CA2554440A1 - Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of breast cancer - Google Patents
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Abstract
Novel markers for breast cancer that are both sensitive and accurate. These markers are overexpressed in breast cancer specifically, as opposed to normal breast tissue. The measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of breast cancer. The markers of the present invention, alone or in combination, show a high degree of differential detection between breast cancer and non-cancerous states.
Description
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
NOVEL NUCLEOTIDE AND AMINO .~~C:ID SEQUENCES, AND ASSAYS AND
METHODS OF USE THEREOF FC~F;. DIAGNOSIS OF BREAST CANCER
FIELD OF THE INVENTION
The present invention is related to novel nucleotide and protein sequences that are diagnostic markers for breast cancer, and assays and methods of use thereof.
BACKGROUND OF THE INVENTION
Breast cancer is the most commonly occurring cancer in women, comprising almost a third of all malignancies in females. It is the leading cause of death for women between the ages 40-55 in the United States and one out of 8 females in the United States wilt develop breast cancer at some point in her life.
The death rate from breast cancer has been slowly declining over the past decade, partially due do the usage of molecular markers that facilitate the discovery, tumor typing (and therefore choice of treatment), response to treatment and recurrence.
The most widely used serum markers for breast cancers are Mucin 1 (measured as CA
15-3) and CEA (CarcinoEmbryonic Antigen). Mucin 1 (MUC1) is present on the apical surface of normal epithelial cells. Its extracellular domain consists of a heavily Q
linked glycosylated peptide core made up of variable number of multiple repeats of 20 amino acid sequence referred to as VNTR (Vari ble Number Tandem Repeat). This variability results in natural polymorphism of MUC 1. Each VNTR has five potential 4lmkage sites. The breast cancer disease state alters the enzymes which glycosylate Mucin 1 and therefore the polysaccharide side chains of tumor associated MUC 1 are generally shorter than those on the normally expressed molecule. Both aberrant and up-regulated expression of MUC1 are features of malignancy and MUCl related markers are based on it. Though CA 15-3 is a broadly used marker for breast cancer, a combination of CA 15-3 and CEA is more sensitive than using a single marker.
For the purpose of monitoring therapeutic response, CA 15-3, CEA and ESR
(Erythrocyte Sedimentation Rate) are used as a panel, leading to over 90% of patients biochemically assessable. Serum markers used to monitor therapeutic response in patients with metastatic breast cancer are associated with the "spike phenomenon". It is an initial transient rise of tumor marker levels which can be seen in up to 30% of responders in the first 3 months of commencing a therapy. It is important not to interpret this as a sign of disease progression leading to premature change of an effective therapy.
CA 27.29 is a new monoclonal antibody directed against a different part of MUCI and it is a newer marker than CA IS-3. It detects a different glycosylation pattern of MUC1, as compared with CA 15-3. CA 27.29 is the first FDA-approved blood test for breast cancer recurrence. Because of superior sensitivity and specificity, CA 27.29 has supplanted CA 15-3 as the preferred tumor marker in breast cancer. The CA 27.29 level is elevated in approximately one third of women with early stage breast cancer (stage I or Il] and in two thirds of women with late-stage disease (stage III or IV). CA 27.29 lacks predictive value in the earliest stages of breast cancer and thus has no role in screening for or diagnosing the malignancy. CA 27.29 also can be found in patients with benign disorders of the breast, liver, and kidney, and in patients with ovarian cysts. However, CA 27.29 levels higher than 100 units per mL are rare in benign conditions.
Recently Estrogen 2 (beta) was shown to have a diagnostic role in breast cancer. It has been shown that the expression of the 'cx' variant of Estrogen 2 is correlated with response to Hormone adjuvant therapy. In addition it has been shown it may assist in better characterization of ER 1 positive breast cancers (together with progesterone receptor).
HER 2 (also known as GerbB2) is a membrane proto-oncogene with intrinsic tyrosine kinase activity. Tumor expressing HER 2 are associated with shorter survival, shorter time-to-relapse and an overall worse prognosis. Tumors expressing HER 2 can be targeted with Trastuzumab - a biological adjuvant therapy which blocks the growth promoting action of HER
2. The ImmunoHistoChemistry (IHC) and Fluorescence In Situ Hybridization (FISH) tests are used to detect HER2: 1.IHC: The most common test used to check HER2 status is an ImmunoHistoChemistry (IHC) test. The IHC test measures the protein made by the HER2 gene.
2. FISH: This test measures the number of copies of the HER2 gene present in the tumor cell.
Measurement of the extracellular domain of HER 2 has been reported to show a better assessment of response to chemotherapy than a biochemical index score based on measurement of CA 15.3, CEA and ESR in a small series of patient. That finding is yet to be confirmed in a larger group of patient with HER 2 expressing tumors.
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
NOVEL NUCLEOTIDE AND AMINO .~~C:ID SEQUENCES, AND ASSAYS AND
METHODS OF USE THEREOF FC~F;. DIAGNOSIS OF BREAST CANCER
FIELD OF THE INVENTION
The present invention is related to novel nucleotide and protein sequences that are diagnostic markers for breast cancer, and assays and methods of use thereof.
BACKGROUND OF THE INVENTION
Breast cancer is the most commonly occurring cancer in women, comprising almost a third of all malignancies in females. It is the leading cause of death for women between the ages 40-55 in the United States and one out of 8 females in the United States wilt develop breast cancer at some point in her life.
The death rate from breast cancer has been slowly declining over the past decade, partially due do the usage of molecular markers that facilitate the discovery, tumor typing (and therefore choice of treatment), response to treatment and recurrence.
The most widely used serum markers for breast cancers are Mucin 1 (measured as CA
15-3) and CEA (CarcinoEmbryonic Antigen). Mucin 1 (MUC1) is present on the apical surface of normal epithelial cells. Its extracellular domain consists of a heavily Q
linked glycosylated peptide core made up of variable number of multiple repeats of 20 amino acid sequence referred to as VNTR (Vari ble Number Tandem Repeat). This variability results in natural polymorphism of MUC 1. Each VNTR has five potential 4lmkage sites. The breast cancer disease state alters the enzymes which glycosylate Mucin 1 and therefore the polysaccharide side chains of tumor associated MUC 1 are generally shorter than those on the normally expressed molecule. Both aberrant and up-regulated expression of MUC1 are features of malignancy and MUCl related markers are based on it. Though CA 15-3 is a broadly used marker for breast cancer, a combination of CA 15-3 and CEA is more sensitive than using a single marker.
For the purpose of monitoring therapeutic response, CA 15-3, CEA and ESR
(Erythrocyte Sedimentation Rate) are used as a panel, leading to over 90% of patients biochemically assessable. Serum markers used to monitor therapeutic response in patients with metastatic breast cancer are associated with the "spike phenomenon". It is an initial transient rise of tumor marker levels which can be seen in up to 30% of responders in the first 3 months of commencing a therapy. It is important not to interpret this as a sign of disease progression leading to premature change of an effective therapy.
CA 27.29 is a new monoclonal antibody directed against a different part of MUCI and it is a newer marker than CA IS-3. It detects a different glycosylation pattern of MUC1, as compared with CA 15-3. CA 27.29 is the first FDA-approved blood test for breast cancer recurrence. Because of superior sensitivity and specificity, CA 27.29 has supplanted CA 15-3 as the preferred tumor marker in breast cancer. The CA 27.29 level is elevated in approximately one third of women with early stage breast cancer (stage I or Il] and in two thirds of women with late-stage disease (stage III or IV). CA 27.29 lacks predictive value in the earliest stages of breast cancer and thus has no role in screening for or diagnosing the malignancy. CA 27.29 also can be found in patients with benign disorders of the breast, liver, and kidney, and in patients with ovarian cysts. However, CA 27.29 levels higher than 100 units per mL are rare in benign conditions.
Recently Estrogen 2 (beta) was shown to have a diagnostic role in breast cancer. It has been shown that the expression of the 'cx' variant of Estrogen 2 is correlated with response to Hormone adjuvant therapy. In addition it has been shown it may assist in better characterization of ER 1 positive breast cancers (together with progesterone receptor).
HER 2 (also known as GerbB2) is a membrane proto-oncogene with intrinsic tyrosine kinase activity. Tumor expressing HER 2 are associated with shorter survival, shorter time-to-relapse and an overall worse prognosis. Tumors expressing HER 2 can be targeted with Trastuzumab - a biological adjuvant therapy which blocks the growth promoting action of HER
2. The ImmunoHistoChemistry (IHC) and Fluorescence In Situ Hybridization (FISH) tests are used to detect HER2: 1.IHC: The most common test used to check HER2 status is an ImmunoHistoChemistry (IHC) test. The IHC test measures the protein made by the HER2 gene.
2. FISH: This test measures the number of copies of the HER2 gene present in the tumor cell.
Measurement of the extracellular domain of HER 2 has been reported to show a better assessment of response to chemotherapy than a biochemical index score based on measurement of CA 15.3, CEA and ESR in a small series of patient. That finding is yet to be confirmed in a larger group of patient with HER 2 expressing tumors.
Other molecular markers, mainly used for the diagnosis for cancers other than breast cancer were shown to have a diagnostic potential in breast cancer. For example, CA125 which is a major marker for ovarian cancer is also associated with breast cancer. High levels of CA 19-9, a major marker for colorectal and pancreatic cancers, can be found in breast cancer. Overall, S these markers are not frequently used for the detection of breast cancer to due their inferiority compared with other markers already described.
Panels of markers for the diagnosis and typing of breast cancer are being used by pathologists, including both markers described above and additional markers, such as immunohistochemistry markers that have been shown to have a beneficial value for the diagnosis of breast cancer, including PCNA and I~67 are maybe the most important and highly used immunohistochemistry markers for breast cancer. Other markers as ~Cadherin, Cathepsin D and TFF1 are also used for that purpose.
Despite relevant research efforts and the identification of many putative good prognosticators, few of them are proving clinically useful for identifying patients at minimal risk of relapse, patients with a worse prognosis, or patients likely to benefit from specific treatments.
Most of them, such as epidermal growth factor receptor, cyclin E, p53 (this mutation is present in approximately 40% of human breast cancers as an acquired defect), bcl-2, vascular endothelial growth factor, urokinase-type plasminogen activator- I and the anti-apoptosis protein survivin, are suggested for possible inclusion in the category of biomarkers with a high level of clinico-laboratory effectiveness. However, no single biomarker was able to identify those patients with the best (or worst) prognosis or those patients who would be responsive to a given therapy. High level cyclin E expression has been associated with the initiation or progression of different human cancers, in particular breast cancer but also leukemia, lymphoma and others.
Cyclin-E expression level in the breast cancer was found to be a very strong indicator for prognosis, stronger than any other biological marker.
There are some non-cancerous pathological conditions which represent an increased risk factor for development breast cancer. Non limiting examples of these conditions include:
- Ductal hyperplasia without atypia. It is the most frequently encountered breast biopsy result that is associated with increased risk of future development of breast cancer (2 fold increased risk). In particular, the loss of expression of transforming growth factor beta receptor II in the affected epithelial cells is associated with an increased risk of invasive breast cancer.
- Atypical hyperplasia. Women having atypical hypezplasia with over-expression of HER 2 have a greater than 7-fold increased risk of developing invasive breast S carcinoma, as compared with women with non-proliferative benign breast lesions and no evidence of HER-2 amplification.
These pathological conditions should be effectively diagnosed and monitored in order to facilitate early detection of breast cancer.
SUMMARY OF THE INVENTION
The background art does not teach or suggest markers for breast cancer that are su~ciently sensitive and/or accurate, alone or in combination.
The present invention overcomes these deficiencies of the background art by providing novel markers for breast cancer that are both sensitive and accurate. These markers are 1S overexpressed in breast cancer specifically, as opposed to normal breast tissue. The measurement of these markers, alone or in combination, in patient (biological) samples provides information that the diagnostician can correlate with a probable diagnosis of breast cancer. The markers of the present invention, alone or in combination, show a high degree of differential detection between breast cancer and non-cancerous states.
According to preferred embodiments of the present invention, examples of suitable biological samples which may optionally be used with preferred embodiments of the present invention include but are not limited to blood, serum, plasma, blood cells, urine, sputum, saliva, stool, spinal fluid or CSF, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, milk, neuronal tissue, breast tissue, any human organ or tissue, 2S including any tumor or normal tissue, any sample obtained by lavage (for example of the bronchial system or of the breast ductal system), and also samples of in vivo cell culture constituents. In a preferred embodiment, the biological sample comprises breast tissue andlor a serum sample and/or a urine sample and/or a milk sample and/or any other tissue or liquid sample. The sample can optionally be diluted with a suitable eluant before contacting the sample to an antibody and/or performing any other diagnostic assay.
Information given in the text with regard to cellular localization was determined according to four different software programs: (i) tmhmm (from Center for Biological Sequence Analysis, Technical University of Denmark DTU, S http://www.cbs.dtu.dklservices/TMI-PVIM/ThZEIMM2.Ob.guide.php) or (ii) tmpred (from EMBnet, maintained by the ISREC Bionformatics group and the LICR Information Technology Office, Ludwig Institute for Cancer Research, Swiss Institute of Bioinfozmatics, hitp://www.ch.embnet.org/software/TMPRED form.hhnl) for transmembrane region prediction; (iii) signalp_hmm or (iv) signalp_nn (both from Center for Biological Sequence Analysis, Technical University of Denmark DTU, http://www.cbs.dtu.dklservices/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 1 S 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 positions) on amino acid sequence", representing fa position of a known mutation on amino acid sequence.
SNPs may optionally be used as diagnostic markers according to the present invention, alone or in combination with one or more other SNPs and/or any other diagnostic marker.
Preferred embodiments of the present invention comprise such SNPs, including but not limited to novel SNPs on the known (WT or wild type) protein sequences given below, as well as novel nucleic acid and/or amino acid sequences formed through such SNPs, and/or any SNP on a variant amino acid and/or nucleic acid sequence described herein.
Information given in the text with regard to the Homology to the 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=blosum 100 Information is given with regard to overexpression of a cluster in cancer based on ESTs.
A key to the p values with regard to the analysis of such overexpression is as follows;
- librar~based statistics: P-value without including the level of expression in cell-lines (PI) - 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 (SP 1) - 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.
Information is given with regard to overexpression of a cluster in cancer based on microarrays. As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured.
There are two types of microarray results: those from microarrays prepared according to a design by the present inventors, for which the microarray fabrication procedure is described in detail in Materials and Experimental Procedures section herein; and those results from microarrays using Affymetrix technology. As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured. 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, CA, USA (see for example data regarding the Human Genome U133 (HG-U133) Set at www.affymetrix.cone/products/arrays/specific/hgu133.affx; GeneChip Human Genome U133A
2.0 Array at www.affymetrix.com/products/arrayslspecific/hgul33av2.affx; and Human Genome U133 Plus 2.0 Array at www.affymetrix.comlproducts/arrays/specific/hgul33plus.affx). The probe names follow the Affymetrix naming conventicn. 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.govlgeo/query/acc.cgi?acc=GSE 1133 for the Series GSE 1133 database (published on March 2004); a reference to these results is as follows: Su et al (Proc Natl Acad Sci LJ S A. 2004 Apr 20;101 (16):6062-7. Epub 2004 Apr 09). The probes designed according to the present inventors are listed below.
>Z21368 0_0 61857 (SEQ 117 N0:895) AGTTCATCCTTCTTCAGTGTGACCAGTAAATTCTTCCCATACTCTTGAAG
>HLJMGRPSE_0_0_I6630 (SEQ ID N0:896) GCTGATATGGAAGTTGGGGAATCTGAATTGCCAGAGAATCTTGGGAAGAG
>HLTMGRPSE 0 2 0 (SEQ ID N0:897) TCTCATAGAAGCAAAGGAGAACAGAA.ACCACCAGCCACCTCAACCCAAGG
>HSENA78 0_1 0 (SEQ ID N0:898) TGAAGAGTGTGAGGAAAACCTATGTTTGCCGCTTA.AGCTTTCAGCTCAGC
>M85491_0_0_25999 (SEQ ID N0:899) GACATCTTTGCATATCATGTCAGAGGTATAACATCATTGTGGAGAAGCTC
>M85491 0_14_0 (SEQ ID N0:900) GTCATGAAAATCAACACCGAGGTGCGGAGCTTCGGACCTGTGTCCCGCAG
>HSSTROL3_0_0_12518 (SEQ ID NO: 901) ATGAGAGTAACCTCACCCGTGCACTAGTTTACAGAGCATTCACTGCCCCA
>HSSTROL3_0 0_12517 (SEQ ID N0:902) CAGAGATGAGAGCCTGGAGCATTGCAGATGCCAGGGACTTCACAAATGAA
>HLTMCA1XIA_0 0_14909 (SEQ ID N0:903) GCTGCAATCTAAGTTTCGGAATACTTATACCACTCCAGAAATAATCCTCG
>HITMCA 1XIA_0_ 18_0 (SEQ ID N0:904) TTCAGAACTGTTAACATCGCTGACGGGAAGTGGCATCGGGTAGCAATCAG
>R20779_0_0_30670 (SEQ ID N0:905) CCGCGTTGCTTCTAGAGGCTGAATGCCTTTCAAATGGAGAAGGCTTCCAT
>HSS 100PCB_0_0_12280 (SEQ ID N0:906) CTCA.A.AATGAAACTCCCTCTCGCAGAGCACAATTCCAATTCGCTCTAAAA
>HSCOC4_0 0 9892 (SEQ ID N0:907) AAGGACCAGAGTCCATGCCAAGACCACCCTTCAGCTTCCAAGGCCCTCCA
>HSCOC4_0 39 0 (SEQ ID N0:908) ATCCTCCAGCCATGAGGCTGCTCTGGGGGCTGATCTGGGCATCCAGCTTC
>HSCOC4_0 0 9883 (SEQ ID N0:909) CCTGTTTGCTCTGACACCAACTTCCTACCCTCTCAGCCTCAA.AGT'%~»A.CTC
>HSCOC4_0_0_9885 (SEQ >D N0:910) GCTGAGGTGTGGCCGAGGACCTGACCATCTGGAAGTGTGAAAATCCCCTT
>TI I628 0_9_0 (SEQ D7 N0:911) ACAAGATCCCCGTGAAGTACCTGGAGTTCATCTCGGAATGCATCATCCAG
>T 11628_0_0 45174 (SEQ ID N0:912) TAAACAATCAAAGAGCATGT'TGGCCTGGTCCTTTGCTAGGTACTGTAGAG
>T11628 0_0 45161 (SEQ ID NO:913) TGCCTCGCCAGAATGGCACCTGCCCTAAAATAGCTTCCCATGTGAGGGCT
>M78076_0_7_0 (SEQ ID N0:914) GAGAAGATGAACCCGCTGGAACAGTATGAGCGAAAGGTGAATGCGTCTGT
>HSMUC 1 A_0 37_0 (SEQ ID N0:915) AAA.AGGAGACTTCGGCTACCCAGAGAAGTTCAGTGCCCAGCTCTACTGAG
>HSMUC 1 A_0_0_11364 (SEQ ID N0:916) AAAGGCTGGCATAGGGGGAGGTTTCCCAGGTAGAAGAAGAAGTGTCAGCA
>HSMUC1A_0 0_11365 (SEQ ID N0:917) AATTAACCCTTTGAGAGCTGGCCAGGACTCTGGACTGATTACCCCAGCCT
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.
