQ ID NO: 382):
    TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAG
    ACGGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG
    HPRT1 (GenBank Accession No. NM_000194 (SEQ ID NO:
    379),
    HPRT1 Forward primer (SEQ ID NO: 560):
    TGACACTGGCAAAACAATGCA
    HPRT1 Reverse primer (SEQ ID NO: 561):
    GGTCCTTTTCACCAGCAAGCT
    HPRT1-amplicon (SEQ ID NO: 380):
    TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATA
    ATCCAAAGATGGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
  • The sequences of the housekeeping genes measured in all the examples on normal tissue samples panel were as follows:
  • RPL19 (GenBank Accession No. NM_000981 (SEQ ID NO:
    369))
    RPL19Forward primer (SEQ ID NO: 570):
    TGGCAAGAAGAAGGTCTGGTTAG
    RPL19Reverse primer (SEQ ID NO: 571):
    TGATCAGCCCATCTTTGATGAG
    RPL19-amplicon (SEQ ID NO: 368):
    TGGCAAGAAGAAGGTCTGGTTAGACCCCAATGAGACCAATGAAATCGCCA
    ATGCCAACTCCCGTCAGCAGATCCGGAAGCTCATCAAAGATGGGCTGATC
    A
    TATAbox (GenBank Accession No. NM_003194 (SEQ ID
    NO:371)),
    TATA box Forward primer (SEQ ID NO: 572):
    CGGTTTGCTGCGGTAATCAT
    TATA box Reverse primer (SEQ ID NO: 573):
    TTTCTTGCTGCCAGTCTGGAC
    TATA (SEQ ID NO:370) box amplicon:
    CGGTTTGCTGCGGTAATCATGAGGATAAGAGAGCCACGAACCACGGCACT
    GATTTTCAGTTCTGGGAAAATGGTGTGCACAGGAGCCAAGAGTGAAGAAC
    AGTCCAGACTGGCAGCAAGAAA
    Ubiquitin (GenBank Accession No. BC000449 (SEQ ID
    NO:366))
    Ubiquitin Forward primer (SEQ ID NO: 568):
    ATTTGGGTCGCGGTTCTTG
    Ubiquitin Reverse primer (SEQ ID NO: 569):
    TGCCTTGACATTCTCGATGGT
    Ubiquitin-amplicon (SEQ ID NO: 367):
    ATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGACAA
    TGCAGATCTTCGTGAAGACTCTGACTGGTAAGACCATCACCCTCGAGGTT
    GAGCCCAGTGACACCATCGAGAATGTCAAGGCA
    SDHA (GenBank Accession No. NM_004168 (SEQ ID NO:
    364),)
    SDHA Forward primer (SEQ ID NO: 556):
    TGGGAACAAGAGGGCATCTG
    SDHA Reverse primer (SEQ ID NO: 557):
    CCACCACTGCATCAAATTCATG
    SDHA-amplicon (SEQ ID NO: 365):
    TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGT
    ATCCAGTAGTGGATCATGAATTTGATGCAGTGGTGG
  • Cluster HSFLT
  • Cluster HSFLT features at least 15 transcript(s) and at least 58 segment(s) of interest, the names for which are described in Tables 6 and 7, respectively and certain protein variants are described in table 8.
  • TABLE 6
    Transcripts
    Transcript Name
    HSFLT_T7 (SEQ ID NO: 1)
    HSFLT_T8 (SEQ ID NO: 2)
    HSFLT_T9 (SEQ ID NO: 3)
    HSFLT_T10 (SEQ ID NO: 4)
    HSFLT_T13 (SEQ ID NO: 5)
    HSFLT_T14 (SEQ ID NO: 6)
    HSFLT_T17 (SEQ ID NO: 7)
    HSFLT_T19 (SEQ ID NO: 8)
    HSFLT_T20 (SEQ ID NO: 9)
    HSFLT_T21 (SEQ ID NO: 10)
    HSFLT_T22 (SEQ ID NO: 11)
    HSFLT_T23 (SEQ ID NO: 12)
    HSFLT_T24 (SEQ ID NO: 13)
    HSFLT_T25 (SEQ ID NO: 14)
    HSFLT_T26 (SEQ ID NO: 15)
  • TABLE 7
    Segments
    Segment Name
    HSFLT_N0 (SEQ ID NO: 32)
    HSFLT_N5 (SEQ ID NO: 33)
    HSFLT_N6 (SEQ ID NO: 34)
    HSFLT_N8 (SEQ ID NO: 35)
    HSFLT_N9 (SEQ ID NO: 36)
    HSFLT_N11 (SEQ ID NO: 37)
    HSFLT_N14 (SEQ ID NO: 38)
    HSFLT_N15 (SEQ ID NO: 39)
    HSFLT_N17 (SEQ ID NO: 40)
    HSFLT_N19 (SEQ ID NO: 41)
    HSFLT_N20 (SEQ ID NO: 42)
    HSFLT_N24 (SEQ ID NO: 43)
    HSFLT_N26 (SEQ ID NO: 44)
    HSFLT_N30 (SEQ ID NO: 45)
    HSFLT_N38 (SEQ ID NO: 46)
    HSFLT_N41 (SEQ ID NO: 47)
    HSFLT_N42 (SEQ ID NO: 48)
    HSFLT_N44 (SEQ ID NO: 49)
    HSFLT_N46 (SEQ ID NO: 50)
    HSFLT_N48 (SEQ ID NO: 51)
    HSFLT_N52 (SEQ ID NO: 52)
    HSFLT_N59 (SEQ ID NO: 53)
    HSFLT_N63 (SEQ ID NO: 54)
    HSFLT_N68 (SEQ ID NO: 55)
    HSFLT_N74 (SEQ ID NO: 56)
    HSFLT_N82 (SEQ ID NO: 57)
    HSFLT_N93 (SEQ ID NO: 58)
    HSFLT_N98 (SEQ ID NO: 59)
    HSFLT_N100 (SEQ ID NO: 60)
    HSFLT_N103 (SEQ ID NO: 530)
    HSFLT_N3 (SEQ ID NO: 502)
    HSFLT_N22 (SEQ ID NO: 503)
    HSFLT_N28 (SEQ ID NO: 504)
    HSFLT_N32 (SEQ ID NO: 505)
    HSFLT_N34 (SEQ ID NO: 506)
    HSFLT_N36 (SEQ ID NO: 507)
    HSFLT_N50 (SEQ ID NO: 508)
    HSFLT_N55 (SEQ ID NO: 509)
    HSFLT_N57 (SEQ ID NO: 510)