"BONE" for "bone";
"COL" for "colon";
"EPI" for "epithelial";
"GEN" for "general";
"LIVER" for "liver";
"LUN" for "lung' ;
"LYMPH" for "lymph nodes";
"MARROW" for "bone marrow";
"OVA" for "ovary";
"PANCREAS" for "pancreas";
"PRO" for "pzostate";
"STOMACH" for "stomach' ;
"TCELL" for "T cells' ;
Panels of markers for the diagnosis and typing of breast cancer are being used by pathologists, including both markers described above and additional markers, such as immunohistochemistry markers that have been shown to have a beneficial value for the diagnosis of breast cancer, including PCNA and I~67 are maybe the most important and highly used immunohistochemistry markers for breast cancer. Other markers as ~Cadherin, Cathepsin D and TFF1 are also used for that purpose.
Despite relevant research efforts and the identification of many putative good prognosticators, few of them are proving clinically useful for identifying patients at minimal risk of relapse, patients with a worse prognosis, or patients likely to benefit from specific treatments.
Most of them, such as epidermal growth factor receptor, cyclin E, p53 (this mutation is present in approximately 40% of human breast cancers as an acquired defect), bcl-2, vascular endothelial growth factor, urokinase-type plasminogen activator- I and the anti-apoptosis protein survivin, are suggested for possible inclusion in the category of biomarkers with a high level of clinico-laboratory effectiveness. However, no single biomarker was able to identify those patients with the best (or worst) prognosis or those patients who would be responsive to a given therapy. High level cyclin E expression has been associated with the initiation or progression of different human cancers, in particular breast cancer but also leukemia, lymphoma and others.
Cyclin-E expression level in the breast cancer was found to be a very strong indicator for prognosis, stronger than any other biological marker.
There are some non-cancerous pathological conditions which represent an increased risk factor for development breast cancer. Non limiting examples of these conditions include:
- Ductal hyperplasia without atypia. It is the most frequently encountered breast biopsy result that is associated with increased risk of future development of breast cancer (2 fold increased risk). In particular, the loss of expression of transforming growth factor beta receptor II in the affected epithelial cells is associated with an increased risk of invasive breast cancer.
- Atypical hyperplasia. Women having atypical hypezplasia with over-expression of HER 2 have a greater than 7-fold increased risk of developing invasive breast S carcinoma, as compared with women with non-proliferative benign breast lesions and no evidence of HER-2 amplification.
These pathological conditions should be effectively diagnosed and monitored in order to facilitate early detection of breast cancer.
SUMMARY OF THE INVENTION
The background art does not teach or suggest markers for breast cancer that are su~ciently sensitive and/or accurate, alone or in combination.
The present invention overcomes these deficiencies of the background art by providing novel markers for breast cancer that are both sensitive and accurate. These markers are 1S overexpressed in breast cancer specifically, as opposed to normal breast tissue. The measurement of these markers, alone or in combination, in patient (biological) samples provides information that the diagnostician can correlate with a probable diagnosis of breast cancer. The markers of the present invention, alone or in combination, show a high degree of differential detection between breast cancer and non-cancerous states.
According to preferred embodiments of the present invention, examples of suitable biological samples which may optionally be used with preferred embodiments of the present invention include but are not limited to blood, serum, plasma, blood cells, urine, sputum, saliva, stool, spinal fluid or CSF, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, milk, neuronal tissue, breast tissue, any human organ or tissue, 2S including any tumor or normal tissue, any sample obtained by lavage (for example of the bronchial system or of the breast ductal system), and also samples of in vivo cell culture constituents. In a preferred embodiment, the biological sample comprises breast tissue andlor a serum sample and/or a urine sample and/or a milk sample and/or any other tissue or liquid sample. The sample can optionally be diluted with a suitable eluant before contacting the sample to an antibody and/or performing any other diagnostic assay.
Information given in the text with regard to cellular localization was determined according to four different software programs: (i) tmhmm (from Center for Biological Sequence Analysis, Technical University of Denmark DTU, S http://www.cbs.dtu.dklservices/TMI-PVIM/ThZEIMM2.Ob.guide.php) or (ii) tmpred (from EMBnet, maintained by the ISREC Bionformatics group and the LICR Information Technology Office, Ludwig Institute for Cancer Research, Swiss Institute of Bioinfozmatics, hitp://www.ch.embnet.org/software/TMPRED form.hhnl) for transmembrane region prediction; (iii) signalp_hmm or (iv) signalp_nn (both from Center for Biological Sequence Analysis, Technical University of Denmark DTU, http://www.cbs.dtu.dklservices/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 1 S 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 positions) on amino acid sequence", representing fa position of a known mutation on amino acid sequence.
SNPs may optionally be used as diagnostic markers according to the present invention, alone or in combination with one or more other SNPs and/or any other diagnostic marker.
Preferred embodiments of the present invention comprise such SNPs, including but not limited to novel SNPs on the known (WT or wild type) protein sequences given below, as well as novel nucleic acid and/or amino acid sequences formed through such SNPs, and/or any SNP on a variant amino acid and/or nucleic acid sequence described herein.
Information given in the text with regard to the Homology to the 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=blosum 100 Information is given with regard to overexpression of a cluster in cancer based on ESTs.
A key to the p values with regard to the analysis of such overexpression is as follows;
- librar~based statistics: P-value without including the level of expression in cell-lines (PI) - 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 (SP 1) - 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.
Information is given with regard to overexpression of a cluster in cancer based on microarrays. As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured.
There are two types of microarray results: those from microarrays prepared according to a design by the present inventors, for which the microarray fabrication procedure is described in detail in Materials and Experimental Procedures section herein; and those results from microarrays using Affymetrix technology. As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured. 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, CA, USA (see for example data regarding the Human Genome U133 (HG-U133) Set at www.affymetrix.cone/products/arrays/specific/hgu133.affx; GeneChip Human Genome U133A
2.0 Array at www.affymetrix.com/products/arrayslspecific/hgul33av2.affx; and Human Genome U133 Plus 2.0 Array at www.affymetrix.comlproducts/arrays/specific/hgul33plus.affx). The probe names follow the Affymetrix naming conventicn. 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.govlgeo/query/acc.cgi?acc=GSE 1133 for the Series GSE 1133 database (published on March 2004); a reference to these results is as follows: Su et al (Proc Natl Acad Sci LJ S A. 2004 Apr 20;101 (16):6062-7. Epub 2004 Apr 09). The probes designed according to the present inventors are listed below.
>Z21368 0_0 61857 (SEQ 117 N0:895) AGTTCATCCTTCTTCAGTGTGACCAGTAAATTCTTCCCATACTCTTGAAG
>HLJMGRPSE_0_0_I6630 (SEQ ID N0:896) GCTGATATGGAAGTTGGGGAATCTGAATTGCCAGAGAATCTTGGGAAGAG
>HLTMGRPSE 0 2 0 (SEQ ID N0:897) TCTCATAGAAGCAAAGGAGAACAGAA.ACCACCAGCCACCTCAACCCAAGG
>HSENA78 0_1 0 (SEQ ID N0:898) TGAAGAGTGTGAGGAAAACCTATGTTTGCCGCTTA.AGCTTTCAGCTCAGC
>M85491_0_0_25999 (SEQ ID N0:899) GACATCTTTGCATATCATGTCAGAGGTATAACATCATTGTGGAGAAGCTC
>M85491 0_14_0 (SEQ ID N0:900) GTCATGAAAATCAACACCGAGGTGCGGAGCTTCGGACCTGTGTCCCGCAG
>HSSTROL3_0_0_12518 (SEQ ID NO: 901) ATGAGAGTAACCTCACCCGTGCACTAGTTTACAGAGCATTCACTGCCCCA
>HSSTROL3_0 0_12517 (SEQ ID N0:902) CAGAGATGAGAGCCTGGAGCATTGCAGATGCCAGGGACTTCACAAATGAA
>HLTMCA1XIA_0 0_14909 (SEQ ID N0:903) GCTGCAATCTAAGTTTCGGAATACTTATACCACTCCAGAAATAATCCTCG
>HITMCA 1XIA_0_ 18_0 (SEQ ID N0:904) TTCAGAACTGTTAACATCGCTGACGGGAAGTGGCATCGGGTAGCAATCAG
>R20779_0_0_30670 (SEQ ID N0:905) CCGCGTTGCTTCTAGAGGCTGAATGCCTTTCAAATGGAGAAGGCTTCCAT
>HSS 100PCB_0_0_12280 (SEQ ID N0:906) CTCA.A.AATGAAACTCCCTCTCGCAGAGCACAATTCCAATTCGCTCTAAAA
>HSCOC4_0 0 9892 (SEQ ID N0:907) AAGGACCAGAGTCCATGCCAAGACCACCCTTCAGCTTCCAAGGCCCTCCA
>HSCOC4_0 39 0 (SEQ ID N0:908) ATCCTCCAGCCATGAGGCTGCTCTGGGGGCTGATCTGGGCATCCAGCTTC
>HSCOC4_0 0 9883 (SEQ ID N0:909) CCTGTTTGCTCTGACACCAACTTCCTACCCTCTCAGCCTCAA.AGT'%~»A.CTC
>HSCOC4_0_0_9885 (SEQ >D N0:910) GCTGAGGTGTGGCCGAGGACCTGACCATCTGGAAGTGTGAAAATCCCCTT
>TI I628 0_9_0 (SEQ D7 N0:911) ACAAGATCCCCGTGAAGTACCTGGAGTTCATCTCGGAATGCATCATCCAG
>T 11628_0_0 45174 (SEQ ID N0:912) TAAACAATCAAAGAGCATGT'TGGCCTGGTCCTTTGCTAGGTACTGTAGAG
>T11628 0_0 45161 (SEQ ID NO:913) TGCCTCGCCAGAATGGCACCTGCCCTAAAATAGCTTCCCATGTGAGGGCT
>M78076_0_7_0 (SEQ ID N0:914) GAGAAGATGAACCCGCTGGAACAGTATGAGCGAAAGGTGAATGCGTCTGT
>HSMUC 1 A_0 37_0 (SEQ ID N0:915) AAA.AGGAGACTTCGGCTACCCAGAGAAGTTCAGTGCCCAGCTCTACTGAG
>HSMUC 1 A_0_0_11364 (SEQ ID N0:916) AAAGGCTGGCATAGGGGGAGGTTTCCCAGGTAGAAGAAGAAGTGTCAGCA
>HSMUC1A_0 0_11365 (SEQ ID N0:917) AATTAACCCTTTGAGAGCTGGCCAGGACTCTGGACTGATTACCCCAGCCT
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.
"BONE" for "bone";
"COL" for "colon";
"EPI" for "epithelial";
"GEN" for "general";
"LIVER" for "liver";
"LUN" for "lung' ;
"LYMPH" for "lymph nodes";
"MARROW" for "bone marrow";
"OVA" for "ovary";
"PANCREAS" for "pancreas";
"PRO" for "pzostate";
"STOMACH" for "stomach' ;
"TCELL" for "T cells' ;
5 "THYROID" for "Thyroid";
"MAM" for "breast";
"BRAIN" for "brain";
"UTERUS" for "uterus";
"SKIN" for "skin";
10 "KIDNEY" for "kidney";
"MUSCLE" for "muscle";
"ADREN" for "adrenal";
"HEAD" for "head and neck";
"BLADDER" for "bladder";
It should be noted that the terms "segment", "seg" and "node" 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 below. They are also the building blocks that were used to construct complete nucleic acid sequences as described in greater detail below. 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.
As used herein the phrase "breast cancer" refers to cancers of the breast or surrounding tissue, including but not limited to ductal carcinoma (in-situ or invasive), lobular carcinoma (in-situ or invasive), inflammatory breast cancer, mucinous carcinoma, tubular carcinoma, or Paget's disease of the nipple, as well as conditions that are indicative of a higher risk factoz for later development of breast cancer, including but not limited to ductal hyperplasia without atypia and atypical hyperplasia, referred to herein collectively as "indicative conditions".
The term "marker" in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from subjects (patients) having breast cancer (or one of the above indicative conditions) as compared to a comparable sample taken from subjects who do not have breast cancer (or one of the above indicative conditions).
The phxase "differentially present" refers to differences in the quantity of a marker present in a sample taken from patients having breast cancer (or one of the above indicative conditions) as compared to a comparable sample taken from patients who do not have breast cancer (or one of the above indicative 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.
As used herein 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.
As used herein the phrase "diagnosing" refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease andlor prospects of recovery. The term "detecting" may also optionally encompass any of the above.
Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide 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 rer,ncrved from the subject, as described in greater detail below.
As used herein, the term "level" refers to expression levels of RNA andlor 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 biopsylsample 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 andlor amplification and/or a decreased expression, of the variant as opposed to the normal tissues.
A "test amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of breast cancer (or one of the above indicative conditions). A test amount can be either in absolute amount (e.g., microgramlml) or a relative amount (e.g., relative intensity of signals).
A "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a patient with breast cancer (or one of the above indicative conditions) or a person without breast cancer (or one of the above indicative conditions). A
control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
"Detect" refers to identifying the presence, absence or amount of the object to be detected.
A "label" includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means. For example, useful labels include 3zP, 3ss~
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. Itlausner, 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," when referring to a protein or peptide (or other epitope), refers 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 tZVO 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). Typicallyy a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name T10888_PEA_1 T1 T10888_PEA_1T4 T1088SPEA_1 TS
T10888_PEA_1T6 a nucleic acid sequence comprising a sequence in the table below:
SegmentName T10888_PEA_1 node_11 TI0888_PEA_1 12 node_ T10888_PEA_1 17 node_ T10888PEA_1 4 node T 10888_PEA_1 6 node T10888_PEA_1 7 node_ T10888PEA_1 9 node T10888PEA_1 15 node_ According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below amino acid sequence comprising a sequence in the table below:
Proteii,~
Nat~e T 10888_PEA_1P2 T 10888_PEA_P4 T10888_PEA_1PS
5 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name a nucleic acid sequence comprising a sequence in the table below:
Segment Name T39971 node 0 T39971 node 18 T39971 node 21 T399 r~.ode22 i' T39971n:,ode23 T39971~node31 T39971node33 T39971node7 T39971node1 T39971node10 T39971node11 T39971node12 T39971node15 T39971node16 T39971node17 T39971node26 T39971node27 T39971node28 T39971node29 T39971node3 T39971node30 T39971node34 T39971node_35 T39971node36 T39971node4 T39971node5 T39971node8 T39971node9 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below:
Protein ZtTan~ , 39971 P12 , According to preferred embodiments of the present invvention, there is provided an isolated polynucleotide comprising~~a nucleic acid sequence in the table below and/or:
Tran.~cript .
Name I
I
I
a nucleic acid sequence comprising a sequence in the-table below:
Se,gioaerxt Name 221368PEA node Q
221368PEA node 1S
221368PEA node 19 I
221368PEA node 2 221368PEA node 21 I
22.],3,681?EA_1node 33 221368PEA node 36 Z2136S node 37.
PEA
221368PEA node 39 221368PEA node 4 _.
Z21368_PEA_1node 41 221368PEA node 43 Zs 221368PEA_1 node_45 Z21368_PEA_1 node_53 Z21368_PEA_1 node_56 .~213.68~PEA_1 node58 _ .
221368PEA_1 node_66 .:tW..:i. -221368PEA1 node67 Z21368_PEA_1 node_69 Z21368_PEA_1 node_11 Z21368_PEA_1 node_12 221368PEA_1 node_16 Z21368_PEA_1 node_17 Z21368_PEA_1 node_23_ 221368PEA1 node24 Z21368_PEA_1 node_30 221368PEA_1 node_31 221368PEA1 node38 -Z21368PEA1 node47 Z21368_PEA_1 node_49 221368PEA1 node51 Z21368_PEA_1 _61 node Z21368_PEA_I 68 node 221368PEA1 node7 According to preferred embodiments of the present invvention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name 221368 PEA 1~ P2 Z21368_PEA_1 P15 PEA
I
I' l 221368PEAI P:22 221368PEA1 p~23 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name T59832_T8 a nucleic acid sequence comprising a sequence in the table below:
Segment Name , T59832node1 T59832node22 T59832_node_23 T59832node_24 T59832node29 T59832_node39 T59832node7 T59832node10 T59832node11 T59832node12 T59832node14 T59832_node_16 T59832node_19 T59832node2 T59832_node20 2o TS9832node2S
TS9832node26 TS9832node27 TS9832node28 TS9832node3 TS9832node30 TS9832node31 TS9832node32 TS9832node34 TS9832node3S
TS9832node36 TS9832node37 TS9832node3S
TS9832node4 TS9832nodeS
TS9832node._6 TS9832node8 TS9832node9 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name TS9832_PS
TS9832_P18 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transez~pt Name a nucleic acid sequence comprising a sequence in the table below:
Segment ~1'~lame 241644 1 node 0 PEA
241644 1 node_11 PEA_ Z41644_PEA_1 node_12 241644 1 node 15 PEA
241644 1 node 20 PEA
241644 1 node 24 PEA
241644 1_node_1 PEA_ 241644 1 node 10 PEA
241644 1 node_13 PEA_ 241644 1 node 16 PEA
241644 1 node 17 PEA
241644 1 node 19 PEA
Z41644_PEA_1 node_2 241644 1 node 21 PEA
241644 1 node 22 PEA_ 241644 1 node 23 PEA
241644 1 node_25 PEA_ Z41644_PEA_I node 3 241644 1 node 4 PEA
241644 1 node 6 PEA
241644 1 node 9 PEA_ According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript tam HUMGRPSE T4 ~~~
a nucleic acid sequence comprising a sequence in the table below:
Segment Name .