    HSFLT_N61 (SEQ ID NO: 511)
    HSFLT_N65 (SEQ ID NO: 512)
    HSFLT_N66 (SEQ ID NO: 513)
    HSFLT_N70 (SEQ ID NO: 514)
    HSFLT_N72 (SEQ ID NO: 515)
    HSFLT_N75 (SEQ ID NO: 516)
    HSFLT_N77 (SEQ ID NO: 517)
    HSFLT_N78 (SEQ ID NO: 518)
    HSFLT_N79 (SEQ ID NO: 519)
    HSFLT_N84 (SEQ ID NO: 520)
    HSFLT_N88 (SEQ ID NO: 521)
    HSFLT_N91 (SEQ ID NO: 522)
    HSFLT_N92 (SEQ ID NO: 523)
    HSFLT_N94 (SEQ ID NO: 524)
    HSFLT_N95 (SEQ ID NO: 525)
    HSFLT_N97 (SEQ ID NO: 526)
    HSFLT_N99 (SEQ ID NO: 527)
    HSFLT_N101 (SEQ ID NO: 528)
    HSFLT_N102 (SEQ ID NO: 529)
  • TABLE 8
    Proteins and their Corresponding Transcript Descriptions:
    Protein Name Corresponding Transcript(s)
    HSFLT_P6 (SEQ ID NO: 16) HSFLT_T9 (SEQ ID NO: 3)
    HSFLT_P7 (SEQ ID NO: 17) HSFLT_T10 (SEQ ID NO: 4)
    HSFLT_P10 (SEQ ID NO: 18) HSFLT_T13 (SEQ ID NO: 5)
    HSFLT_P11 (SEQ ID NO: 19) HSFLT_T14 (SEQ ID NO: 6)
    HSFLT_P13 (SEQ ID NO: 20) HSFLT_T17 (SEQ ID NO: 7)
    HSFLT_P14 (SEQ ID NO: 21) HSFLT_T19 (SEQ ID NO: 8)
    HSFLT_P15 (SEQ ID NO: 22) HSFLT_T20 (SEQ ID NO: 9)
    HSFLT_P16 (SEQ ID NO: 23) HSFLT_T21 (SEQ ID NO: 10)
    HSFLT_P17 (SEQ ID NO: 24) HSFLT_T22 (SEQ ID NO: 11)
    HSFLT_P18 (SEQ ID NO: 25) HSFLT_T23 (SEQ ID NO: 12)
    HSFLT_P19 (SEQ ID NO: 26) HSFLT_T24 (SEQ ID NO: 13)
    HSFLT_P20 (SEQ ID NO: 27) HSFLT_T25 (SEQ ID NO: 14)
    HSFLT_P21 (SEQ ID NO: 28) HSFLT_T26 (SEQ ID NO: 15)
    HSFLT_P41 (SEQ ID NO: 29) HSFLT_T21 (SEQ ID NO: 10)
    HSFLT_P48 (SEQ ID NO: 30) HSFLT_T7 (SEQ ID NO: 1)
    HSFLT_P49 (SEQ ID NO: 31) HSFLT_T8 (SEQ ID NO: 2)
  • The sequences listed in Tables 8 comprise variants of the known protein Vascular endothelial growth factor receptor 1 precursor (SwissProt accession identifier VGR1_HUMAN (SEQ ID NO: 359); known also according to the synonyms EC 2.7.1.112; VEGFR-1; Vascular permeability factor receptor; Tyrosine-protein kinase receptor FLT; Flt-1; Tyrosine-protein kinase FRT; Fms-like tyrosine kinase 1)), and may be referred to herein as “the corresponding native protein”.
  • Protein Vascular endothelial growth factor receptor 1 precursor is associated with the following function(s): it is a receptor for VEGF, VEGFB and PGF, has tyrosine-protein kinase activity. The VEGF-kinase ligand/receptor signaling system plays a key role in vascular development and regulation of vascular permeability. Isoform SFlt1 may have an inhibitory role in angiogenesis. The sequence for protein Vascular endothelial growth factor receptor 1 precursor is given at the end of the application, as “Vascular endothelial growth factor receptor 1 precursor amino acid sequence”. Known polymorphisms for this sequence include an SNP at amino acid position 779, having an L to F substitution.
  • According to this aspect of the invention and in some embodiments, the polypeptides related thereto, and polynucleotides encoding the same, may be useful in applications in the following: angiogenesis inhibition; angiogenesis stimulation; endothelial growth factor agonism; endothelial growth factor receptor kinase inhibition, or combinations thereof.
  • In some embodiments, related polypeptides/polynucleotides of this invention will accordingly have the following therapeutic indication: anticancer, cardiovascular; growth stimulation; antidiabetic; vulnerary, or others.
  • The following GO Annotation(s) apply to the previously known protein. The following annotation(s) were found: positive regulation of cell proliferation; pregnancy; transmembrane receptor protein tyrosine kinase signaling pathway, which are annotation(s) related to Biological Process; receptor activity; vascular endothelial growth factor receptor activity, which are annotation(s) related to Molecular Function; and extracellular space; integral to plasma membrane, which are annotation(s) related to Cellular Component.
  • The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from <http://www.expasy.ch/sprot/>; or Locuslink, available from <http://www.ncbi.nlm.nih.gov/projects/LocusLink/>.
  • According to some embodiments of the present invention, variants of this cluster according to the present invention (amino acid and/or nucleic acid sequences of HSFLT) may optionally have one or more of the following utilities, as described below. It should be noted that these utilities are optionally and preferably suitable for human and non-human animals as subjects, except where otherwise noted.
  • A non-limiting example of such a diagnostic utility is detection of various cancer tumors. Vascular endothelial growth factor (VEGF) levels are associated with increased angiogenesis and aggressive tumor growth. In addition, it may serve as a marker for the early detection of coronary artery disease (CAD): experimental data show abnormal angiogenesis (VEGF and sFlt-1) in the patients with CAD.
  • Placental Growth Factor (HSPLGF), a member of the vascular endothelial growth factor (VEGF) family, competes with VEGF for binding to VEGF Receptor-1 (VEGFR1) (Am J Physiol Heart Circ Physiol. Apr. 24, 2003). Another non-limiting example of diagnostic utility of one or more HSFLT variants according to the present invention may optionally be related to one or more of the utilities of the HSPLGF placental growth factor, described herein (see the “Table of Utilities for Variants of HSPLGF, related to placental growth factor”, herein). Therefore, variants of HSFLT cluster according to the present invention (amino acid and/or nucleic acid sequences of HSFLT) could be used as molecular marker for conditions including but not limited to the following: inflammation, pathological angiogenesis, monocyte recruitment that underlie chronic inflammatory disease.
  • Another non-limiting example of the utility of the variants of HSFLT cluster according to the present invention (amino acid and/or nucleic acid sequences of HSFLT) is using this marker as a surrogate marker for determining the efficacy of treatment for modulators, preferably inhibitors, of the VEGF-kinase ligand/receptor signaling system, which plays a key role in vascular development and regulation of vascular permeability. Blocking this system may be used to block angiogenesis, for example for treating cancer. The system may also optionally be modulated for treating cardiovascular conditions, peripheral vascular disease; ulcers; and ischaemia. This marker could also be used as a surrogate marker for determining the efficacy of treatment for modulators of the above conditions. Its suitability for treatment of the above conditions was described in PCT Application No. WO 05/072340 and hence is presence is clearly related to the mechanism of action of the above system in the body.
  • As noted above, cluster HSFLT features 15 transcript(s), which were listed in Table 6 above. These transcript(s) encode for protein(s) which are variant(s) of protein Vascular endothelial growth factor receptor 1 precursor. A description of each variant protein according to the present invention is provided as follows:
  • Variant protein HSFLT_P6 (SEQ ID NO:16) according to the present invention has an amino acid sequence as provided in the sequence listing; and is encoded by transcript(s) HSFLT_T9 (SEQ ID NO:3). An alignment is provided with respect to the known protein (Vascular endothelial growth factor receptor 1 precursor) in the alignment table on the attached CD-ROM.
  • A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • 1. Comparison Report Between HSFLT_P6 (SEQ ID NO:16) and VGR1_HUMAN_V1 (SEQ ID NO: 575):
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P6 (SEQ ID NO:16), 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 MTAP (SEQ ID NO: 459) corresponding to amino acids 1-4 of HSFLT_P6 (SEQ ID NO:16), and a second amino acid sequence being at least 90% homologous to FPLDTNIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTL VLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKS VNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKD VTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHP CNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGR NISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQKMAITKEHSITLNL TIM VSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFK NNHKIQQEPGIILGPGSSTLFIERVTEEDEGVYHCKATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLF WLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDEQCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASA FGIKKSPTCRTVAVKMLKEGATASEYKALMTELKILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSN YLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQGKKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEP ITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNILLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMA PESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPGVQMDEDFCSRLREGMRMPRAPEYSTPEIYQIMLDCWBRDP KERPRFAELVEKLGDLLQANVQQDGKDYIPINAILTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNA FKFMSLERIKTFEELLPNATSMFDDYQGDSSTLLASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPS FCHSSCGHVSEGKRRFTYDHAELERKIACCSPPPDYNSVVLYSTPPI corresponding to amino acids 172-1338 of VGR1_HUMAN_V1 (SEQ ID NO: 575), which also corresponds to amino acids 5-1171 of HSFLT_P6 (SEQ ID NO:16), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for a head of HSFLT_P6 (SEQ ID NO:16), 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 MTAP (SEQ ID NO: 459) of HSFLT_P6 (SEQ ID NO:16).
  • It should be noted that the known protein sequences VGR1_HUMAN (SEQ ID NO: 359) and NP002010 (SEQ ID NO: 531) have one or more changes than the sequence for VGR1_HUMAN_V1 (SEQ ID NO: 575). These changes were previously known to occur and are listed in table 9.
  • TABLE 9
    Changes to VGR1_HUMAN_V1 (SEQ ID NO: 575)
    SNP position on amino
    acid sequence Type of change
    779 conflict
  • 3. Comparison Report Between HSFLT_P6 (SEQ ID NO:16) and P17948-2 (SEQ ID NO:360)
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P6 (SEQ ID NO:16), 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 MTAP (SEQ ID NO: 459) corresponding to amino acids 1-4 of HSFLT_P6 (SEQ ID NO:16), a second amino acid sequence being at least 90% homologous to FPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGBLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTL VLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKS VNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKD VTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHP CNBNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGR NISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQKMAITKEHSITLNL TIMNVSLQDSGTYACRARNVYTGEEILQKKEITIR corresponding to amino acids 172-656 of P17948-2 (SEQ ID NO:360), which also corresponds to amino acids 5-489 of HSFLT_P6 (SEQ ID NO:16), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNKIQQEPGIILGPGSSTLFIERVTEEDEGVYHCK ATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDE QCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAVKMLKEGATASEYKALMTELK ILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQG KKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNI LLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPG VQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAI LTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLL ASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPP DYNSVVLYSTPPI (SEQ ID NO: 460) corresponding to amino acids 490-1171 of HSFLT_P6 (SEQ ID NO:16), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for a head of HSFLT_P6 (SEQ ID NO:16), 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 MTAP (SEQ ID NO: 459) of HSFLT_P6 (SEQ ID NO:16).