HUMGRPSE node 0 HUMGRPSE node 2 HUMGRPSE node g-_ HUMGRPSE node 3 HUMGRPSE_rbade_'7 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name GRPSE_P5 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Naive AA155578 PEA_1 T13 .4A155578 PEA 1 T8 a nucleic acid sequence comprising a sequence in the table below:
Segtnettt Name AA155575PEA_1 node_11 AA155578PEA 1 node12 AA155578_PEA_1 node_14 AA155578PEA_1 node_19 AA155578PEA_1 node21 AA155578PEA_1 node_23 AA155578PEA_1 node24 AA 155578PEA_1 node25 AA155578PEA_1 node4 AA15557bPEA_1 node_7 AA155578-PEA_1 node_I5 AA155578PEA_1 node_1S
AA155578PEA_1 node22 AA15557RPEA _1 6 node AA 15 PEA_1 node_8 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protei~a Name AA155575PEA_1 P6 AA15S578PEA_1 P8 AA 155578_PEA_1 P9 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
'T'ransc,,ript Name a nucleic acid sequence comprising a sequence in the table below:
S~egrnsnt Name HSENA78 node 0 HSENA7S nude HSENA78 node 6 HSENA7S node 9 HSENA78 node 3 HSENA78 node 4 HSENA78 node a According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below:
Pxotein Name According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcz-i~t Name , 'I T94936_PEA_1 T2 a nucleic acid sequence comprising a sequence in the table below:
Segnnent Name T94936PEA1 nodeI4 T94936_PEA_1 node_16 T9493 PEA_1 _node_2 6_ T94936_PEA_1 node20 T94936PEA1 node23 T94936PEA1 node0 T94936_PEA_ 1 node_11 T94936_PEA_1 node_13 T94936_PEA_1 node_17 T94936PEAI node6 T94936PEA1 node8 T94936PEA1 node9 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name-T94936_PEA_1 P3 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Trattscript Narne M85491_PEA_1 T20 a nucleic acid sequence comprising a sequence in the table below:
Segment Name M85491 PEA_1 node_0 2b node node node M85491PEA_I 24 node M85491_PEA_1 8 node M85491_PEA_1 9 node node MS5491PEA_1 18 node_ node M85491PEA_1 6 node According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name M8549I PEA_1 PI4 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcripx Name a nucleic acid sequence comprising a sequence in the table below:
Segment Name HSSTROL3 node 6 HSSTROL3 node10 HSSTROL3 node13 HSSTROL3 node15 HSSTROL3 node19 HSSTROL3 node21 HSSTROL3 node HSSTROL3 node25 HSSTROL3 node HSSTROL3 node28 HSSTROL3 node29 HSSTROL3 node11 HSSTROL3 node17 HSSTROL3 node_18 HSSTROL3 node20 HSSTROL3 node According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Proteui .Name:
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name 2s a nucleic acid sequence comprising a sequence in the table below:
Segment Names AY180924_PEA_1 node 3 AY 180924 PEA_ 1 node 0 AY180924_PEA_I node 2 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein N~txte ,. , According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name R75793_PEA_1 T5 a nucleic acid sequence comprising a sequence in the table below:
node node node node R75793_PEA_1 4 node 875793PEA_1 5 node R7S793_PEA_1 6 node_ 875793PEA _1 8 node 875793 PEA_1 node_13 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Proteain Name 875793 PEA_1 P2 875793 PEA_1 PS
875793 PEA_1 P6 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
T~anseript I'Jame HLTMC.A 1 XIA_T 16 HUMCAIh'IA T19 HC1NICA1~ T2ll a nucleic acid sequence comprising a sequence in the table below:
Segment-Name HL1MCA 13~IA_node_0 HLiT4CA 1 XIA_node2 HUIVICAI~IA
node 4 node HUIVICA 1 ~'IA 8 node HUMCAIZIA_node 9 HUMCA1~IA node _18 node HUMCAIXIA-node 55 node HUMCAI XIA. 11 node node node node node HUMCA 1 XIA-node_25 node HUMCA1XIA node29 node_ XIA node HjJMCA 1 XIA 35 node node node node 41 node 43 HUMCA1XIA node HUMCA1XIA node HUMCA1XIA node_49 HUMCA1XIA node51 HUMCAIXIA node_S7 node HUMCA1XIA node62 node HUMCA1XIA node66 HUIvICAIXIA 68 node I-IUMCA1XIA_node70 HUMCA 1 XIA-node_72 XIA node 74 I-IUMCA I XIA_node_76 HLTMCA1XIAnode78 HLTMCA node81 HUMCA 1 node83 XIA
node -.
HUMCA1XTA node87 HUMCA1XIA nodeS9 HLTMCA node 1 XIA_ 91 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Prot~iu Name HLTMCA 1 XIA P 17 -__ According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name' a nucleic acid sequence comprising a sequence in the table below:
Segment Na.nn node 820779 2 -_ node node node node node_ 820779node24 820779node27 820779node28 820779node30 820779node3I
820779node32 820779node1 820779node3 820779nodeIO
820779node1l 820779nodeI4 820779node17 820779node19 820779node20 820779node22 820779node23 820779node25 820779node29 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence according to R20779_P2.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name ' ' a nucleic acid sequence comprising a sequence in the table below:
Segnent Name HSS 100PCB_node_3 HSS 100PCB node 4 HSS100PCB node_5 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence according to HSS100PCB
P3.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name HSCOC4_PEA_1 T3 HSCOC4_PEA_1 T4 HSCOC4 PEA_1 T8 HSCOC4 PEA_1 T1?
HSCOC4 PEA_1 T15 HSCOC~ PEA_1 T21 HSCOC4_PEA_1 T25 HSCOC4_PEA_1 T30 HSCOC4_PEA_1 T31 a nucleic acid sequence comprising a sequence in the table below:
Segmenf Name HSCOC4 _PEA_1 1 node_ HSCOC4 _PEA_1 5 node_ HSCOC4 PEA_1 7 node HSCOC4 _PEA_1 30 node HSCOC4 PEA_1 33 node_ node node node node node node node node node PEA node node node node node node node node node node node node node node HSCOC4_PEA_1 2 node_ node HSCOC4_PEA_ 1 10 node_ HSCOC4_PEA_ 1 12 node_ HSCOC4_PEA_1 14 node_ HSCOC4_PEA_1 17 node_ HSCOC4_PEA _1 19 node_ node HSGOC4_PEA_1 22 node node HSCOC4 PEA_1 29 node node node HSCOC4_PEA_ 1 47 node node node node node node node nude node PEA node PEA node node node node node PEA node PEA node HSCOC4_PEA_1 S3 node_ node node node node PEA node node PEA node node node node node node node node node node node node node HSCOC4 _PEA_1 111 node_ According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below protein Name PEA
PEA
HSCOC4 PEA 1 P4?
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name HUIvTTREFAC PEA 2 T4 a nucleic acid sequence comprising a sequence in the table below:
Segment Name HUMTREFAC PEA node 0 HUI~~ITREFACPEA node 9 HUMTREFAC PEA node 2 HLJMTREFAC PEA node 3 HUMTREFAC PEA node 4 HLThrITREFACPEA node 5 HUMTREFAC PEA node 8 According to preferred embodiments of the present invention, there is provided t2x~~
isolated polypeptide comprising an amino acid sequence in the table below Prat~in Name HUMTREFAC_PEA_2 P7 HUMTREFAC_PEA_2_P 8 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name HUMOSTRO PEA_1 PEA_1_T14 ~ HUMOSTRO PEA_1 PEA_1 T16 ~ HUMOSTRO PEA_1 PEA_1 T30 a nucleic acid sequence comprising a sequence in the table below:
'Segnnent .
Natn~
HUMOSTRO PEA_1 1_node0 PEA_ HUMOSTRO PEA_1 1 10 PEA_ node_ HUMOSTRO_ PEA_1 1 16 PEA_ node_ HUNIOSTRO_PEA_1 1 23 PEA_ nods HUMOSTRO PEA_1 1_node_31 PEA_ PEA_ PEA_ node_43 HLTMOSTRO_PEA_ I 1 3 PEA_ node_ HUMOSTRO_PEA_ 1 1 _5 PEA_ node HUMOSTRO_ PEA_1 1_node_7 PEA_ HCTI~~IOSTROPEA_1 1_node8 PEA_ HUMOSTRO_ PEA_1 1_node_15 PEA_ HUMOSTRO PEA_1 1 _17 PEA_ node HLJMOSTRO PEA_1 1 20 PEA_ node PEA_ PEA_ node _ )-ItTMOSTRO_PEA_1 1 22 PEA_ node HLTMOSTRO PEA_1 1 24 PEA_ node_ HUMOSTRO_PEA_ 1 1 26 PEA_ node HUMOSTRO_PEA_ I 1 PEA_ node HUIvI0STR0_PEA_1 1 PEA_ node HLTMOSTRO_PEA_1 1 29 PEA_ node HUMOSTRO PEA_ I 1 30 PEA_ node_ HUMOSTRO PEA_ 1 1 32 PEA_ node HUMOSTRO_PEA_ I I 34 PEA_ node HUMOSTRO PEA_ 1 1 36 PEA_ node HLTMOSTRO PEA_1 1 37 PEA_ node )-IZTMO STRO 1 1 _3 S
PEA_ PEA_ node HLTMOSTRO_PEA_1 _1 35~
PEA rode HUMOSTRO PEA_ 1 1 _40 PEA_ node HUMOSTRO_PEA_ 1 1 PEA_ node HUMOSTRO PEA_ 1 1 PEA_ node_42 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name HUMOSTRO PEA_I PEA_1 P21 HUMOSTRO_PEA_1 PEA_1 P25 HUhZOSTRO PEA_1 PEA_1 P30 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: Rl 1723 PEA_1 TIS, 811723 PEA_1 T17, R11723_PEA_1 T19, 811723 PEA_I T20, R11723_PEA_1 T5, or R11723_PEA_1 T'6.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a mde having a sequence selected from the group consisting of : 811723 PEA_1 node_13, R11723_PEA_1 node_16, 811723 PEA_1 node_19, 811723 PEA_1 node 2, R11723_PEA_1 node 22, R11723_PEA_1 node 31, 5 R11723_PEA_1 node_10, 811723 PEA_1 node_l l, 811723 PEA_1 node_15, 811723 PEA_1 node_18, 811723 PEA_1 node 20,, _811723 PEA_1 node 21, R11723_PEA_1 node_23, 811723 PEA_1 node 24, 811723 PEA_1 node 25, 811723 PEA_1 node_26, 811723 PEA_1 node 27, 811723 PEA_1 node 28, 811723 PEA_1 node 29, 811723 PEA_1 node 3, R11723_PEA_1 node 30, 10 811723 PEA_1 node 4, R11723_PEA_1 node_5, 811723 PEA_1 node 6, 811723 PEA_1 node_7 or 811723 PEA_1 node 8.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of : 811723 PEA_1 P2, _ _811723 PEA_1 P6, 811723 PEA_1 P7, 15 811723 PEA_ 1 P 13, or 811723 PEA_ 1 P 10.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name, PEA
T4698d-PEA_1T3 T46984_PEA_1T12 PEA
PEA_ PEA_ PEA
T46984_PEA_1T23 PEA_ T46984_PEA_1T32 T46984 _1T34 PEA
T46984PEA_1 T40 T46984_PEA_1 T42 T46984PEA_1 T43 T4698~PEA_1 T46 T469S4_PEA_1 T47 T46984PEA_1 T48 T46984PEA_1 T51 T46984PEA_1 T52 T46984_PEA_1 T54 a nucleic acid sequence comprising a sequence in the table below:
Seg~ient Name T46984_PEA_1 2 node_ T46984_PEA_1 4 node T46984_PEA_1 6 node T46984PEA_1 12 node_ T46984_PEA_1 14 node_ T469R4_PEA_1 25 node_ T46934PEA_1 29 node T46984PEA_1 34 node T46984_PEA_1 _46 node T46984PEA_1 47 node T46984PEA_1 52 node T46984PEA_1 65 node T46984PEA_1 69 node T469S4_PEA_1 _75 node T46984PEA_1 86 node T46984_IV'1.;.A_1 9 node_ T469841'1:A1 13 node T46984~'EA_1 19 node_ node T46984_PEA_1 22 node_ T46984_PEA_1 26 node_ T46984_PEA_1 28 node_ T46984PEA_1 31 node T46984_PEA_1 32 node T46984_PEA_1 38 node_ T46984_PEA_1 39 node T46984_PEA_1 40 node T46984_PEA_1 42 node T46984PEA_1 43 node T46984_PEA_1 48 node T46984_PEA_1 49 node T46984_PEA_1 _S0 node T46984_PEA_1 S1 node T46984_PEA_1 _53 node T46984_PEA_1 S4 node T46984_PEA_I SS
node T46984PEA_1 S7 node T46984-PEA_1 60 node T46984-PEA_1 62 node T46984PEA _1 66 node T46984PEA _1 67 node T46984_PEA_ _70 node T46984PEA _1 71 node T46984PEA _1 72 node T46984_PEA_1 73 node T46984_PEA_1 node-74 T46984 1 node_83 PEA_ T46984_PEA_1 node84 T46984 1 node85 PEA
According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein.
Na><axe PEA
PEA
PEA
PEA
T46984 1 P1~
PEA
PEA
PEA
PEA
PEA
PEA
T46984_PEA_1 P3S
PEA
PEA
PEA
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name T116?8 PEA 1 TS
a nucleic acid sequence comprising a sequence in the table belov,~:
Seg~r~.ent I'Jarn~
T11628_PEA_1_node_7 node T11628_PEA_1 16 node_ node node node node rx~de node node node T11628PEA_1 _14 node node node node node node node T11628_PEA_1 29 node node node T11628PEA_1 33 node .~ s T11628 PEA_1_~~~.:o-le 34 T11628_PEA_1 i~oaie 35 T11628_PEA_1 r~~acfe 36 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Frotex~
Name PEA
T11628_PEA_1 PS
PEA
T11628_PEA_1 P10 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name, M78076_PEA_1 M78076_PEA_1 M78076_PEA_1 TS
M78076_PEA_1 M78076_PEA_1 M7807d 1 PEA_ T23 M78076_PEA_1 M78076_PEA_1 M78076_PEA_1 a nucleic acid sequence comprising a sequence in the table below:
Segn~aent Name node node node node node node node node node node node node node M78076_PEA_1 54 node node node M78076_PEA_1 _3 node node node M78076_PEA_1 _12 node node node M78076_PEA_1 30 node M78076_PEA_1 31 node node node node M78076_PEA_1 42 node node M78076_PEA_1 45 node M78076_PEA_1 node_49 M78076 _PEA_1 node_50 M78076 _PEA_1 node_51 M78076 _PEA_1 node_52 M78076 _PEA_1 node53 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name M78076 PEA_1_P3 M78076_PEA_1 P4 M78076_PEA_1 P12 M78076_PEA_1 P14 M78076_PEA_1_P21 M78076_PEA_1 P24 M78076 _PEA_1 P2 M78076 _PEA_1 P25 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name HSMUC1A PEA_1 T12 HSMUC 1 A PEA_T26 HSMCTC1A_PEA_1T28 HSMUC1A PEA_1 T29 HSMUC1A_PEA_1 T30 HSMLTC 1 A_PEA_T31 HSMUC1A_PEA_1 T33 HSMUC1A_PEA_1 T34 4a HSMUC1A_ PEA_1 T35 HSMIJC1A_PEA_1 T36 HSIvfUCIA_PEA_1 T40 HSMLTC _PEA_1 T42 HSMUC1A _PEA_1 T43 HSMUC PEA_1 T47 a nucleic acid sequence comprising a sequence in the table below:
Se;rrn~nt Name 1 A node HSMUCIA PEA_1 14 node_ node 1 A_PEA_ node HSMUC1A_ PEA_1 35 node HSMCTC1A PEA_1 38 node HSMUC1A_ PEA_1 3 node 1 A node PEA node HSMUC1A_ PEA_1 6 node HSMUCIA PEA_1 7 node HSMUCIA_PEA_ 1 17 node_ HSMf.JCIA_PEA_1 _18 node HSMUC1A_ PEA_1 20 node HSMUCIA_PEA_ 1 21 node HSMUCIA_ PEA_1 23 node HSMCJC1A_PEA_ 1 26 node HSMUC PEA_1 27 1 A_ node node HSMUC 1 A PEA 1 node 34 HSMUC 1 A PEA 1 node 36 HSMCJC1A_PEA_1 node 37 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name HSMLJC1APEA 1 P~5 -. _ HSMUC1A PEA 1 P?9 PEA
_ _ -. _ According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSMLJC 1 A PEA_ 1 P63, comprising a first amino acid sequence being at least 90 % homologous to MTPGTQSPFFLLLLLTVLTWTGSGHASSTPGGEkETSATQRSSV corresponding to amino acids 1 - 45 of MUC 1 HUMAN, which also corresponds to amino acids 1 - 45 of HSMUC1A PEA_1 P63, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence so EEEVSADQVSVGASGVLGSFKEARNAPSFLSWSFSMGPSK corresponding to amino acids 46 - 85 of HSI~fUCIA PEA_1 P63, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSIvfUCIA_PEA_1 P63, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence EEEVSADQVSVGASGVLGSFKEARNAPSFLSWSFSMGPSK in HSMUC1A_PEA_1 P63.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTILHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAKItACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNR1'HVPVVWPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVGDVFELNFMNVKF'SSGYYDFLVEVEGDN
RYIANTVELRVKISTEVGITNVDLSTVDKDQSIAPKTTRVTYPAKAKGTFIADSHQNFAL
FFQLVDVNTGAELTPHQTFVRLHNQKTGQEVVFVAEPDNhNVYKFELDTSERKIEFDS
ASGTYTLY'LIIGDATLKNPILWNV corresponding to amino acids 1 - 498 of RIB2 HUMAN, which also corresponds to amino acids 1 - 498 of T46984_PEA_1 P~, 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 VCA corresponding to amino acids 499 - 501 of T46984_PEA_1 P2, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P3, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAKKACT1'IRSNLDPSNVDSLF1'AAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIS.'HAVAALSGFGLPLASQEALSALTARLSkEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIhEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRY'HVPVVVVPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAhSVASRATVLQKTSFTPVGDVFELNFMNVhF'SSGYYDFLVEVEGDN
RYLANTVELRVILISTEVGITNVDLSTVIaKDQSIAPItTTRVTYPAKAKGTFIADSHQNFAL
FFQLVDVNTGAELTPHQ corresponding to amino acids 1 - 433 of RIB2_HL1MAN, which also corresponds to amino acids 1 - 433 of T46984_PEA_1 P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ICHIWKLIFLP corresponding to amino acids 434 - 444 of T46984_PEA_I P3, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984 PEA_1 P3, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ICHIWKLIFLP in T46984 PEA_1 P3.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P10, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLhASLDRPFTNLESAFIrSIVGLSSL
GAQVPDAKKACT1'IRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAV SEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRYHVPV VVVPEGSASDTHEQ.AILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVGDVFELNFMNVKFSSGYYDFLVEVEGDN
RYIANTVELRVKISTEVGITNVDLSTVDKDQSIAPKTTRVTYPAKAKGTFIADSHQNFAL
FFQLVDVNTGAELTPHQTFVRLHNQKTGQEVVFVAEPDNhNVYKFELDTSERKIEFDS
ASGTYTLYLIIGDATLKNPILVVNV corresponding to amino acids 1 - 49S of RIB2 I-iUNIAN, which also corresponds to amino acids 1 - 498 of T46984 PEA_1 P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence LMDQK corresponding to amino acids 499 - 503 of T46984_PEA_1 P10, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984_PEA_1 P10, comprising a polyypeptide 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 LMDQK
in T46984 PEA 1 P 10.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P1 l, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKA.SLDRPFTNLESAFYSIVGLSSL
GAQVPDAKKACTI'IRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFShKNFESLSEAFSVASAAAVLSHNRYHVPVVVVPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVGDVFELNFMNVKFSSGYYDFLVEVEGDN
RYIANTVELRVKISTEVGITNVDLSTVDILDQSIA.PKTTRVTYPAKAKGTFIADSHQNFAL
FFQLVDVNTGAELTPHQTFVRLHNQKTGQEVVFVAEPDNKNVYKFELDTSERKIEFDS
ASGTYTLYYLIIGDATLKNPILWNVADWThFPEEEAPS'TVL,SQNLFTPKQEIQHLFREPEK
RPPTWSNTFTALILSPLLLLFALWIRIGANVSNFTFAPSTIIFHLGHA.4MLGLMYVYWT
QLNMFQTLKYLAILGSVTFLAGNRMLAQQAVIt.R corresponding to amino acids 1 - 6~8 of RIB2 I-IUIvIAN, which also corresponds to amino acids 1 - 628 of T46984 PEA_1 P11.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P 12, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKASLDRPFTNLESAFIrSIVGLSSL
GAQVPDAKKACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETILDLLLAAVVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTAI,f'V'AAT'YKLMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRYHVI' ~'VWPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVG'f~VFELNFMN corresponding to amino acids 1 - 338 ofRIB2_HZJMAN, which also corresponds to amino acids 1 - 33S of T4698~1 PEA_1 P12, and a second amino acid sequence being at least 70%, optionally at least SO%, preferably at least SS%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence SQDLH corresponding to amino acids 339 -343 of T4698~ PEA_1 P12, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T469S4 PEA_1 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90°~'o and most preferably at least about 95% homologous to the sequence SQDLH in T469S4 PEA 1 P 12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P21, 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 M
corresponding to amino acids 1 - 1 of T46984_PEA_1 P21, and a second amino acid sequence being at least 90 % homologous to KACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSSVTQI~.~iAV
AALSGFGLPLASQEALSALTARLSKEE'TVL,ATVQALQTASHLSQQADLRSIVEEIEDLVA
RLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNAIFSKKNFES
LSEAFSVASAAAVLSHNRYHVPVWVPEGSASDTHEQAIL,RLQVTNVLSQPLTQATVKL
EHAhSVASRATVLQILTSFTPVGDVFELNFMNVkFSSGYYYDFLVEVEGDNRYIANTVEL
RVKISTEVGITNVDLSTVDKDQSIAPhTTRVTYPAkAhGTFIADSHQNFALFFQLVDVNT
GAELTPHQTFVRLHNQKTGQEV VFVAEPDNKNVYKFELDTSERKIEFDSASGTYTLY~LII
GDATLhNPILWNVADVVIKFPEEEAPSTVLSQNLFTPKQEIQHLFREPEKRPPTVVSNTF
TAL.u,SPLLLLFAL~~~IRIGANVSNFTFAPSTIIFHLGHAAMLGLMYVI'WTQLNMFQTLK.Y
LAILGSVTFLAGNRMLAQQAVhRTAH corresponding to amino acids 70 - 631 of RIB2_I-ICTMAN, which also corresponds to amino acids 3 - 563 of T46984 PEA_1 P21, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T4698~ PEA_1 P27, comprising a first amino acid 'segue nce being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKI~VERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDA_KKACTI'IRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHL,SQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKIMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRYHVPVVIIVPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVkLEHAhSVASRATVLQhTSFTPVGDVFELNFMNVKFSSG~'~'DFLVEVEGDN
RYIANTVELRVKISTEVGITNVDLSTVDKDQSIAPKTTRVTYPAKAKGTFIADSHQNFA
corresponding to amino acids 1 - 415 of RIB?_HUMAN, which also corresponds to amino acids 1 - 415 of T46984_PEA_1 P27, 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 FGSGLVPMSPTSLLLLARLYFTWDMLLCWDSCMSTGLSSTCSRP corresponding to amino acids 416 - 459 of T46984 PEA_1 P27, wherein said first amino acid sequence and second amino acid sequence axe contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984_PEA_1 P27, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence FGSGLVPMSPTSLLLLARLYFTWDMLLCWDSCMSTGLSSTCSRP in T46984 PEA 1 P27.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1_P32, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTH~.'LTKHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAhHACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQII'~I-IAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFSkKNFESLSEAFSVASAAAVLSHNR1'HVPVWVPEGSASDTHEQAIL,RLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVGDVFELNFMNVKFSSGYYDFLVEVEGDN
RYIANTVE corresponding to amino acids 1 - 364 of R1B2_HUMAN, which also corresponds 5 to amino acids 1 - 364 of T46984_PEA_1 P32, 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 GQVRWLTPVIPALWEAKAGGSPEVRSSIL,AWPT corresponding to amino acids 365 - 397 of T46984_PEA_I P32, wherein said first amino acid sequence and second amino acid 10 sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T469S4_PEA_1 P32, 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 15 GQVRWLTPVIPALWEAKAGGSPEVRSSILAWPT in T46984 PEA_1 P32.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P34, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKASLDRPFTNLESAFYSIVGLSSL
20 GAQVPDAKKACTI'IRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSILEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQWQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRYHVPVVVVPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVG corresponding to amino acids 1 - 329 of 25 RIB2_I-ItJMAN, which also corresponds to amino acids 1 - 329 of T4698~
PEA_1 P34.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P35, comprising a first amino acid sequence being at least 90 % homologous to GAQVPDAKhACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQII'I3AVAALSGFGLPLASQEALSALTARLSKEET'VLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVI'LQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNR.YHVPWVVPEGSASDTHEQAI corresponding to amino acids 1 - 2S7 of RIB? HUMAN, which also corresponds to amino acids 1 -287 of T469S4_PEA_1 P35, 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 GCWPSRQSREQHISSRRKNIEILKTECQEKESRTIHSMR.RIi.MEKKNFI corresponding to amino acids 2S8 - 334 of T469S4_PEA_1_P35, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T469S~ PEA_1 P35, comprising a polypeptide being at least 70°io, optiorally at least about SO%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GCWPSRQSREQHISSRRKMEILKTECQEKESRTIHSMRRI~MEKKNFI in T46984 PEA 1 P35.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P38, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHY'I,TKHDVERLIkASLDRPFTNLESAFYSIVGLSSL
GAQVPDAKKACTYIRSNLDPSIVVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEAL corresponding to amino acids 1 - 145 of RIB2 HUMAN, which also corresponds to amino acids I - 145 of T4698~ PEA_I P38, and a second amino acid sequence being at least 70%, optionally at least SO%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MDPDWCQCLQLHFCS corresponding to amino acids 146 - 160 of T46984_PEA_1 P38, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984_PEA_1 P3S, 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 MDPDWCQCLQLHFCS in T46984_PEA_1 P3S.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T4698~ PEA_1 P39, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSST~rFLLALTIIASTWALTPTI-IY~LTILHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAI:KACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAV SEDSS
VTQIY'HAVAALSGFGLPLASQEALSALTARLSKEETVLA corresponding to amino acids 1 -160 of RIB2 HTJMAN, which also corresponds to amino acids 1 - 160 of T46984_PEA_1 P39.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T469g4 PEA_1 P45, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTIiY'LTKHDVERLILASLDRPFTNLESAFI'SIVGLSSL
GAQVPDAhKACTI'IRSNLDPSNVDSLFYAAQASQALSGCE corresponding to amino acids 1 - 101 of RIB2_I-ILT(vIAN, which also corresponds to amino acids 1 - 101 of T46984_PEA_1 P45, 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°,~0 homologous to a polypeptide having the sequence NSPGSADSIPPVPAG corresponding to amino acids 102 - 116 of T4698~ PEA_1 P45, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984 PEA_1 P45, 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 NSPGSADSIPPVPAG in T46984 PEA 1 P45.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P46, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAh corresponding to amino acids 1 - 69 of RIB2_HUMAN, which also corresponds to amino acids 1 - 69 of T46984 PEA_1 P46, 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 NSPGSADSIFPVPAG corresponding to amino acids 70 - 84 of T46984_PEA_1 P46, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is pnwided an isolated polypeptide encoding for a tail of T46984_PEA_ 1 P46, 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 NSPGSADS1PPVPAG in T46984 PEA 1 P46.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T11628_PEA_1 P2, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MGLSDGEWQLVLNVWGhVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDE
corresponding to amino acids 1 - 55 of T11628 PEA_1 P2, and a second amino acid sequence being at least 90 % homologous to MKASEDLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKII'VKYLEFISECIIQV
LQSKHPGDFGADAQGAMMtALELFRKDMASNYKELGFQG corresponding to amino acids 1 - 99 of QBVW~H6, which also corresponds to amino acids 56 - 154 of T11628_PEA_1 P2, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of T11628_PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFhGHPETLEKFDKFKHLKSEDE of T11628 PEA 1 P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T1162S_PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to I\~IKKASEDLKKHGATVLTALGGIL,KKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQV
LQSKHPGDFGADAQGAMNIiAL,ELFRKDMASNYKELGFQG corresponding to amino acids 56 - 154 of M5.'G_HIIMAN_V 1, which also corresponds to amino acids 1 -99 of T1162S PEA 1 P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T11628_PEA_1 P7, comprising a first amino acid sequence being at least 90 % homologous to MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMK
ASEDLKKHGATVLTALGGILKKKGI-~IEAEIKPLAQSHA.TKHKIPVKYLEFIS~ECIIQVLQ
SKHPGDFGADAQGA.MNK corresponding to amino acids 1 - 134 of IVtYG_HUM.AN V 1, which also corresponds to amino acids 1 - 134 of T11628_PEA_1 P7, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90°~o and most preferably at least 95% homologous to a polypeptide having the sequence G
corresponding to amino acids 135 - 135 of T11628_PEA_1 P7, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T11628_PEA_1 P10, comprising a first amino acid sequence being at least 70°~0, 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 MGLSDGEW QLVLN VWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDE
corresponding to amino acids 1 - 55 of TI 1628_PEA_1 P10, and a second amino acid sequence being at least 90 % homologous to MKASEDLKILHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQV
LQSKHPGDFGADAQGAMNKALELFRKDMASN~'KELGFQG corresponding to amino acids 1 - 99 of Q8WVH6, which also corresponds to amino acids 56 - 154 of T11628_PEA_1 P10, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polygeptide encoding for a head of T11628_PEA_1 PIO, 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 5 MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPET'I,EkFDKFKHLKSEDE of T11628 PEA 1 P10.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076_PEA_I P3, comprising a first amino acid sequence being at least 90 % homologous to CGRLTLHRDLRTGRWEPDPQRSRRCLRDPQRVLEYCRQMYPELQIARVEQATQAIPME
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQGESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGKVTPTPRPTDGV
15 DIYFGMPGEISEHEGFLRAItMDLEERRMRQINEVIvIREWAMA.DNQSKNLPKADRQALN
EHFQSILQTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYLRAEQK.EQRHTLRHYQHVAAVDPEKAQQMRFQVHTHLQVIEERVNQSLGLLD
QNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKD corresponding to amino acids I - 517 of APP1_HUMAN, which also corresponds to amino acids 1 -517 of 20 M78076_PEA_1 P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence GE corresponding to amino acids 51 S - 519 of M78076_PEA_1 P3, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
25 According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M7S076_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
CGRLTLHRDLRTGRWEPDPQRSRRCLRDPQRVLEIfCRQMYPELQIARVEQATQAIPME
30 RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRR_HQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVE1~1CCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGK'~~Tj~TPRPTDGV
DIYFGMPGEISEHEGFLRAI~IvIDLEERI2MRQINEVMREWAMADNQSKNL h'KADRQALN
EHFQSILQTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQl~Li'PQAERVLL
ALRRYLRAEQKEQRHTLRHYQHVAAVDPEKAQQMRFQVHTHLQVIEERVNQSLGLLD
QNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKDD1'PMTLPKG
corresponding to amino acids 1 - 526 of APP 1 ~IUMAN, which also corresponds to amino acids 1 - 526 of M78076_PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence ECLTVNPSLQIPLNP corresponding to amino acids 527 - 541 of M78076_PEA_1 P4, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ECLTVNPSLQIPLNP in M7S076_PEA_1 P4.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076_PEA_1 P12, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGhVTPTPRPTDGV
DIYFGMPGEISEHEGFLRAKN>DLEERRMRQINE~WREWAIViADNQSKNLPKADRQALN
EHFQSII,QTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYI,RAEQKEQRHTLRHYQHVAAVDPEKAQQMRFQVHTHLQVIEERVNQSLGLLD
QNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKDDTPMTLPKG
corresponding to amino acids 1 - 526 of APP1 HUMAN, which also corresponds to amino acids 1 - 536 of M78076_PEA_1 P12, and a second amino acid sequence being at least 70%, b2 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 ECVCSKGFPFPLIGDSEG corresponding to amino acids 527 - 544 ofM78076_PEA_1 P12, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°'0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ECVCSKGFPFPLIGDSEG in M78076 PEA 1 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076_PEA_1 P14, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
CGRLTLHRDLRTGRWBPDPQRSRRCLRDPQRVLEYCRQMYPELQIARVEQATQAIP1~IE
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEf'VCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAWGKVTPTPRPTDGV
DIYFGMPGEISEHEGFLRAI~LEERRIVfRQINEVMREWAMADNQSKNLPKADRQALN
EHFQSILQTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYLRAEQKEQRHTLRHYQHVAAVDPEKAQQMRFQVHTHLQ~~ERVNQSLGLLD
QNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKDDTPMTLPIiGST
EQDAASPEKEKMNPLEQYERKVNASVPRGFPFHSSEIQRDEL corresponding to amino acids 1 - 570 of APP 1 HUMAN, which also corresponds to amino acids 1 - 570 of M78076_PEA_1 P14, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence VRGGTAGYLGEETRGQRPGCDSQSHTGPSKKPSAPSPLPAGTSWDRGVP corresponding to amino acids 571 - 619 of M78076_PEA_1_P14, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P14, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRGGTAGYLGEETRGQRPGCDSQSHTGPSKKPSAPSPLPAGTSWDRGVP in M78076 PEA 1 P14.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076_PEA_1 P21, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
CGRLTLHRDLRTGRWEPDPQRSRRCLRDPQRVLEYCRQMYPELQIARVEQATQAIPME
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSVVPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGILVTPTPRPTDGV
DIYFGMPGEISEHEGFLRAILMDLEERRMRQINEVMREWAMADNQSKNLPKADRQALN
E corresponding to amino acids 1 - 352 of APP1 HUMAN, which also corresponds to amino acids 1 - 352 of M78076_PEA_1 P21, and a second amino acid sequence being at least 90 homologous to AERVLLALRRYLRAEQKEQRHTLRHYQHVAAVDPEILAQQMRFQVHTHLQVIEERVNQ
SLGLLDQNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKDDTPMT
LPKGSTEQDAASPEKEkMNPLEQYERhVNASVPRGFPFHSSEIQRDELAPAGTGVSREA
VSGLLII~~GAGGGSLIVLSMLLLRRhhPYGAISHGVVEVDPMLTLEEQQLRELQRHGYE
NPTYRFLEERP corresponding to amino acids 406 - 650 of APP1 HUMAN, which also corresponds to amino acids 353 - 597 of M78076_PEA_1 P21, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding fox an edge portion of M78076_PEA_1 P21, 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 EA, having a structure as follows: a sequence starting from any of amino acid numbers 353-x to 352; ~,aul ending at any of amino acid numbers 353+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M7807~ PEA_1 P24, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
CGRLTLHRDLRTGRWEPDPQRSRRCLRDPQRVLEYCRQMYPELQIAR.VEQATQAIPME
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGKVTPTPRPTDGV
DIYF'GMPGEISEHEGFLRAICMDLEERRMRQINEVMREWAMADNQSKNLPKADRQALN
EHFQSIL,QTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYLRAEQKEQRHTLRHYQHVAAVDPEIkAQQMRFQVHTHLQVIEERVNQSLGLLD
QNPHLAQELRPQI corresponding to amino acids 1 - 481,of APP1HUMAN, which also corresponds to amino acids 1 - 481 of M78076 PEA_1 P24, 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 RECLLPWLPLQISEGRS corresponding to amino acids 482 - 498 of M78076 PEA_1 P24, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P24, 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 RECLLPWLPLQISEGRS in M78076 PEA_1 P24.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076 PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGKVTPTPRPTDGV
DIYFGMPGEISEHEGFLRAI~MDLEERRMRQINEVMREWAMADNQSKNLPKADRQALN
ALRRYLRAEQKEQRHTLRHYQHVAAVDPEKAQQMRFQV corresponding to amino acids 1 - 449 of APP1 HLJIvIAN, which also corresponds to amino acids 1 - 449 of M78076_PEA_1 P2, 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%
10 homologous to a polypeptide having the sequence LTSFQLPNAPLFLRRPRLRLFSCPLDPLSVSWTPSYPLNTASLPLPSLSAQLPDPETWTLT
CCVFDPCFLALGFLLPPPSILCSVVPWIFTAFPRIVFFFFFFLRQVLALSPRQESSVRSWLIAT
STSWVQAIL,LPQPLE corresponding to amino acids 450 - 588 of M78076 PEA_1 P2, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a 15 sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076 PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about S5%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence CCVFDPCFLALGFLLPPPSILCSVPWIFTAFPRNFFFFFFLRQVLALSPRQESSVRSWLIAT
STSVi~VQAILLPQPLE in M78076_PEA_1 P2.