  • C. An isolated polypeptide encoding for an edge portion of HSFLT_P6 (SEQ ID NO:16), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNB QQEPGIILGPGSSTLFIERVTEEDEGVYHCK ATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDE QCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAVKMLKEGATASEYKALMTELK ILTHIMHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQG KKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNI LLSENNVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPG VQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAI LTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLL ASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPP DYNSVVLYSTPPI (SEQ ID NO: 460) of HSFLT_P6 (SEQ ID NO:16).
  • The location of the variant protein was determined via the use of a number of different software programs and analyses, as described and including analyses from SignalP and other specialized programs.
  • In some embodiments of the invention, the variant protein is located in or in association with the cell membrane.
  • Variant protein HSFLT_P6 (SEQ ID NO:16) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 10, (lists the position(s) within the sequence and the alternative amino acid(s); the presence of known SNPs in variant protein HSFLT_P6 (SEQ ID NO:16) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • TABLE 10
    Amino acid mutations
    SNP position(s) on amino acid
    sequence Alternative amino acid(s)
    83 L -> P
    117 Q -> R
    176 V -> A
    227 D -> G
    593 I -> V
    680 F -> S
  • The glycosylation sites of variant protein HSFLT_P6 (SEQ ID NO:16), as compared to the known protein Vascular endothelial growth factor receptor 1 precursor, are described in Table 11 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 11
    Glycosylation site(s)
    Position(s) on known Position(s) on
    amino acid sequence Present in variant protein? variant protein
    29 Yes 29
    84 Yes 84
    100 No
    156 Yes 156
    164 No
    235 Yes 235
    250 Yes 250
    307 Yes 307
    380 Yes 380
    430 Yes 430
    453 Yes 453
    458 Yes 458
    499 Yes 499
  • The phosphorylation sites of variant protein HSFLT_P6 (SEQ ID NO:16), as compared to the known protein, are described in Table 12 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the phosphorylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 12
    Phosphorylation site(s)
    Position(s) on known amino Position(s) on
    acid sequence Present in variant protein? variant protein
    886 Yes 886
    1002 Yes 1002
    1046 Yes 1046
    1075 Yes 1075
    1160 Yes 1160
    1166 Yes 1166
  • The variant protein has the following domains, as determined by using InterPro. The domains are described in Table 13:
  • TABLE 13
    InterPro domain(s)
    Domain description Analysis type Position(s) on protein
    Protein kinase BlastProDom 661-760, 826-992
    Vascular endothelial growth FPrintScan 17-27, 75-87, 223-240,
    factor receptor, VEGFR 281-295
    Vascular endothelial growth FPrintScan 57-80, 106-123, 183-203,
    factor receptor 1, VEGFR1 208-222
    Immunoglobulin-like HMMPfam 78-146, 403-471, 508-566
    Protein kinase HMMPfam 660-987
    Tyrosine protein kinase HMMSmart 660-987
    Serine HMMSmart 660-991
    Immunoglobulin V-type HMMSmart 80-146, 405-471
    Immunoglobulin C2 type HMMSmart 76-151, 181-245, 401-476,
    506-571
    Immunoglobulin subtype HMMSmart 70-162, 177-258, 272-386,
    395-491, 500-582
    Protein kinase ProfileScan 660-991
    Immunoglobulin-like ProfileScan 63-160, 182-237, 261-386,
    389-487, 494-580
    Protein kinase ScanRegExp 666-694
    Tyrosine protein kinase, ScanRegExp 851-863
    active site
    Receptor tyrosine kinase, ScanRegExp 719-732
    class III
  • Variant protein HSFLT_P6 (SEQ ID NO:16) is encoded by the following transcript(s): HSFLT_T9 (SEQ ID NO:3), for which the coding portion begins at position 1113 and ends at position 4625. The transcript also has the following SNPs as listed in Table 14 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HSFLT_P6 (SEQ ID NO:16) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • TABLE 14
    Nucleic acid SNPs
    Polymorphism: Position(s) on nucleotide sequence
    T -> C 1360; 1639; 3151; 3815; 4250
    A -> G 1462; 1622; 1792; 2889; 3683; 5718; 7237
    T -> G 6143; 6148; 7266
    C -> T 1830; 4975
    G -> A 2315
    C -> A 3362
    T -> 4774
    G -> T 5896
    A -> C 6984
  • In one embodiment, a variant protein HSFLT_P7 according to the present invention has an amino acid sequence as set forth in (SEQ ID NO:17). In one embodiment, it is encoded by a p HSFLT_T10 polynucleotide (SEQ ID NO:4), and an alignment of the variant to the known protein (Vascular endothelial growth factor receptor 1 precursor) is presented in the alignment table on the attached CD-ROM. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • 1. Comparison Report Between HSFLT_P7 (SEQ ID NO:17) and VGR1_HUMAN_V1 (SEQ ID NO: 575):
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) corresponding to amino acids 1-6 of HSFLT_P7 (SEQ ID NO:17), and a second amino acid sequence being at least 90% homologous to FPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGBLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTL VLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKS VNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKD VTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHP CNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGR NISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQKMAITKEHSITLNL TIMUVSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFK NNHKIQQEPGIILGPGSSTLFIERVTEEDEGVYHCKATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLF WLLLTLFIRKMKSSSEIKTDYLSIIMDPDEVPLDEQCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASA FGIKKSPTCRTVAVKMLKEGATASEYKALMTELKILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSN YLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQGKKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEP ITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNILLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMA PESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPGVQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWBRDP KERPRFAELVEKLGDLLQANVQQDGKDYIPINAILTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNA FKFMSLERIKTFEELLPNATSMFDDYQGDSSTLLASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPS FCHSSCGHVSEGKRRFTYDHAELERKIACCSPPPDYNSVVLYSTPPI corresponding to amino acids 172-1338 of VGR1_HUMAN_V1 (SEQ ID NO: 575), which also corresponds to amino acids 7-1173 of HSFLT_P7 (SEQ ID NO:17), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for a head of HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) of HSFLT_P7 (SEQ ID NO:17).