According to preferred embodiments of the present invention, there is provided an 25 isolated chimeric polypeptide encoding for M78076 PEA_1 P25, comprising a first amino acid sequence being at least 90 % homologous to n~IGPASPAARGLSRRPGQPPLP LLLPLLLLLLRAQPAIGSLAGGSPGA.AEAPGSAQVAGL
CGRLTLHRDLRTGRVVEPDPQRSRRCLRDPQRVL,EYCRQMYPELQIARVEQATQAIPME
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERIvIDQCESSTRRHQ
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAWGKVTPTPRPTDGV
DIYFGIvfPGEISEHEGFLRAI~MDLEERRNiRQINEVIviREWAIVIADNQSKNLPKADRQALN
EHFQSILQTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYLRAEQILEQRHTLRH~'QHVAAVDPEhAQQMRFQ corresponding to amino acids 1 - 448 of APPIHiCIMAN, which also corresponds to amino acids 1 - 448 of M78076_PEA_1 P25, 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°'0 homologous to a polypeptide having the sequence PQNPNSQPRA.AGSLEVIISHPFVRRLEILISPFQFQNSIPKNSQIVPAASPRGTSSP
corresponding to amino acids 449 - 505 of M78076_PEA_1 P25, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P25, comprising a polypeptide being at least 70°'0, 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 PQNPNSQPRAAGSLEVIISHPFVRRLEILISPFQFQNSIPfNSQIVPAASPRGTSSP in M78076 PEA 1 P25.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M85491 PEA_1 P13, comprising a first amino acid sequence being at least 90 % homologous to MALRRLGAALLLLPLLAAVEETLMDSTTATAELGWMVHPPSGWEEVSGYDENMNTIR
TYQVCNVFESSQNNWLRTKFIRRRGAHRIHVEIVIILFSVRDCSSIPSVPGSCKETFNLYYY
EADFDSAThTFPNWMENPWVIiVDTIAADESFSQVDLGGRVMICINTEVRSFGPVSRSGF
YLAFQDYGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVAARGSCIANAEEVD
VPIKLYCNGDGEWLVPIGRCMCItAGFEAVENGTVCRGCPSGTFhANQGDEACTHCP1N
SRTTSEGATNCVCRNGYYRADLDPLDMPCTTIPSAPQAVISSVNETSLI~~ILEWTPPRDSG
GREDL~~.'I~1IICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYISDLLAHTQYTFEIQAV
NGVVTDQSPFSPQFASVNITTNQAAPSAVSIIVIIQVSRTVDSITLSWSQPDQPNGVILDI~L
QYYEIk corresponding to amino acids 1 - 476 of EPB2 HU1~~IAN, which also corresponds to amino acids 1 - 476 of M85491 PEA_1 P13, and a second amino acid sequence being at least 70%, optionally at least SO%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VPIGWVLSPSPTSLRAPLPG corresponding to amino acids 477 - 496 of M85491 PEA_1 P13, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M85491 PEA_1 P13, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VPIGWVLSPSPTSLRAPLPG in MS5491 PEA 1 P13.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M85491 PEA_1 P14, comprising a first amino acid sequence being at least 90 % homologous to MALRFLGAALLLLPLLAAVEETLMDSTTATAELGWI\~IVHPPSGWEEV SGYDENMNTIR
TYQVCNVFESSQNNW~LRTKFIRR.RGAIiRIHVEMKFSVRDCSSIPSVPGSCILETFNLYYY
EADFDSATKTFPNWMENPWVIkVDTIAADESFSQVDLGGRVMK1NTEVRSFGPVSRSGF
Y'LAFQDYGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVVAARGSCIANAEEV'D
VPIKLYCNGDGEWLVPIGRCMCKAGFEAVENGTVCR corresponding to amino acids 1 -270 of EPB2 HLJN1AN, which also corresponds to amino acids 1 - 270 of M85491 PEA_1 P14, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence ERQDLTMLSRLVLNSWPQA~IILPPQPPKVL,EL corresponding to amino acids 271 - 301 of M85491 PEA_1 P14, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M85491 PEA_1_P14, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ERQDLTMLSRLVLNS~VPQMILPPQPPKVL,EL in M85491 PEA_1 P14.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSSTROL3 P4, comprising a first amino acid sequence Ia;ing at least 90 % homologous to MAPAA~%~n..RSAAARALLPPMLLLLLQPPPLLARALPPDVHLILHAERRGPQPW~LAALPSS
PAPAPA1 QLAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRiZRFP
WQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEGRADIIv>mFARYW corresponding to amino acids 1 - 163 of MM 11 HC>MAN, which also corresponds to amino acids I -163 of HSSTROL3_P4, a bridging amino acid H corresponding to amino acid 164 of HSSTROL3_P4, a second amino acid sequence being at least 90 °'o homologous to GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFYTFRYPLSLSPDDCRGVQHLYGQP~VPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGF~rWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQGAQYWVYDGEKPVLGPAPLTELGLVRFPVHAALVWGPE
KNHIYFFRGRDYWRFHPSTRRVDSPVPRRATDWRGVPSEIDAAFQDADG corresponding to amino acids 165 - 445 of MMI I HLJIVIAN, which also corresponds to amino acids 165 - 445 of HSSTROL3_P4, 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 ALGVRQLVGGGHSSRFSHLVVAGLPHACHRKSGSSSQVLCPEPSALLSVAG
corresponding to amino acids 446 - 496 of HSSTROL3_P4, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSSTROL3 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ALGVRQLVGGGHSSRFSHLVVAGLPHACHRKSGSSSQVLCPEPSALLSVAG in HSSTROL3 P4.
According to preferred embodiments of the present invertion, there is provided an isolated chimeric polypeptide encoding for HSSTROL3_P5, comprising a first amino acid sequence being at least 90 % homologous to MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWHAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRIL,RFP
WQLVQEQVRQTMAEALKVWSDVTPLTFT'EVHEGRADIIVImFARYW corresponding to amino acids 1 - 163 of MM 11 HUMAN, which also corresponds to amino acids 1 -163 of HSSTROL3_P5, a bridging amino acid H corresponding to amino acid 164 of HSSTROL3 P5, a second amino acid sequence being at least 90 % homologous to GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAF1'TFRYPLSLSPDDCRGVQHLI'GQPWPTVTSRTP.ALGPQAG>DTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQ corresponding to amino acids 165 - 358 of MM11 HUMAN, which also corresponds to amino acids 165 - 358 of HSSTROL3_P5, 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 ELGFPSSTGRDESLEHCRCQGLHK corresponding to amino acids 359 - 382 of HSSTROL3_P5, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSSTROL3 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ELGFPSSTGRDESLEHCRCQGLHK in HSSTROL3_P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSSTROL3_P7, comprising a first amino acid sequence being at least 90 % homologous to MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWHAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRILRFP
WQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEGRADIMIDFARYW corresponding to amino acids 1 - 163 of MM 11 HUMAN, which also corresponds to amino acids 1 -163 of HSSTROL3_P7, a bridging amino acid H corresponding to amino acid 164 of HSSTROL3_P7, a second amino acid sequence being at least 90 % homologous to GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGH~TL,G
LQHTTAAhALMSAFYTFRYPLSLSPDDCRGVQHL1'GQPWPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFhAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQG corresponding to amino acids 165 - 359 of MM11 HU~~iA.N, which also corresponds to amino acids 165 - 359 of HSSTROL3_P7, 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 TTGVSTPAPGV corresponding to amino acids 360 - 370 of HSSTROL3_P7, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an 10 isolated polypeptide encoding for a tail of HSSTROL3 P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°'0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence TTGVSTPAPGV in HSSTROL3 P7.
According to preferred embodiments of the present invention, there is provided an 15 isolated chimeric polypeptide encoding for HSSTROL3_P8, comprising a first amino acid sequence being at least 90 % homologous to MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWI-IAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRILRFP
WQLVQEQVRQTMAEALhVWSDVTPLTFTEVHEGRADIMI)7FARYW corresponding to 20 amino acids 1 - 163 of MM11 HLTIVIAN, which also corresponds to amino acids 1 - 163 of HSSTROL3 P8, a bridging amino acid H corresponding to amino acid 164 of HSSTROL3_P8, a second amino acid sequence being at least 90 % homologous to GDDLPFDGPGGILAHAFFPIiTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFYTFRYPLSLSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN
25 EIAPLE corresponding to amino acids 165 - 286 of MM 11 HUI\~IAN, which also corresponds to amino acids 165 - 286 of HSSTROL3 P8, and a third amino acid sequence being at least 70°~0, 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 VRPCLPVPLLLCWPL corresponding to amino acids 287 - 301 of HSSTROL3_P8, wherein 30 said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to lyreferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSSTROL3 P8, comprising a polypeptide being at least 70%, optionally- at least about 80%, preferably at least about 85°./0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRPCLPVPLLLCWPL in HSSTROL3 P8.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSSTROL3_P9, comprising a first amino acid sequence being at least 90 % homologous to IvfAPAAWLRSAAARALLPPMLLLLLQPPPLLAR.ALPPDUHHI_,HAERRGpQpWHAALpSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSA.RNRQK corresponding to amino acids 1 -96 of MM11 HUMAN, which also corresponds to amino acids 1 - 96 of HSSTROL3_P9, a second amino acid sequence being at least 90 % homologous to RILRFPWQLVQEQVRQTMAEALKVW SDVTPLTFTE VHEGRADII'TIDFARYW
corresponding to amino acids 113 - 163 of MM 11 HUMAN, which also corresponds to amino acids 97 - 147 of HSSTROL3_P9, a bridging amino acid H corresponding to amino acid 148 of HSSTROL3 P9, a third amino acid sequence being at least 90 % homologous to GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFhTFRYPLSLSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQG corresponding to amino acids 165 - 359 of 1\w11 1 HUMAN, which also corresponds to amino acids 149 - 343 of HSSTROL3 P9, and a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence TTGVSTPAPGV corresponding to amino acids 344 - 354 of HSSTROL3_P9, wherein said first amino acid sequence, second amino acid sequence, bridging amino acid, third amino acid sequence and fourth amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of HSSTROL3_P9, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about ?0 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 ILR, having a structure as follows: a sequence starting from any of amino acid numbers 96-x to 96; and ending at any of amino acid numbers 97+ ((n-2) - x), in which x varies from 0 to n-?.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSSTR0L3 P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence TTGVSTPAPGV in HSSTROL3 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for AY180924 PEA_1 P3, comprising a first amino acid sequence being at least 90 % homologous to 1\~ILNVSGLFVLLCGLLVSSSAQEVLAGVSSQLLN corresponding to amino acids 1 - 33 of LATH_HUMAN, which also corresponds to amino acids 1 - 33 of AY180924 PEA_1_P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GETVLLWVMQNPEPMPVIkFSLAKYLGHNEHY
corresponding to amino acids 34 - 64 of AY 180924_PEA_1 P3, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of AI'180924_PEA_1 P3, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GETVLLWVMQNPEPMPVkFSLAKYLGI-LNEH1T in AY1809?4_PEA_1 P3.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for 875793 PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MItFLAVLVLLGVSIFLVSAQNPTTAAPADTYPATGPADDEAPDAETTAAATTATTAAPT
TATTAASTTARItDIP corresponding to amino acids 1 - 74 of Q96DR8, which also corresponds to amino acids 1 - 74 of 875793 PEA_1 P2, 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 AP
corresponding to amino acids 75 - 76 of R75793_PEA_1 P?, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HCTI\~iCAI~ P14, comprising a first amino acid sequence being at least 90 % homologous to MEPWSSRWKTICRWLWDFTVTTLALTFLFQAREVRGAAPVDVLKALDFHNSPEGISKTT
GFCTNRKNSKGSDTAl'RVShQAQLSAPThQLFPGGTFPEDFSILFTVItPKKGIQSFLLSIY
NEHGIQQIGVEVGRSPVFLFEDHTGKPAPEDYPLFRTVNIADGKWHRVAISVEIkKTVTM
IVDCKKKTTHPLDRSERAIVDTNGITVFGTRIL,DEEVFEGDIQQFLITGDPKAAYDYCEH
YSPDCDSSAPKAAQAQEPQIDEYAPEDIIEYDYEYGEAEYKEAESVTEGPTVTEETIAQT
EANIVDDFQEYNYGTMESIrQTEAPRHV SGTNEPNPVEEIFTEEYLTGEDYDSQRKNSED
TLY'ENKEIDGRDSDLLVDGDLGEYDFYEYKEYEDKPTSPPNEEFGPGVPAETDITETSIN
GHGAYGEKGQKGEPAVVEPGMLVEGPPGPAGPAGIMGPPGLQGPTGPPGDPGDRGPPG
RPGLPGADGLPGPPGTMLMLPFR1'GGDGSKGPTISAQEAQAQAILQQARIALRGPPGPM
GLTGRPGPVGGPGSSGAhGESGDPGPQGPRGVVQGPPGPTGKPGKRGRPGADGGRGMP
GEPGAI~GDRGFDGLPGLPGDKGHRGERGPQGPPGPPGDDGMRGEDGEIGPRGLPGEAG
PRGLLGPRGTPGAPGQPGMAGVDGPPGPKGNMGPQGEPGPPGQQGNPGPQGLPGPQG
PIGPPGEKGPQGKPGLAGLPGADGPPGHPGILEGQSGEKGALGPPGPQGPIGYPGPRGVIL
GADGVRGLKGSKGEKGEDGFPGFKGDMGLKGDRGEVGQIGPRGEDGPEGPKGRAGPT
GDPGPSGQAGEKGKLGVPGLPGYPGRQGPKGSTGFPGFPGANGEKGARGVAGKPGPR
GQRGPTGPRGSRGARGPTGKPGPKGTSGGDGPPGPPGERGPQGPQGPVGFPGPhGPPGP
PGKDGLPGHPGQRGETGFQGKTGPPGPGGWGPQGPTGETGPIGERGHPGPPGPPGEQG
LPGAAGKEGAKGDPGPQGISGKDGPAGLRGFPGERGLPGAQGAPGLKGGEGPQGPPGP
V corresponding to amino acids 1 - 1056 of CA1B_HUMAN_V5, which also corresponds to amino acids 1 - 1056 of HW~ICAI~iIA_P14, and a second amino acid sequence being at least 70%, optionallyy at least 80%, preferably at least 85%, more preferably at least 90°ro and most preferably at least 95°,~o homologous to a polypeptide having the sequence VSMMIINSQTIMWNYSSSFITLML corresponding to amino acids 1057 - 1081 of HLTMCA1XIA_P14, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HUMCA 1 ~ P 14, comprising a polypeptide being at least 70%, options lly 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 VSI~wIIINSQTI1'SSSFITLML in HCTMCA1.~IA P14.