  • 2. Comparison Report Between HSFLT_P7 (SEQ ID NO:17) and NP002010_V1 (SEQ ID NO: 574):
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) corresponding to amino acids 1-6 of HSFLT_P7 (SEQ ID NO:17), and a second amino acid sequence being at least 90% homologous to FPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTL VLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKS VNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKD VTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHP CNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGR NISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQKMAITKEHSITLNL TIMNVSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFK NNHKIQQEPGIILGPGSSTLFIERVTEEDEGVYHCKATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLF WLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDEQCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASA FGIKKSPTCRTVAVKMLKEGATASEYKALMTELKILTHIGHBLNVVNLLGACTKQGGPLMVIVEYCKYGNLSN YLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQGKKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKBP ITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNILLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMA PESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPGVQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDP KERPRFAELVEKLGDLLQANVQQDGKDYIPINAILTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNA FKFMSLERIKTFEELLPNATSMFDDYQGDSSTLLASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPS FCHSSCGHVSEGKRRFTYDHAELERKIACCSPPPDYNSVVLYSTPPI corresponding to amino acids 172-1338 of NP002010_V1 (SEQ ID NO: 574), which also corresponds to amino acids 7-1173 of HSFLT_P7 (SEQ ID NO:17), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for a head of HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) of HSFLT_P7 (SEQ ID NO:17).
  • 3. Comparison Report Between HSFLT_P7 (SEQ ID NO:17) and P17948-2 (SEQ ID NO:360)
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) corresponding to amino acids 1-6 of HSFLT_P7 (SEQ ID NO:17), a second amino acid sequence being at least 90% homologous to FPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTL VLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKS VNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKD VTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHP CNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGR NISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQKMAITKEHSITLNL TIMNVSLQDSGTYACRARNVYTGEEILQKKEITIR corresponding to amino acids 172-656 of P17948-2 (SEQ ID NO:360), which also corresponds to amino acids 7-491 of HSFLT_P7 (SEQ ID NO:17), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGIILGPGSSTLFIERVTEEDEGVYHCK ATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDE QCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAVKMLKEGATASEYKALMTELK ILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQG KKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNI LLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPG VQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAI LTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLL ASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPP DYNSVVLYSTPPI (SEQ ID NO: 460) corresponding to amino acids 492-1173 of HSFLT_P7 (SEQ ID NO:17), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for a head of HSFLT_P7 (SEQ ID NO:17), 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 MPLPFQ (SEQ ID NO: 576) of HSFLT_P7 (SEQ ID NO:17).
  • C. An isolated polypeptide encoding for an edge portion of HSFLT_P7 (SEQ ID NO:17), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNMIQQEPGIILGPGSSTLFIERVTEEDEGVYHCK ATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDE QCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAVKMLKEGATASEYKALMTELK ILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQG KKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNI LLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPG VQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAI LTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLL ASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPP DYNSVVLYSTPPI (SEQ ID NO: 460) of HSFLT_P7 (SEQ ID NO:17).
  • The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is membrane.
  • Variant protein HSFLT_P7 (SEQ ID NO:17) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 15, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the presence of known SNPs in variant protein HSFLT_P7 (SEQ ID NO:17) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • TABLE 15
    Amino acid mutations
    SNP position(s) on amino acid
    sequence Alternative amino acid(s)
    85 L -> P
    119 Q -> R
    178 V -> A
    229 D -> G
    595 I -> V
    682 F -> S
  • The glycosylation sites of variant protein HSFLT_P7 (SEQ ID NO:17), as compared to the known protein Vascular endothelial growth factor receptor 1 precursor, are described in Table 16 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 16
    Glycosylation site(s)
    Position(s) on known amino Position(s) on
    acid sequence Present in variant protein? variant protein
    31 Yes 31
    86 Yes 86
    100 No
    158 Yes 158
    164 No
    237 Yes 237
    252 Yes 252
    309 Yes 309
    382 Yes 382
    432 Yes 432
    455 Yes 455
    460 Yes 460
    501 Yes 501
  • The phosphorylation sites of variant protein HSFLT_P7 (SEQ ID NO:17), as compared to the known protein, are described in Table 17 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the phosphorylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 17
    Phosphorylation site(s)
    Position(s) on known amino Position(s) on
    acid sequence Present in variant protein? variant protein
    888 Yes 888
    1004 Yes 1004
    1048 Yes 1048
    1077 Yes 1077
    1162 Yes 1162
    1168 Yes 1168
  • The variant protein has the following domains, as determined by using InterPro. The domains are described in Table 18:
  • TABLE 18
    InterPro domain(s)
    Domain description Analysis type Position(s) on protein
    Protein kinase BlastProDom 663-762, 828-994
    Vascular endothelial growth factor FPrintScan 19-29, 77-89, 225-242, 283-297
    receptor, VEGFR
    Vascular endothelial growth factor FPrintScan 59-82, 108-125, 185-205, 210-224
    receptor 1, VEGFR1
    Immunoglobulin-like HMMPfam 80-148, 405-473, 510-568
    Protein kinase HMMPfam 662-989
    Tyrosine protein kinase HMMSmart 662-989
    Serine HMMSmart 662-993
    Immunoglobulin V-type HMMSmart 82-148, 407-473
    Immunoglobulin C2 type HMMSmart 78-153, 183-247, 403-478, 508-573
    Immunoglobulin subtype HMMSmart 72-164, 179-260, 274-388, 397-493, 502-584
    Protein kinase ProfileScan 662-993
    Immunoglobulin-like ProfileScan 65-162, 184-239, 263-388, 391-489, 496-582
    Protein kinase ScanRegExp 668-696
    Tyrosine protein kinase, active site ScanRegExp 853-865
    Receptor tyrosine kinase, class III ScanRegExp 721-734
  • Variant protein HSFLT_P7 (SEQ ID NO:17) is encoded by the following transcript(s): HSFLT_T10 (SEQ ID NO:4), for which the coding portion begins at position 448 and ends at position 3966. The transcript also has the following SNPs as listed in Table 19 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the presence of known SNPs in variant protein HSFLT_P7 (SEQ ID NO:17) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • TABLE 19
    Nucleic acid SNPs
    Polymorphism nucleotide position
    T -> C 701, 980, 2492, 3156, 3591
    A -> G 803, 963, 1133, 2230, 3024, 5059, 6578
    T -> G 5484, 5489, 6607
    C -> T 1171, 4316
    G -> A 1656
    C -> A 2703
    G -> T 5237
    A -> C 6325
    T -> 4115
  • In some embodiments, HSFLT_P10 of the present invention has an amino acid sequence homologous to or as set forth in SEQ ID NO:18, and may be encoded by transcript(s) HSFLT_T13 (SEQ ID NO:5). An alignment of HSFLT_P10 to known protein vascular endothelial growth factor receptor 1 precursor is provided in the alignment table on the attached CD-ROM. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • 1. Comparison report between HSFLT_P10 (SEQ ID NO:18) and VGR1_HUMAN (SEQ ID NO: 359):
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P10 (SEQ ID NO:18), comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESE RLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIHMT EGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKM QNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDG LPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSR QILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVV ADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRT MHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAI SSSTTLDCHANGVPEPQITWFKNNHKIQQEP corresponding to amino acids 1-705 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-705 of HSFLT_P10 (SEQ ID NO:18), 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 ELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 462) corresponding to amino acids 706-733 of HSFLT_P10 (SEQ ID NO:18), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for an edge portion of HSFLT_P10 (SEQ ID NO:18), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 462) of HSFLT_P10 (SEQ ID NO:18).
  • 2. Comparison Report Between HSFLT_P10 (SEQ ID NO:18) and P17948-2 (SEQ ID NO:360)
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P10 (SEQ ID NO:18), comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESE RLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMT EGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFHSNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKM QNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDG LPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSR QILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVV ADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRT MHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIR corresponding to amino acids 1-656 of P17948-2 (SEQ ID NO:360), which also corresponds to amino acids 1-656 of HSFLT_P10 (SEQ ID NO:18), 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 DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 463) corresponding to amino acids 657-733 of HSFLT_P10 (SEQ ID NO:18), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for an edge portion of HSFLT_P10 (SEQ ID NO:18), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 463) of HSFLT_P10 (SEQ ID NO:18).
  • 3. Comparison Report Between HSFLT_P10 (SEQ ID NO:18) and NP002010 (SEQ ID NO: 531):
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P10 (SEQ ID NO:18), comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESE RLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMT EGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKM QNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDG LPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSR QILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVV ADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRT MHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAI SSSTTLDCHANGVPEPQITWFKNNHKIQQEP corresponding to amino acids 1-705 of NP002010 (SEQ ID NO: 531), which also corresponds to amino acids 1-705 of HSFLT_P10 (SEQ ID NO:18), 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 ELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 462) corresponding to amino acids 706-733 of HSFLT_P10 (SEQ ID NO:18), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for an edge portion of HSFLT_P10 (SEQ ID NO:18), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ELYTSTSPSSSSSSPLSSSSSSSSSSSS (SEQ ID NO: 462) of HSFLT_P10 (SEQ ID NO:18).
  • The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is secreted.
  • Variant protein HSFLT_P10 (SEQ ID NO:18) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 20, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the presence of known SNPs in variant protein HSFLT_P10 (SEQ ID NO:18) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • TABLE 20
    Amino acid mutations
    SNP position(s) on amino acid
    sequence Alternative amino acid(s)
    250 L -> P
    284 Q -> R
    343 V -> A
    394 D -> G
  • The glycosylation sites of variant protein HSFLT_P10 (SEQ ID NO:18), as compared to the known protein Vascular endothelial growth factor receptor 1 precursor, are described in Table 21 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 21
    Glycosylation site(s)
    Position(s) on known Position(s) on
    amino acid sequence Present in variant protein? variant protein
    100 Yes 100
    164 Yes 164
    196 Yes 196
    251 Yes 251
    323 Yes 323
    402 Yes 402
    417 Yes 417
    474 Yes 474
    547 Yes 547
    597 Yes 597
    620 Yes 620
    625 Yes 625
    666 Yes 666
  • The phosphorylation sites of variant protein HSFLT_P10 (SEQ ID NO:18), as compared to the known protein, are described in Table 22 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the phosphorylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 22
    Phosphorylation site(s)
    Position(s) on known Position(s) on
    amino acid sequence Present in variant protein? variant protein
    1053 No
    1169 No
    1213 No
    1242 No
    1327 No
    1333 No
  • The variant protein has the following domains, as determined by using InterPro. The domains are described in Table 23:
  • TABLE 23
    InterPro domain(s)
    Domain description Analysis type Position(s) on protein
    Vascular endothelial growth factor FPrintScan 89-107, 125-136, 184-194, 242-254, 390-407,
    receptor, VEGFR 448-462
    Vascular endothelial growth factor FPrintScan 26-41, 79-93, 130-155, 224-247, 273-290,
    receptor 1, VEGFR1 350-370, 375-389
    Immunoglobulin-like HMMPfam 245-313, 570-638, 675-731
    Immunoglobulin V-type HMMSmart 247-313, 572-638
    Immunoglobulin C2 type HMMSmart 149-214, 243-318, 348-412, 568-643,
    673-732
    Immunoglobulin subtype HMMSmart 38-129, 143-224, 237-329, 344-425, 439-553,
    562-658
    Immunoglobulin-like ProfileScan 32-107, 230-327, 349-404, 428-553, 556-654,
    661-733
  • Variant protein HSFLT_P1 (SEQ ID NO:18) is encoded by the following transcript(s): HSFLT_T13 (SEQ ID NO:5), for which the coding portion starts at position 315 and ends at position 2513. The transcript also has the following SNPs as listed in Table 24 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the presence of known SNPs in variant protein HSFLT_P10 (SEQ ID NO:18) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • TABLE 24
    Nucleic acid SNPs
    SNP position(s) on nucleotide
    Polymorphism sequence
    -> C  823
    T -> C 1063, 1342
    A -> G 1165, 1325, 1495
    C -> T 1533
    G -> A 2018
    G -> T 3301
  • Variant protein HSFLT_P11 (SEQ ID NO:19) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HSFLT_T14 (SEQ ID NO:6). An alignment is given to the known protein (Vascular endothelial growth factor receptor 1 precursor) in the alignment table on the attached CD-ROM. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • 1. Comparison Report Between HSFLT_P11 (SEQ ID NO:19) and VGR1_HUMAN (SEQ ID NO: 359):
  • A. An isolated chimeric polypeptide as set forth in a chimeric HSFLT_P11 (SEQ ID NO:19) polypeptide comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESE RLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMT EGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKM QNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEWWLKDG LPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSR QILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVV ADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRT MHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAI SSSTTLDCHANGVPEPQITWFKNNHKIQQEPG corresponding to amino acids 1-706 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-706 of HSFLT_P11 (SEQ ID NO:19), 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 SANTAVNKKTEI (SEQ ID NO: 464) corresponding to amino acids 707-718 of HSFLT_P11 (SEQ ID NO:19), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for an edge portion of HSFLT_P11 (SEQ ID NO:19), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SANTAVNKKTEI (SEQ ID NO: 464) of HSFLT_P11 (SEQ ID NO:19).