According to preferred embodiments of the present invent ion, there is provided an isolated chimeric polypeptide encoding for HUMCA1~IA-P15, comprising a first amino acid sequence being at least 90 °,%o homologous to MEPWSSRWKTKRWLWDFTVTTLALTFLFQAREVRGAAPVDVLKALDFHNSPEGISKTT
GFCTNRKNSKGSDTAYRVSKQAQLSAPTKQLFPGGTFPEDFSILFTVKPKKGIQSFLLSIY
NEHGIQQIGVEVGRSPVFLFEDHTGKPAPEDYPLFRTVNIADGKWHRVAISVEKkTVTM
IVDCKKKTTKPLDRSERAIVDTNGITVFGTRILDEEVFEGDIQQFLITGDPKAAYDYCEH
YSPDCDSSAPKAAQAQEPQIDEYAPEDIIEYDYEYGEAEYhEAESVTEGPTVTEETIAQT
EANIVDDFQEYNYGTMESYQTEAPRHVSGTNEPNPVEEIFTEEYLTGEDYDSQRItNSED
TLYENKEIDGRDSDLLVDGDLGEYDFYEYKEYEDhPTSPPNEEFGPGVPAETDITETSIN
GHGAYGEKGQKGEPAVVEPGMLVEGPPGPAGPAGIMGPPGLQGPTGPPGDPGDRGPPG
RPGLPGADGLPGPPGTMLMLPFRYGGDGSKGPTISAQEAQAQAILQQARIALRGPPGPM
GLTGRPGPVGGPGSSGAKGESGDPGPQGPRGVQGPPGPTGKPGKRGRPGADGGRGMP
GEPGAKGDRGFDGLPGLPGDhGHRGERGPQGPPGPPGDDGI'~fRGEDGEIGPRGLPGEAG
PRGLLGPRGTPGAPGQPGIvIAGVDGPPGPKGNMGPQGEPGPPGQQGNPGPQGLPGPQG
PIGPPGEIL corresponding to amino acids 1 - 714 of CA1B hII)MAN, which also corresponds to amino acids 1 - 714 of HLTMCA1~IA P15, 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 MCCNLSFGIL1PLQK corresponding to amino acids 715 - 729 of HUMCA1~A_P15, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding fur a tail of HLTMCA1XIA_P15, comprising a polypeptide being at least 70°./0, optionally at least about SO%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MCCNLSFGILIPLQK in HUMCA 1 ~ P 15.
According to preferred embodiments of the present invention, there is pravided an 5 isolated chimeric polypeptide encoding for HITMCA1~IA P16, comprising a first amino acid sequence being at least 90 % homologous to MEPWSSRWIiTKRWLWDFTVTTLALTFLFQAREVRGAAPVDVLKALDFHNSPEGISKTT
GFCTNRKNSKGSDTAYRVSKQAQLSAPTKQLFPGGTFPEDFSILFTVKPKKGIQSFLLSIY
NEHGIQQIGVEVGRSPVFLFEDHTGKPAPEDYPLFRTVNTADGKWHRVAISVEKKTVTM
10 IVDCK.KKTTKPLDRSERAIVDTNGITVFGTRILDEEVFEGDIQQFLITGDPKAAYDYCEH
YSPDCDSSAPKAAQAQEPQIDEYAPEDIIEYDYEYGEAEI'KEAESVTEGPTVTEETIAQT
EANIVDDFQEYNYGTMESYQTEAP RHVSGTNEPNPVEEIFTEEYLTGEDYDSQRKNSED
TLYENKEIDGRDSDLLVDGDLGEYDF1'EYKEYEDKPTSPPNEEFGPGVPAETDITETSIN
GHGAYGEKGQKGEPAVVEPGMLVEGPPGPAGPAGIMGPPGLQGPTGPPGDPGDRGPPG
GLTGRPGPVGGPGSSGAKGESGDPGPQGPRGVQGPPGPTGKPGKRGRPGADGGRGI\R' GEPGAKGDRGFDGLPGLPGDKGHRGERGPQGPPGPPGDDGMRGEDGETGPRGLPGEA
corresponding to amino acids 1 - 648 of CA1B_HUMAN, which also correspands to amino acids 1 - 64S of HUMCA1XIA P16, a second amina acid sequence being at least 90 20 homologous to GMAGVDGPPGPKGNMGPQGEPGPPGQQGNPGPQGLPGPQGPIGPPGEK
corresponding to amina acids 667 - 714 of CA1B HTJMAN, which also corresponds to amino acids 649 - 696 of HUMCA1XIA-P16, and a third amina 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% homologaus to a polypeptide having the sequence 25 VSFSFSLFYKKVIKFACDICRFVGRHDERKVVKLSLPLY'LIYE carrespanding to amino acids 697 - 738 of HUMCAIs~ P16, wherein said first amino acid sequence, second amino acid sequence and third arnina acid sequence are contiguaus and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of HUMCA1~:IA_P16, comprising a 30 palypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionallyy 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 AG, having a structure as follows: a sequence starting from any of amino acid numbers 64S-x to 648; and ending at any of amino acid numbers 649+ ((rr2) - x), in which x varies from 0 to rr2.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HUMCA1~ P16, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°~0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VSFSFSLFYKKVIKFACDKRFVGRHDERKWKLSLPLYLIYE in HUMCA1~A P16.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HLTMCA1~IA_P17, comprising a first amino acid sequence being at least 90 % homologous to MEPWSSRWKTKRWLWDFTVTTLALTFLFQAREVRGAAPVDVLKALDFHNSPEGISKTT
GFCTNRKNSKGSDTAYRV SKQAQLSAPTKQLFPGGTFPEDFSILFTVKPKKGIQSFLLSIIr NEHGIQQIGVEVGRSPVFLFEDHTGKPAPEDYPLFRTVNIADGKWHRVAISVEKKTVTM
IVDCKKKTTKPLDRSERAIVDTNGITVFGTRILDEEVFEGDIQQFLITGDPKAAYDYCEH
YSPDCDSSAPIkAAQAQEPQIDE corresponding to amino acids I - 260 of CAIB HUMAN, which also corresponds to amino acids 1 - 260 of HUMCA1~ P17, and a second amino acid sequence being at least 70°,~0, 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 VRSTRPEKVFVF'Q corresponding to amino acids 261 - 273 of HUMCAI~ P17, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HUMCA11IA P17, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRSTRPEKVF'VFQ in HUMCA1~ P17.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for R20779_P2, comprising a first amino acid sequence being at least 90 % homologous to MCAERLGQFMTLALVLATFDPARGTDATNPPEGPQDRSSQQKGRLSLQNTAEIQHCLV
NAGDVGCGVFECFENNSCEIRGLHGICMTFLHNAGKFDAQGKSFIKDALKCKAHALRH
RFGCISRKCPAIREMVSQLQRECYLKHDLCAAAQENTRVIVEMII-iF'KDLLLHE
corresponding to amino acids 1 - 169 of STC2_HCTMAN, which also corresponds to amino acids 1 - 169 of R2077Q P2, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence CYKIEITMPKRRKVKLRD
corresponding to amino acids 170 - 187 of R20779_P2, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of R20779_P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°.'0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence CYKIEITMPKRRKVhLRD in R20779_P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for HSCOC4 PEA_1 P3, comprising a first amino acid sequence being at least 90 °,% homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSWHL,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKhEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIY'GKPVQGV AYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVhVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSH1'~YI'MILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRhILRNVNFQK_AINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPIVISVRRFEQLELRPVLYIVYLDKNLTV corresponding to amino acids 1 -865 of CO~ HUMAN, which also corresponds to amino acids 1 - 865 of HSCOC~
PEA_1 P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence RPHRSLSIQELGEPGPSEGWGG corresponding to amino acids 866 - 887 of HSCOC~ PEA_1 P3, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding fox a tail of HSCOC4 PEA_1 P3, comprising a polypeptide being at least 70°~0, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90°~o and most preferably at least about 95% homologous to the sequence RPHRSLSIQELGEPGPSEGWGG in HSCOC4 PEA_1 P3.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_I P5, comprising a first amino acid sequence being at least 90 % homologous to MRLLWWGLIWASSFFTLSLQKPRLLLFSPSWIiLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLkDAhSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVTtk.kE«~'SSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKI'VL
PNFEVKITPGKPYILTVPGI-IL,DEMQLDIQARl'IYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSkAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSW1'FVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
SRPPRVGDTLNLNLRAVGSGATFSH~'YYMILSRGQIVFMNREPILRTLTSVSVFVDHHLA
PSF1'FVAFYYHGDHPVANSLRVDVQAGAGEGKLELSVDGAKQI'RNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPIs~EKTTRICILRNVNFQKAINEKLGQI'ASPTAKR.CCQDGVTR
LPMhTRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQIL,TLWLPDSLTTWEIFiGLSLSKTKG corresponding to amino acids 1 - 818 of C04_HL1MAN, which also corresponds to amino acids 1 -818 of HSCOC4 PEA_1 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at lca:wk 90% and most preferably at least 95%
homologous to a polypeptide having the sequence L7'~-'TLSGPQVTLLPFPCTPAPCSLCS
corresponding to amino acids 819 - S43 of HSCOC 3~ ~EA_1 P5, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95°ib homologous to the sequence DVTLSGPQVTLLPFPCTPAPCSLCS in HSCOC4 PEA_1 P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P6, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQWKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVfLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQG1NLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQkMRPSTDTITVMV
ENSHGLRVRKKEVI'MPSSIFQDDFVIPDISEPGTWILISARFSDGLESNSSTQFEVILK~'VL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKILTFFR
LTSWYFVSSPFSLDLSKTKRF-ILVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYZ'MILSRGQIVFMNREPHRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVVQAGACEGKLELSVDGAKQYRNGESVKL,HLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEA1~~1SYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPhwIRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALELLQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAIHREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASR1'LDKTEQWSTLPPETKDHAVDLIQKG corresponding to amino acids 1 - 1052 of C04 HUMAN, which also corresponds to amino acids 1 -1052 of sa HSCOC4 PEA_1 P6, 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°.~0 homologous to a polypeptide having the sequence SGCKGKQEGGQERTVTGRWTAQEATEGKKGGP corresponding to amino acids 1053 -1084 of HSCOC~ PEA_1 P6, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SGCKGKQEGGQERTVTGRWTAQEATEGIVKGGP in HSCOC4 PEA_1 P6.
Accarding to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P1?, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQIiPRLLLFSPSWHLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
ENSHGLRVRKICEVI'MPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
GLESQTKLVNGQSHISLSKAEFQDALEItLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKThRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHY-YYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAI~~INSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKICEZNVNFQKAINEKLGQYASPTAh:RCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRILKSRDILGQAGLQRALEII,QEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTIS.GLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLI'NYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGS.ARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAI13REEL
VY~ELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTIviIYLAPTLAASRI'LDKTEQWSTLPPEThDHAVDLIQKG1'MRIQQFRIi ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISK.ASS
FLGEKASAGLLGAH.AAAITAYALTLTKAPADLRGVAHI~NLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKV corresponding to amino acids 1 - 1380 of C04 HLTM.~ V1, which also corresponds to amino acids 1 - 1380 ofHSCOC4 PEA_1 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence RAREGVGPGTGGGEGVE corresponding to amino acids 1381 - 1397 of HSCOC4 PEA_1 P12, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence R.AREGVGPGTGGGEGVE in HSCOC~ PEA_1 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1~,P15, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHL,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRK~EVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMILSRGQIVFIvINREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGShSHKPLNIViGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRhkRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPItwIRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRD)LGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQIL,TLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDhNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAI~-IREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTI~I~APTLAASRY'I,DKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEIkASAGLLGAHA AAITAYALTLTKAPADLRGVAHNNLIvIAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAI'WIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQ corresponding to amino acids 1 - 1359 of C04_HUMAN_V 1, which also corresponds to amino acids 1 - 1359 of HSCOC4_PEA_1 P15, 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 VNHSLVNHSLAWVARTPGPRGQARSRPQPPTRGIPAALLPGVFGGRLTSWLRDLEL
corresponding to amino acids 1360 - 1415 of HSCOC4_PEA_1 P15, wherein said first amino ?0 acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4_PEA_1 P15, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VNHSLVNHSLAWVARTPGPRGQARSRPQPPTRG1PAALLPGVFGGRLTSWLRDLEL in HSCOC4 PEA 1 P15.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P16, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSGGLHQLLRGPEVQLVAHSPWLK
a3 DSLSRTTNTQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRICKEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVIL.ITPGKPYIL,TVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYT~IILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHK.PLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKILRNVNFQKAINEKLGQYY ASPTAILRCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQIL,TLW LPDSLTTVVEIHGLSLShTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAII-IR.EEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRIt ADGSYYAAWLSRDSSTWLTAFVLKVL,SLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTILAPADLRGVAI-INNLMAMIAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IR.GLEEELQFSLGSKINVItVGGNSKGTLKVLRT~'NVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDYEYDELPAKDDPDAPLQPVTPLQLFEGRRNRRR.REAPK corresponding to amino acids 1 - 1457 of C04_HLTMAN_V 1, which also corresponds to amino acids 1 - 1457 of HSCOC4_PEA_1_P16, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence AERQGGAVWHGHRGRHPPEWIPRPAC corresponding to amino acids 1458 - 1483 of HSCOC4 PEA_1 P16, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P16, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90°,/° and most preferably at least about 95%
homologous to the sequence AERQGGAVWHGHRGRHPPEWIPRPAC in HSCOC4 PEA_1 P16.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P20, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLG~TPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRIiKEVYMPSSTFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYIL,TVPGHLDEMQLDIQARI'IYGKPVQGVAYVRFGLLDEDGIS.KTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYY~~SRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAG ACEGILLELSVDGAKQYRNGESVhLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEANINSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRI::RLSCPKEKTTRkKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEII,QEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTIS'_GLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAIHREEL
V1'ELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTVvrL,TAFVLKVL,SLAQEQVGGSPEKLQETSN'VLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAI-IAAAITAYALTLTKAPADLRGVAHNNLMAIvIAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQ corresponding to amino acids 1 - 1303 of C04 HLJMAN_V 1, which also e5 corresponds to amino acids 1 - 1303 of HSCOC4_PEA_1 P20, 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 VGAVPGLWRGWVVL,RPRACLSPGSTSLGHGDCPGCPVCLLDCLPHH corresponding to amino acids 1304 - 1349 of HSCOC4 PEA_1 P20, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P20, 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 VGAVPGLWRGWVULRPRACLSPGSTSLGHGDCPGCPVCLLDCLPHI-i in HSCOC4 PEA 1 P20.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P9, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIVVASSFFTLSLQKPRLLLFSPSVVHI,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRI~EV~'MPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDiQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSVVYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHCrDHPVANSLRVDVQAGACEGKLELSVDGAKQ1'RNGESVItLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNS1'DLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPIviMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVL1'NYLDhNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGA114R.EEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRI'LDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSI\~IQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTIiAPADLRGVAHNNLMAMAQETGDNL~'WGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDI'EYDELPAKDDPDAPLQPVTPLQLFEGRF~NRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
1'FDSV corresponding to amino acids 1 - 1529 of CO~ HLTMAN_V 1, which also corresponds to amino acids 1 - 1529 of HSCOC4 PEA_1 P9, and a second amino acid sequence being at least 70%, optionally at least SO%, preferably at least SS%, more. preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SGER
corresponding to amino acids 1530 - 1533 of HSCOC~ PEA_1 P9, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P9, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SGER in HSCOC4 PEA 1 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P22, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSWHL,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPILVDFTLSSERDFALLSLQVFLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKhEV~~'SSIFQDDFVIPDISEPGTVvThISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKhTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRL1'VAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
s7 EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGCy't'C'rrLSLERPD
SRPPRVGDTLNLNLRAVGSGATFSHYY1'MiLSRGQIVFMNREPKRTLTSV;i'~FVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESV~;..LHLETDS
LALVALGALDTALYAAGSIkSHKPLNMGKVFEAMNSYDLGCGPGGGDSAL,QVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPIviNIRSCEQRAARV QQPDCREPFLSCCQFAESLRKhSRDKGQAGLQRALEIL,QEEDLID
EDDIPVRSFFPENWLWRVETV'DRFQII,TLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYN'~'LDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVI,QIEKEGAIHREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMTYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGS1'AAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITA~'ALTLTKAPADLRGVAHNNLMAMAQETGDNL1TWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDYEI'DELPAKDDPDAPLQPVTPLQLFEGRF~NRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
YFDSVPTSRECVGFEAVQEVPVGLVQPASATLYDYZ'1VPERRCSVFYGAPSKSRLLATLC
SAEVCQCAEGKCPRQRRALERGLQDEDGYRMKFACYYPRVEYGFQVKVLREDSRAAF
RLFETKITQVLHF corresponding to amino acids 1 - 1653 of C04 HUMAN V1, which also corresponds to amino acids 1 - 1653 of HSCOC4_PEA_1 P22, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SMKQTGEAGRAGGRQGG corresponding to amino acids 1654 - 1670 of HSCOC~ PEA_1 P22, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1_P22, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about 85%, more preferably ss at least about ~0% and most preferably at least about 95% homologous to the sequence SMKQTGEAGRAGGRQGG in HSCOC4 PEA_1 P22.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC~ PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologots to MRLLWGL1WASSFFTLSLQILPRLLLFSPSV~~HI,GVPLSVGVQLQDVPRGQVVKGSVF'LR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTIT~VMV
ENSHGLRVRKKEVYIviPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSFFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYY~YMILSRGQIVFMNREPKRTLTSVSVF'VDHHLA
PSFYFVAFYYI-iGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKICRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEIL,QEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLY~NYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAA.AVSLKVVARGSFEFPVGDAVShVLQIEKEGAIHREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSY~VRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYL,APTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISkASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVAHNNLMAMAQETGDNLYWGSV
TGSQSNAV SPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDI~~NTTCQDLQIEVTVKGHVE
YTMEANEDY~EDYEYDELPAKDDPDAPLQPVTPLQLFEGRRNRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
YFDSVPTSRECVGFEAVQEVPVGLVQPASATLYDY~~NPERRCSVFYGAPShSRLLATLC
SAEVCQCAEGKCPRQRRALERGLQDEDGYRMKFACYYPRVEYG corresponding to amino acids 1 - 1626 of C04_HIJMAN_V 1, which also corresponds to amino acids 1 - 1626 of HSCOC4_PEA_1 P23, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90°.~o and most preferably at least 95% homologous to a polypeptide having the sequence QSSHRGPGLTLPRGPAVLVSLGVACSSYRSCTQPVCSDTNFLPSQPQSNSPFPLLLTPS
corresponding to amino acids 1627 - 1685 of HSCOC~ PEA_1 P23, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4_PEA_1 P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence QSSHRGPGLTLPRGPAVLVSLGVACSSYRSCTQPVCSDTNFLPSQPQSNSPFPLLLTPS in HSCOC4 PEA 1 P23.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P24, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQV~~ICGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLIkDAhSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKKEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKK1'VL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEIvIEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVhVSA'f~jSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSH1'YY~MILSRGQIVFMNREPKRTLTSVSVFVDHHI,A
PSFYFVAFY~f'HGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQRAARVQQPDCR.EPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATP ~Ii~L, RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGI,!~.Q
QVLVPAGSARPVAFSVVPTAAAAVSLKWARGSFEFPVGDAVSKVLQIEKEGAIFiR~Ei, VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
ADGSY AAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVA~INNLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDI'~EYDELPAkDDPDAPLQPVTPLQLFEGRFSJRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
1'FDS corresponding to amino acids I - 1528 of C04 HUl~ V1, which also corresponds to 15 amino acids 1 - 1528 of HSCOC4 PEA_1 P24, 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 SADVLCFTGHQVR.ADSWPPCVLLILSASVLRGSALASVAPWSGVCRTRMATG
corresponding to amino acids 1529 - 1579 of HSCOC4_PEA_1 P24, wherein said first amino 20 acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P24, 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 25 SADVLCFTGHQVRADSWPCVLLKSASVLRGSALASVAPWSGVCRTRMATG in HSCOC4 PEA 1 P24.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P25, comprising a first amino acid sequence being at least 90 % homologous to NPSRNNVPCSPKWFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTIVIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKKEVYMPSSIFQDDFVIPDISEPGTWhISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTkLUNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAlIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMIL,SRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGIhLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAIIVEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQR.AARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLm EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRF'EQLELRPVLYNYLDhNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAffiREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMII'LAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLILVLSLAQEQVGGSPEKL.QETSNWLLSQQQADGSFQ
DPCPVLDRSIVIQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVAHNNLMAMAQETGDNLYWGS V
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
1'TI~~ANEDY'EDY'EYDELPAKDDPDAPLQPVTPLQLFEGRFS~IItRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
YFDSVPTSRECVGFEAVQEVPVGLVQPASATLYDYYNPERRCSVFYGAPSKSRLLATLC
SAEVCQCAEG corresponding to amino acids 1 - 1593 of C04_HUMAN_V1, which also corresponds to amino acids 1 - 1593 of HSCOC4_PEA_1 P25, 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 ETEGLGRGSGGGMAGAPPTLSDGFPNFREVPSPASRPGAGSAGRGWLQDEVCLLLPPC
GVRLPG corresponding to amino acids 1594 - 1657 of HSCOC4 PEA_1 P25, wwherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is pro~~ided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P35, 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 ETEGLGRGSGGGMAGAPPTLSDGFPNFREVPSPASRPGAGSAGRGWLQDEVCLLLPPC
GVRLPG in HSCOC4 PEA 1 P25.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P?6, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPS~~GVPLSVGVQ LQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
ENSHGLRVRKKEV~'MPSSIFQDDFVIPDISEPGTWhISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTI~.LVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAA.AIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSH1'1'~c'IvIILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNS1'DLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPh:EKTTRKICRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMNIRSCEQR.AARVQQPDCREPFLSCCQFAESLRhhSRDKGQAGLQRALEILQEEDLII7 EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSWPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGA)TIREEL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAVhL.SRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISIiASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVAI-INNLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDYEYDELPAKDDPDAPLQPVTPLQLFEGR.RNRRRREAPKVVEEQESRV
YFDSVPTSRECVGFEAVQEVPVGLVQPASATLYDYYNPERRCSVFYGAPSKSRLLATLC
SAEVCQCAEG corresponding to amino acids 1 - 1593 of C04_HLTIVI~_V1, which also corresponds to amino acids 1 - 1593 of HSCOC4_PEA_1 P26, 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 ETEGLGRGSGGGMAGAPPTLSDGFPNFREVPSPASRPGAGSAGRGWLQDEVCLLLPPC
GVRSVFPPRPWPDPPSGTGCFGLSGCSLLLLQVMHAACLL corresponding to amino acids 1594 - 1691 of HSCOC4_PEA_1 P26, wherein said first amino acid sequence and second I S amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P26, 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 ETEGL.GRGSGGGMAGAPPTLSDGFPNFREVPSPASRPGAGSAGRGWLQDEVCLLLPPC
GVRSVFPPRPWPDPPSGTGCFGLSGCSLLLLQVMHAACLL in HSCOC4 PEA_1 P26.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P30, comprising a first amino acid sequence being at least 90 % homologous to I~~LLWGLIWASSFFTLSLQKPRLLLFSPSWHLGVPLSVGVQLQDVPRGQVVICGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQG1NLLFSSRRGHLFLQTDQPIYNPGQRVR1'RVFALDQKMRPSTDTITVMV
ENSHGLRVRKKEVYn~IPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVILKY'VL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLShAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEIvIEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQF:( _rIjGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGD T I,NLNLRAVGSGATFSHYYYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFI'FVAF ;''~.c iiGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRICI~E~NVNFQKAINEILLGQYASPTAKRCCQDGVTR
EDDIPVRSFFPENWLWRVETVDRFQIL,TLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGA)HREEEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSY'VRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVAHNNLMAMAQETGDNLl'WGS
corresponding to amino acids 1 - 1232 of C04 HLIMAN_V3, which also corresponds to amino acids 1 - 1232 of HSCC~C4 PEA_1 P30, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90°ro and most preferably at least 95% homologous to a polypeptide having the sequence RNPVRLLQPRAQMFCVLRGTK corresponding to amino acids 1233 - 1253 of HSCOC4 PEA_1 P30, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P30, 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 RNPVRLLQPRAQIvfFCVLRGTK in HSCOC4 PEA_1 P30.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSC(aC4 PEA_1 P38, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTIVIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQIhMRPSTDTITVMV
ENSHGLRVRKKEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVhITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEI'~EAE
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHY~1'MILSRGQNFMIVREPKRTLTSVSVFVDHHLA
PSFYFVAFY1'HGDHPVANSLRVDVQAGACEGkLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTAL1'AAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
10 LAFSDGDQWTL.SRKRLSCPKEKTTRKKRNVNFQK.AINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQR.AARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQIL,TLWLPDSLTTWEIHGLSLSKTKG corresponding to amino acids 1 - 818 of CO4 HUMAN, which also corresponds to amino acids 1 -818 of HSCOC4 PEA_1 P38, and a second amino acid sequence being at least 70%, optionally at 15 least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DVTLSGPQVTLL.PFPCTPAPCSLCS
corresponding to amino acids 819 - 843 of HSCOC~ PEA_1_P38, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an 20 isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P3S, comprising a polypeptide being at least 70%, optionally at least about SO°'/°, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DVTLSGPQVTLLPFPCTPAPCSLCS in HSCOC4 PEA_1 P38.
According to preferred embodiments of the present invention, there is provided an 25 isolated chimeric polypeptide encoding for HSCOC4_PEA_1_P39, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQILPRLLLFSPSV~~,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTT'NIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
30 ENSHGLRVRKKEVI'1VIPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQ corresponding to amino acids 1 - 387 of C04_ _ _HUMAN, which also corresponds to amino acids 1 - 387 of HSCOC4 PEA_1 P39, 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 VSSRGEG corresponding to amino acids 388 - 394 of HSCOC4 PEA_I P39, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4_PEA_1 P39, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VSSRGEG in HSCOC4_PEA_1 P39.
According to preferred embodiments of the present invention, there is provided an IS isolated chimeric _polypeptide encoding for HSCOC4_PEA_1 P40, comprising a first amino acid sequence being at least 90 % homologous to MRLLVvjGLIWASSFFTLSLQKPRLLLFSPSVVHL,GVPLSVGVQLQDVPRGQV~~kGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRhKEVI'MPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKY
corresponding to amino acids 1 - 236 ofC04_HUMAN, which also corresponds to amino acids I - 236 of HSCOC4_PEA_1 P40, 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 AGEWTEPHFPLKGRVPGRPGEAEYGHY corresponding to amino acids 237 - 263 of HSCOC4_PEA_1 P40, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4_PEA_I P40, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% anc~. a~aost preferably at least about 95% homologous to the sequence AGEWTEPHFPLKGRevfGRPGEAEYGHY in HSCOC4 PEA 1 P40.
According to preferred embodiments of the present invvention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P41, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQIkPRLLLFSPSWHI.GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAItSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRhhEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVIiKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVA1'VRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKICRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMNIRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSWPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAIHREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKG1'MRIQQFRK
ADGSYAAWLSRDSSTWLTAFVL,KVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAI3AA.AITAYALTLTKAPADLRGVAFINNLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
LRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVZDMKNTTCQDLQIEVTVKGHVE
1'TMEANEDYEDYEYDELPAKDDPDAPLQPVTPLQLFEGR.FSJRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
YFDSV _corresponding to amino acids 1 - 1529 of C04 HLTMAN_V 1, which also corresponds to amino acids 1 - 1529 of HSCOC4 PEA_1 P41, 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 SGER
corresponding to amino acids 1530 - 1533 of HSCOC4 PEA_1 P41, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of HSCOC4 PEA_1 P41, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and mast preferably at least about 95% homologous to the sequence SGER in HSCOC4 PEA 1 P41.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for HSCOC4 PEA_1_P42, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGL,HQLLRGPEVQLVAHSPVrL,K
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKKEVYMPSSIFQDDFVIPDISEPGTWILISARFSDGLESNSSTQFEVILKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVhVSATVSSPGSVP
EVQDIQQNTDGSGQVSTPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWWTLSRKRLSCPKEKTTRKI~E~NVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMI\~IRSCEQR.AARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEIL,QEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVL1'NYL,DKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVWARGSFEFPVGDAVSKVLQIEKEGA)T REIREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLThAPADLRGVAHNNLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDYEYDELPAKDDPDAPLQPVTPLQLFEGRTS~RRRREAPKVVEEQESRV
HYTVCIW -corresponding to amino acids 1 - 1473 of C04_HUMAN_V 1, which also corresponds to amino acids 1 - 1473 of HSCOC4_PEA_1 P42, 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 WAPGAALGQGREGRTQAGAGLLEPAQAEPGRQLTRLHR corresponding to amino acids 1474 - 1511 of HSCOC4 PEA_1 P42, a third amino acid sequence being at least 90 homologous to RNGKVGLSGMAIADVTLLSGFHALRADLEK corresponding to amino acids 1474 - 1503 of C04_HLTMAN_V 1, which also corresponds to amino acids 1512 -1541 of HSCOC4 PEA_1 P42, and a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence VWSATQGNPLCPRY corresponding to amino acids 1542 - 1555 of HSCOC4-PEA_1 P42, wherein said first amino acid sequence, second amino acid sequence, third amino acid sequence and fourth amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ -encoding for an edge portion of HSCOC4_PEA_1 P42, 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 encoding for WAPGAALGQGREGRTQAGAGLLEPAQAEPGRQLTRLHR, corresponding to HSCOC4 PEA_1 P42.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of HSCOC~ PEA_1 F42, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about 85%, more preferably at least about 90% and most pref:~~-~hly at least about 95% homologous to the sequence VWSATQGNPLCPRY in HSCOC4i, PEA_1 P4?.
According to preferred eml~o~liments of the present invention, there is provided an isolated chimeric _ _ _polypeptide encoding for HLTMTREFAC PEA 2 P8, comprising a first amino acid sequence being at least 90 % homologous to MAARALCMLGLVLALLSSSSAEEYVGL corresponding to amino acids 1 - 27 of TFF3 HUI\~L~1N, which also corresponds to amino acids 1 - 27 of HUMTREFAC_PEA
2 P8, 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 WkVHLPKGEGFSSG corresponding to amino acids ~8 of HUMTREFAC_PEA_2 P8, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ _encoding for a tail of HLTIvITREFAC PEA 2 P8, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence WKVHLPKGEGFSSG in HLTMTREFAC_PEA 2 P8.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _ _polypeptide encoding for HLTMOSTRO PEA_1 PEA_1 P21, comprising a first amino acid sequence being at least 90 % homologous to MRIAVICFCLLGITCAIPVILQADSGSSEEKQLYNKYPDAVATWLNPDPSQKQNLLAPQ
corresponding to amino acids 1 - 58 of OSTP_I-IIJMAN, which also corresponds to amino acids 1 - 58 of HUMOSTRO PEA_1 PEA_1 P?1, 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 VFLNFS
corresponding to amino acids 59 - 64 of HUMOSTRO PEA_1 PEA_1 P21, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ _encoding for a tail of HUMOSTRO_PEA_1 FEA_1 P21, comprising a polypeptide being at least 70%, optionallyy at least about 80%, preferably at least about S5%, 1oi more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VFLNFS in HLTMOSTRO PEA_1 PEA_1 P21.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HLTMOSTRO PEA_1 PEA_1 P25, comprising a first amino acid sequence being at least 90 % homologous to MRIAVICFCLLGITCAIPVILQADSGSSEEhQ corresponding to amino acids 1 - 31 of OSTP_HTJMAN, which also corresponds to amino acids 1 - 31 of HUMOSTRO _PEA_1 PEA_1 P25, 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 H
corresponding to amino acids 32 - 32 of HLTMOSTRO _PEA_1 PEA_1 P25, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric - - -polypeptide encoding for HUMOSTRO PEA_1 PEA_1 P30, comprising a first amino acid sequence being at least 90 % homologous to NIRIAVICFCLLGITCAIPVKQADSGSSEEKQ corresponding to amino acids 1 - 31 of OSTP_HUMAN, which also corresponds to amino acids 1 - 31 of IiUMOSTRO_PEA_1 PEA_1 P30, 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 VSIFYVFI
corresponding to amino acids 32 - 39 of HUMOSTRO PEA_1 PEA_1 P30, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide - - -encoding for a tail of HLTMOSTRO PEA_1 PEA_1 P30, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VSIFYVFI _ _ -in IiUMOSTRO PEA_1 PEA_1 P30.
According to preferred embodiments of the present invention, there is provided an isolated chimeric - .polypeptide encoding for T10888_PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWIiEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVYPELPhPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
WWVNGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLY
GPDVPTISPSItANYRPGENLNLSCHAASNPPAQYSWF1NGTFQQSTQELFIPNITVNNSGS
YMCQAHNSATGLNRTTVTMITVS co~esponding to amino acids 1 - 319 of CEA6_ -HUMAN, which also corresponds to amino acids 1 - 319 of T10888_PEA_1 P2, 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 DWTRP -corresponding to amino acids 320 - 324 of T10888_PEA_1 P2, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ _encoding for a tail of T10888 PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DWTRP in T1088S_PEA_1 P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for T10888 PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPFSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYIiGERVDGNSLI~~GYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDILDAVAFTCEPEVQNTTYI, WWVNGQSLPVSPRI,QLSNGNMTLTLLSVhRNDAGSYECEIQNPASANRSDPVTLNVL
corresponding to amino acids 1 - 234 of CEA6_HUMAN, which also corresponds to amino acids 1 - 234 of T10888_PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence LLLSSQLWPPSASRLECWPGWL corresponding to amino acids 235 - 256 of T10888_PEA_1 P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T10888_PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LLLSSQLWPPSASRLECWPGWL in T10888_PEA_1 P4.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T10888_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYKGERVDGNSLIVGI'~~IGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFI-IVYPELPIiPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
WW-VNGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNP ASANRSDPVTLNVL
corresponding to amino acids 1 - 234 of Q 13774, which also corresponds to amino acids 1 - 234 of T10888_PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence LLLSSQLWPPSASRLECWPGWL
corresponding to amino acids 235 - 256 of T10888_PEA_1 P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T10888_PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LLLSSQLWPPSASRLECWPGVhL, in T10888_PEA_1 P4.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T108SS PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGhEVLLLAHNLP
QNRIGYS WYKGERVDGNSLIVGYV IGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTY'L
GQSLPVSPRLQLSNGNMTLTLLSVhRNDAGSYECEIQNPASANRSDPVVTLNVLY
GPDVPTISPSK:ANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGS
YMCQAHNSATGLNRTTVTMITVSG corresponding to amino acids 1 - 320 of CEA6_ _ _HUMAN, which also corresponds to amino acids 1 - 320 of T10888 PEA_1 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, mare preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence KWIHEALASHFQVESGSQRRARItICFSFPTCVQGAHANPhFSPEPSQFTSADSFPLVFLFF
VVFCFLISHV _ _corresponding to amino acids 321 - 390 of T10888 PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of T10888_PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence hWIHEALASHFQVESGSQRRARI~FSFFTCVQGAHANPKFSPEPSQFTSADSFPLVFLFF
VVFCFLISHV in T10888 PEA_I P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T10888_PEA_1 P6, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVP'WItEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGILEVLLLA
HNLPQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIY'PNASLLIQNVTQ
NDTGFYTLQVIK.SDLVNEEATGQFHVY
corresponding to amino acids 1 - 141 of CEA6 HUMAN, which also corresponds to amino acids 1 - 141 of T10888_PEA_1 P6, 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 REYFHMTSGCWGSVLLPTYGIVRPGLCLWPSLHYILYQGLDI
corresponding to amino acids 142 - 183 of T10888_PEA_1 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T10888_PEA_1 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 9>".s;, homologous to the sequence REYFHMTSGCWGSVLLPTYGIVRPGLCLWPSLH~'u''LYQGLDI in T10888 PEA_1 P6.