  • 2. Comparison Report Between HSFLT_P11 (SEQ ID NO:19) and P17948-2 (SEQ ID NO:360)
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P11 (SEQ ID NO:19), comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESE RLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMT EGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKM QNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEWWLKDG LPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSR QILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVV ADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRT MHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIR corresponding to amino acids 1-656 of P17948-2 (SEQ ID NO:360), which also corresponds to amino acids 1-656 of HSFLT_P11 (SEQ ID NO:19), 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 DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGSANTAVNKKTEI (SEQ ID NO: 465) corresponding to amino acids 657-718 of HSFLT_P11 (SEQ ID NO:19), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for an edge portion of HSFLT_P11 (SEQ ID NO:19), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGSANTAVNKKTEI (SEQ ID NO: 465) of HSFLT_P11 (SEQ ID NO:19).
  • 3. Comparison Report Between HSFLT_P11 (SEQ ID NO:19) and NP002010 (SEQ ID NO: 531):
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P11 (SEQ ID NO:19), comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESE RLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMT EGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKM QNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDG LPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSR QILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVV ADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRT MHYSISKQKMAITKEHSITLNLTIMSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAI SSSTTLDCHANGVPEPQITWFKNNHKIQQEPG corresponding to amino acids 1-706 of NP002010 (SEQ ID NO: 531), which also corresponds to amino acids 1-706 of HSFLT_P11 (SEQ ID NO:19), 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 SANTAVNKKTEI (SEQ ID NO: 464) corresponding to amino acids 707-718 of HSFLT_P11 (SEQ ID NO:19), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for an edge portion of HSFLT_P11 (SEQ ID NO:19), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SANTAVNKKTEI (SEQ ID NO: 464) of HSFLT_P11 (SEQ ID NO:19).
  • The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is secreted.
  • Variant protein HSFLT_P11 (SEQ ID NO:19) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 25, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the presence of known SNPs in variant protein HSFLT_P11 (SEQ ID NO:19) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • TABLE 25
    Amino acid mutations
    SNP position(s) on amino acid
    sequence Alternative amino acid(s)
    250 L -> P
    284 Q -> R
    343 V -> A
    394 D -> G
  • The glycosylation sites of variant protein HSFLT_P11 (SEQ ID NO:19), as compared to the known protein Vascular endothelial growth factor receptor 1 precursor, are described in Table 26 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 26
    Glycosylation site(s)
    Position(s) on known Position(s) on
    amino acid sequence Present in variant protein? variant protein
    100 Yes 100
    164 Yes 164
    196 Yes 196
    251 Yes 251
    323 Yes 323
    402 Yes 402
    417 Yes 417
    474 Yes 474
    547 Yes 547
    597 Yes 597
    620 Yes 620
    625 Yes 625
    666 Yes 666
  • The phosphorylation sites of variant protein HSFLT_P11 (SEQ ID NO:19), as compared to the known protein, are described in Table 27 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the phosphorylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 27
    Phosphorylation site(s)
    Position(s) on known amino acid
    sequence Present in variant protein?
    1053 No
    1169 No
    1213 No
    1242 No
    1327 No
    1333 No
  • The variant protein has the following domains, as determined by using InterPro. The domains are described in Table 28:
  • TABLE 28
    InterPro domain(s)
    Domain description Analysis type Position(s) on protein
    Vascular endothelial growth factor FPrintScan 89-107, 125-136, 184-194, 242-254, 390-407,
    receptor, VEGFR 448-462
    Vascular endothelial growth factor FPrintScan 26-41, 79-93, 130-155, 224-247, 273-290,
    receptor 1, VEGFR1 350-370, 375-389
    Immunoglobulin-like HMMPfam 245-313, 570-638
    Immunoglobulin V-type HMMSmart 247-313, 572-638
    Immunoglobulin C2 type HMMSmart 149-214, 243-318, 348-412, 568-643,
    673-716
    Immunoglobulin subtype HMMSmart 38-129, 143-224, 237-329, 344-425, 439-553,
    562-658
    Immunoglobulin-like ProfileScan 32-107, 230-327, 349-404, 428-553, 556-654,
    661-718
  • Variant protein HSFLT_P11 (SEQ ID NO:19) is encoded by the following transcript(s): HSFLT_T14 (SEQ ID NO:6), for which the coding portion starts at position 315 and ends at position 2468. The transcript also has the following SNPs as listed in Table 29 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the presence of known SNPs in variant protein HSFLT_P11 (SEQ ID NO:19) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • TABLE 29
    Nucleic acid SNPs
    SNP position(s) on nucleotide
    Polymorphism sequence
    -> C  823
    T -> C 1063, 1342
    A -> G 1165, 1325, 1496
    C -> T 1533
    G -> A 2018
  • Variant protein HSFLT_P13 (SEQ ID NO:20) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HSFLT_T17 (SEQ ID NO:7). An alignment is given to the known protein (Vascular endothelial growth factor receptor 1 precursor in the alignment table on the attached CD-ROM. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • 1. Comparison Report Between HSFLT_P13 (SEQ ID NO:20) and VGR1_HUMAN (SEQ ID NO: 359):
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P13 (SEQ ID NO:20), comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESE RLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMT EGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKM QNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDG LPATEKSARYLTRGYSLII DVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSR QILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVV ADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRT MHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAI SSSTTLDCHANGVPEPQITWFKNNHKIQQEPG corresponding to amino acids 1-706 of VGR1_HUMAN (SEQ ID NO: 359), which also corresponds to amino acids 1-706 of HSFLT_P13 (SEQ ID NO:20), 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 KRLFFLPFIISHLSSAPLSLNSPVTCFQYV (SEQ ID NO: 466) corresponding to amino acids 707-736 of HSFLT_P13 (SEQ ID NO:20), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for an edge portion of HSFLT_P13 (SEQ ID NO:20), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence KRLFFLPFIISHLSSAPLSLNSPVTCFQYV (SEQ ID NO: 466) of HSFLT_P13 (SEQ ID NO:20).
  • 2. Comparison Report Between HSFLT_P13 (SEQ ID NO:20) and P17948-2 (SEQ ID NO:360):
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P13 (SEQ ID NO:20), comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHBINQAGQTLHLQCRGEAAHKWSLPEMVSKESE RLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMT EGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKM QNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDG LPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSR QILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVV ADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRT MHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIR corresponding to amino acids 1-656 of P17948-2 (SEQ ID NO:360), which also corresponds to amino acids 1-656 of HSFLT_P13 (SEQ ID NO:20), 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 DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNEKIQQEPGKRLFFLPFIISHLSSAPLSLNSPVTCF QYV (SEQ ID NO: 467) corresponding to amino acids 657-736 of HSFLT_P13 (SEQ ID NO:20), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for an edge portion of HSFLT_P13 (SEQ ID NO:20), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPGKRLFFLPFIISHLSSAPLSLNSPVTCF QYV (SEQ ID NO: 467) of HSFLT_P13 (SEQ ID NO:20).
  • 3. Comparison Report Between HSFLT_P13 (SEQ ID NO:20) and NP002010 (SEQ ID NO: 531):
  • A. An isolated chimeric polypeptide as set forth in HSFLT_P13 (SEQ ID NO:20), comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESE RLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMT EGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKM QNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEWWLKDG LPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSR QILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIEGKNKMASTLVV ADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRT MHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAI SSSTTLDCHANGVPEPQITWFKNNIKIQQEPG corresponding to amino acids 1-706 of NP002010 (SEQ ID NO: 531), which also corresponds to amino acids 1-706 of HSFLT_P13 (SEQ ID NO:20), 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 KRLFFLPFIISHLSSAPLSLNSPVTCFQYV (SEQ ID NO: 466) corresponding to amino acids 707-736 of HSFLT_P13 (SEQ ID NO:20), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • B. An isolated polypeptide encoding for an edge portion of HSFLT_P13 (SEQ ID NO:20), comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence KRLFFLPFIISHLSSAPLSLNSPVTCFQYV (SEQ ID NO: 466) of HSFLT_P13 (SEQ ID NO:20).
  • The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is secreted.
  • Variant protein HSFLT_P13 (SEQ ID NO:20) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 30, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the presence of known SNPs in variant protein HSFLT_P13 (SEQ ID NO:20) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • TABLE 30
    Amino acid mutations
    SNP position(s) on amino acid
    sequence Alternative amino acid(s)
    250 L -> P
    284 Q -> R
    343 V -> A
    394 D -> G
  • The glycosylation sites of variant protein HSFLT_P13 (SEQ ID NO:20), as compared to the known protein Vascular endothelial growth factor receptor 1 precursor, are described in Table 31 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 31
    Glycosylation site(s)
    Position(s) on known Position(s) on
    amino acid sequence Present in variant protein? variant protein
    100 Yes 100
    164 Yes 164
    196 Yes 196
    251 Yes 251
    323 Yes 323
    402 Yes 402
    417 Yes 417
    474 Yes 474
    547 Yes 547
    597 Yes 597
    620 Yes 620
    625 Yes 625
    666 Yes 666
  • The phosphorylation sites of variant protein HSFLT_P13 (SEQ ID NO:20), as compared to the known protein, are described in Table 32 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the phosphorylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
  • TABLE 32
    Phosphorylation site(s)
    Position(s) on known amino acid
    sequence Present in variant protein?
    1053 No
    1169 No
    1213 No
    1242 No
    1327 No
    1333 No
  • The variant protein has the following domains, as determined by using InterPro. The domains are described in Table 33:
  • TABLE 33
    InterPro domain(s)
    Domain description Analysis type Position(s) on protein
    Vascular endothelial growth FPrintScan 89-107, 125-136,
    factor receptor, VEGFR 184-194, 242-254,
    390-407, 448-462
    Vascular endothelial growth FPrintScan 26-41, 79-93, 130-155,
    factor receptor 1, VEGFR1 224-247, 273-290,
    350-370, 375-389
    Immunoglobulin-like HMMPfam 245-313, 570-638,
    675-734
    Immunoglobulin V-type HMMSmart 247-313, 572-638
    Immunoglobulin C2 type HMMSmart 149-214, 243-318,
    348-412, 568-643,
    673-725
    Immunoglobulin subtype HMMSmart 38-129, 143-224,
    237-329, 344-425,
    439-553, 562-658