According to preferred embodiments of the pre,acnt invention, there is provided an isolated chimeric polypeptide encoding for T39971 P6, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLIL,ALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQY~WRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKG corresponding to amino acids 1 - 276 of VTNC_HLTMAN, which also corresponds to amino acids 1 - 276 of T39971 P6, 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 TQGVVGD corresponding to amino acids 277 - 283 of T39971 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T39971 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologois to the sequence TQGWGD
in T39971 P6.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T39971 P9, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDE1'TVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLIiNGSLFAFR
GQYCI'ELDEKAVRFGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQ1'WRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEI'QFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRT corresponding to amino acids 1 - 325 of VTNC_HUMAN, which also corresponds to amino acids 1 - 325 of T39971 P9, and a second amino acid sequence being at least 90 % homologous to SGMAPRPSLAKKQRFRHIZNRI~GYRSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGA
NNYDDYRMDWLVPATCEPIQSVFFFSGDK1'YRVNLRTRRVDTVDPPYPRSIAQYWLGC
PAPGHL corresponding to amino acids 357 - 478 of VTNC_HUMAN, which also corresponds to amino acids 326 - 447 of T39971 P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of T39971 P9, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise TS, having a structure as follows: a sequence starting from any of amino acid numbers 325-x to 325; and ending at any of amino acid numbers 326 + ((n-2) - x), in which x vanes from 0 to t~-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T39971 P11, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTI~~'EDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHS1'SGRERVYFFKGKQYWEYQFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDLFELLFWGRTS corresponding to amino acids 1 - 326 of VTNC_ -HUMAN, which also corresponds to amino acids 1 - 326 of T39971 PI 1, and a second amino acid sequence being at least 90 % homologous to DKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL corresponding to amino acids 442 - 478 of VTNC'_HLJIvIAN, which also corresponds to amino acids 327 - 363 of T39971 P11,, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for an edge portion of T39971 PI 1, 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 SD, having a structure as follows: a sequence starting from any of amino acid numbers 326-x to 326; and ending at any of amino acid numbers 327 + ((n-2) - x), in which x varies from 0 to rr2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T39971 P1 l, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNA': VHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASkPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQI'WRFEDGV
LDPDYPRIVISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTS corresponding to amino acids 1 - 326 of Q9BSH7, -which also corresponds to amino acids 1 - 326 of T39971 P11, and a second amino acid sequence being at least 90 % homologous to DKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL corresponding to amino acids 442 - 478 of Q9BSH7,, -which also corresponds to amino acids 327 - 363 ofT39971 P11, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of T39971 P11, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise SD, having a structure as follows: a sequence starting from any of amino acid numbers 326-x to 326; and ending at any of amino acid numbers 327 + ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T39971 P12, comprising a first amino acid sequence being at least 90 % homologous to 1\~IAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
1os KPQVTRGDVFTMPEDEI'TVYDDGEEKNNATVHEQVGC?~:1's I.TSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELC'S~~KPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFK corresponding to amino acids 1 - 223 of VTNC_HTJMAN, which also corresponds to amino acids 1 -223 of T39971 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence VPGAVGQGRKHLGRV corresponding to amino acids 224 - 233 of T39971 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T39971 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VPGAVGQGRKHLGRV in T39971 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T39971 P12, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLIL,ALLAWVALADQESChGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCY~ELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFK corresponding to amino acids 1 - 223 of Q9BSH7, _which also corresponds to amino acids 1 - 223 of T39971 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VPGAVGQGRKHLGRV corresponding to amino acids 238 of T39971 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T39971 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VPGAVGQGRKHLGRV in T39971 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for 221366 PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIPvfNIILVLTDDQDVELGSL
QVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKY~r'><~TNNENCSSPSW
QAMFIEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYYIPPGWREWLGLIKNSRFYNYTVCR
NGIKEKHGFDYAKDYFTDLITNESINYFKMSKRW'PHRPVNEVIVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYNYAPNMDKIiWIMQYTGPMLPIHMEFTMLQRKRLQTLMSVDD
SVERLYNMLVETGELENTYIIYTADHGYHIGQFGLVKGKSMP~'DFDIRVPFFIRGPSVEP
GSIVPQIVLNIDLAPTILDIAGLDTPPDVDGKSVLKLLDPEKPGNRFRTNKKAKIWRDTFL
VERGKFLRKKEESSKNIQQSNHLPKYERVKELCQQARYQTACEQPGQKWQCIEDTSGK
LRIHKCKGPSDLLTVRQSTRNLYARGF HDKDKECSCRESGYRASRSQRKSQRQFLRNQ
GTPKYKPRFV~-iTRQTRSLSVEFEGEIYDINLEEEEELQVLQPRNIAkRHDEGHKGPRDLQ
ASSGGNRGRMLADSSNAVGPPTTVRVTHKCFILPNDSIHCERELYQSARAWKDHKAYI
DKEIEALQDKIKNLREVRGHLKRRILPEECSCSKQSYYNKEKGVIkKQEKLKSHLHPFKE
AAQEVDSKLQLFKENNRRRICKERKEKRRQRKGEECSLPGLTCFTHDNNIiWQTAPFWN
corresponding to amino acids 1 - 761 of SUL1 HUMAN, which also corresponds to amino acids 1 - 761 of 221368 PEA_1 P2, 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 PHKYSAHGRTRHFESATRTTNGAQKLSRI corresponding to amino acids 762 - 790 of Z21368_PEA_1 P2, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of 221368 PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence PHKI'SAHGRTRHFESATRTTNGAQKLSRI in 221368 PEA_1 P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ -polypeptide encoding for 221368 PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIIL,VLTDDQDVEL
corresponding to amino acids 1 - 57 of Q7Z2W2, which also corresponds to amino acids 1 - 57 of 221368 PEA_1 P5, second bridging amino acid sequence comprising A, and a third amino acid sequence being at least 90 % homologous to FFGKYLNEYNGSYIPPGWREWLGLIKNSRFYNIrTVCRNGIKEKHGFDYAKDYFTDLITN
ES1NYFKMSKRIvfYPHRPVMIvIVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNM
DKHWIMQYYTGPMLPIHMEFTNILQRKRLQTLMSVDDSWRLYNMLVETGELENTYIIYT
ADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTILDIAGLDT
PPDVDGKSVLkLLDPEKPGNRFRTNKKAKIWRDTFLVERGKFLRKKEESSKNIQQSNHL
PKYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRNLY
ARGFHDIkDKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRF~~I-ITRQTRSLSVEFE
GEIYDINLEEEEELQVLQPRNIAKItHDEGHhGPRDLQASSGGNRGRMLADSSNAVGPPT
TVRVTHKCFILPNDSIHCERELYQSARAWKDHKAYIDKEIEALQDK)kNLREVRGHLKR
RKPEECSCSKQSYYNKEKGVKKQEKLKSHLHPFKEAAQEVDSKLQLFKErTNRRRIiICER
KEKRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNE
THNFLFCEFATGFLEYFDMNTDPYQLTNTVHTVERGIL,NQLHVQLMELRSCQGYILQCN
PRPKNLDVGNKDGGSYDLHRGQLWDGWEG corresponding to amino acids 139 - 571 of Q7Z2W2; which also corresponds to amino acids 59 - 791 of Z2136S PEA_1 P5, wherein said first, second arid third amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ -encoding for an edge portion of 221368 PEA_1 P5, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least three amino acids comprise LAF having a structure as follows (numbering according to Z2136g PEA_1 PS): a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 59 + ((n-2) - x), in which x varies from 0 to n-2.
m According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for 221368_PEA_1 P5, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MKI'SCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIII,VLTDDQDVELAFF
GKYLNBYNGSY1PPGWREWLGLIKNSRFYNYTVCRNG)KF~HGFDYAKDYFTDLITNES
INYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNMDK
HWIMQI'TGPMLPIHIviEF1 NILQRKRLQTLMSVDDSVERLYNNiZVETGELENTYIIYTAD
HGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTILDIAGLDTPP
DVDGKSVLKLLDPEKPGNRFRTNKKAhiWRDTFLVERGKFLRKKEESSKNIQQSNHLP
KYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRNLYA
RGFHDKDKECSCRESG1'RASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEFEGE
RVTHKCFILPNDSIHCERELYQSARAWItDHKAI'IDKELEAL,QDKI~NLREVRGHLKRRK
PEECSCSKQSYYNKEKGVKKQEKLKSHLHPFKEAAQEVDSKLQLFKENNR7f~RItKERhE
KRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNETH
NFLFCEFATGFLEYFDMNTDPYQLT'NTVHTVERGILNQLHVQLME corresponding to amino acids 1 - 751 of Z21368_PEA_1 P5, and a second amino acid sequence being at least 90 % homologous to LRSCQGI'ICQCNPRPKNLDVGNILDGGSYDLHRGQLWDGWEG
corresponding to amino acids 1 - 40 of AAH12997, which also corresponds to amino acids 752 -791 of 221368_PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a head of 221368 PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELAFF
GKYLNEYNGSYIPPGWREWLGLIKNSRFYNYTVCRNGIKEKHGFDYAKDYFTDLIT'NES
IN1'FKMSKRMYPHRFVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNMDK
HWIMQl'TGPMLPII~-iMEFTNILQRKRLQTLMSVDDSVERLI'NMI,VETGELENT'YIIYTAD
HGYHIGQFGLVKGKSIvIPI'DFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTILDIAGLDTPP
DVDGKSVLKLLDPEKPGNRFRTNItKAKIWRDTFLVERGKFLRKKEESSKNIQQSNHLP
KYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRII~hCKGPSDLLTVRQSTRNLYA
RGFHDKDKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEFEGE
I1'DINLEEEEELQVLQPRNL4ICRHDEGHKGPRDLQASSGGNRGRMLADSSNAVGPPTTV
RVTHKCFILPNDSIHCERELYQSARAWKDHKAY'mKEIEALQDKIKNLREVRGHLKRRK
PEECSCSKQSYYNKEKGVKKQEKLKSHLHPFKEAAQEVDSKLQLFKENNRRRKICERKE
ILRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNETH
NFLFCEFATGFLEYFDMNTDPYQLTNT'VHTVERGILNQLHVQLME of Z21368_PEA_1_P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric - -polypeptide encoding for Z2136S PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERhNIRPNIIL,VLTDDQDVEL
corresponding to amino acids 1 - 57 of SL1L 1 HTJMAN, which also corresponds to amino acids 1 - 57 of X21365 PEA_1 P5, and a second amino acid sequence being at least 90 homologous to AFFGKYLNEYNGS1'IPPGWREWLGLIKNSRFYNYTVCRNGIKEhHGFDYAKDYFTDLIT
NESINYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPN
MDhHWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDDSVERLYNIviLVETGELENTYII
YTADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQNLNIDLAPTILDIAGL
DTPPDVDGILSVLKLLDPEKPGNRFRTNKKAhIWRDTFLVERGKFLRKKEESSKNIQQSN
HLPKYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRN
LYARGFHDKDKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVE
FEGEIYDINLEEEEELQVLQPRNL~1KRI-IDEGHKGPRDLQASSGGNRGRMLADSSNAVGP
PTTVRVTHKCFILPNDSIHCERELYQSARAWKDHKAYIDKEIEALQD 'I~NLREVRGHL
KRRKPEECSCSKQSY1'NKEKGVhKQEKLKSHI,HPFKEAAQEVDSKLQLFKENNRRRK
ILERKEKRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRT
VNETHNFLFCEFATGFLEI'FDMNTDPYQLTNTVHTVERGIL,NQLHVQLMELRSCQGYK
QCNPRPKNLDVGNKDGGSYDLHRGQLWDGWEG corresponding to amino acids 138 - 871 of SLTL1 HUT~IAN, which also corresponds to amino acids 58 - 791 of Z2136S
PEr~~._,1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential .order.
According to preferred embodiments of the present invention, there is providc~i an isolated chimeric -polypeptide encoding for an edge portion of Z21368_PEA_1 P5, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more.
preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise LA, having a structure as follows: a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 58 + ((n-2) - x), in which x varies from 0 to n-?.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for Z2136s PEA_1 P15, comprising a first amino acid sequence being at least 90 °~o homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIiLVLTDDQDVELGSL
QVMNkTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYV'I~IHIVVYTNNENCSSPSW
QAMTIEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYIPPGWREWLGLIKNSRFIrNI'TVCR
NGIKEKHGFDYAKDYFTDLITNESINYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYNYAPNMDKHWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDD
SVERLYNNIL,VETGELENTYIIYTADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEP
GSNPQIVT,NIDLAPTILDIAGLDTPPDVDGKSVLKLLDPEKPGNRFRTNKKAkIWRDTFL
VERG corresponding to amino acids 1 - 416 of SL1L1 HCTMAN, which also corresponds to amino acids 1 - 416 of Z2136s PEA_1 P15.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for 221368 PEA_1 P16, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCST'VRSPRFRGRIQQERhNIRPNIILVLTDDQDVELGSL
QVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYYVI~VHNVYYTNNENCSSPSW
QAIVgiEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYIPPGWREWLGLIKNSRFYNYTVCR
NGIKEKHGFDYAKD1'FTDLITNESINYFIiMSKRMYPHRPVMMVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYNYAPNMDKHWIIvIQYYTGPMLPIHI\~FTNILQRKRLQTLMSVDD
SVERLYNNILJVETGELENTYIIYTADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEP
GSIVPQIVLNIDLAPTILDIAGLDTPPDVDGKSVLKLLDPEKPGNR corresponding to amino acids 1 - 397 of SLTL1 HUMAN, which also corresponds to amino acids 1 - 397 of 221368 PEA_1 P16, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence CVIVPPLSQPQIH corresponding to amino acids 398 - 410 of 221368 PEA_1 P16, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of Z21368_PEA_1 P 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 CVIVPPLSQPQIH in Z21368_PEA_1 P16.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for Z21368_PEA_ 1 P22; comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVI,TDDQDVELGSL
QVNINKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYVI3fNHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRTAFFGKYLNEYNGSZ'IPPGVvrREWLGLIItNSRFYNYTVCR
NGIKEhHGFDYAK corresponding to amino acids 1 - 188 of SUL1 HUMAN, which also corresponds to amino acids 1 - 1S8 of Z21368_PEA_1 P22, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ARYDGDQPRCAPRPRGLSPTVF corresponding to amino acids 189 - 210 of Z21368_PEA_1 P22, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of 221368 PEA_1 P22, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ARYDGDQPRCAPRPRGLSPTVF in Z21368_PEA_1 P22.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for Z21368_PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologous to MILYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERIiNIRPNIILVLTDDQDVELGSL
QVMNKTRK~EHGGATFINAFVTTPMCCPSRSSIvIL,TGKYV~-1NHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRT corresponding to amino acids 1 - 137 of Q7Z2W2, which also corresponds to amino acids I - 137 of Z21368_PEA_1 P23, 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 GLLHRLNH _ _corresponding to amino acids 138 - 145 of 221368 PEA_1 P23, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of Z2136S PEA_I P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GLLHRLNH in Z21368_PEA_1 P23.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for Z21368_PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVL,GTELLGSLCSTVRSPRFRGRIQQERhNIRPNIILVLTDDQDVELGSL
QVIvINKTRKIMEHGGATF1NAFVTTPMCCPSRSSMLTGKY~VHNHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRT corresponding to amino acids 1 - 137 of SLTL IHiUMAN, which also corresponds to amino acids 1 - 137 of Z21368_PEA_1 P23, 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 GLLHRLNH _corresponding to amino acids 138 - 145 of Z21368_PEA_1 P23, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of 221368 PEA_1 P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GLLHRLNH in 221368 PEA_I P23.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832_P5, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYK corresponding to amino acids 12 - 55 of GILT_1-IUMAN, which also corresponds to amino acids 1 - 44 of T59832 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VGTATGRAGWREQAPCRGTRLLLSPQTSQGKTRAPRGRCPCRVPGhTLFSSRRCGHTP
SVPFRFRIPHLRGAAASTRLVPPKGSI''lSAYCVLLGQELGSPFVAQGTSS.AAGQGPPACIL, AATLDAFIPARAGLACLWDLLGRCPRG corresponding to amino acids 45 - 189 of T59832_P5, wherein said first and second amino acid~sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T5983? P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VGTATCTRAGWREQAPCRGTRLLLSPQTSQGKTRAPRGRCPCRVPGKTLFSSRRCGHTP
SVPFRFRIPHLRGAAASTRLVPPKGSMSAYCVLLGQELGSPFVAQGTSSAAGQGPPACIL
AATLDAFIPARAGLACLWDLLGRCPRG in T59832 P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832 P7, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLVI~~ILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVEAGVLDELDMELAFLTIVCMEEFEDI~~RSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMIiANAQRTDALQPPHEYVPWVTVNG corresponding to amino acids 12 - 223 of GILT HUMAN, which also corresponds to amino acids 1 - 212 of T59832_P7, 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 VRIFLALSLTLNPWSQGWTRQRDQR corresponding to amino acids 213 - 238 of T59832 P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T59832_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRIFLALSLTLIVPWSQGWTRQRDQR in T59832 P7.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59833 P7, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPV~NYhTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFItC
QHGEEECKFNKVEACVLDELDMELAFLTIVCI\~EFEDMERSLPLCLQLYAPGLSPDTII~i ECAMGDRGMQLMHANAQRTDALQPPHEYVPWVTVNG corresponding to amino acids 1 - 212 of BAC98466, which also corresponds to amino acids 1 - 212 of T59832_P7, 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 VRIFLALSLTLIVPWSQGWTRQRDQR corresponding to amino acids 213 - 238 of T59832_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T59832_P7, comprising a polypeptide being at least 70%, optionallyy 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 VRIFLALSLTLIVPWSQGWTRQRDQR in T59832_P7.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832_P7, comprising a first amino acid sequence being at least 70°.~0, optionally at least 80%, preferably at least 85%, more preferablyy at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ms MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKILSNA
PLVN VTLYYEALCGGCRAFLIRELFPTWLLV corresponding to amino acids 1 - 90 of T59832_P7, and a second amino acid sequence being at least 90 % homologous to MEILNVTLVPYGNAQEQNVSGRWEFhCQHGEEECKFNKVEACVLDELDMELAFLTIVC
MEEFEDMERSLPLCLQLYAPGLSPDT)IvvIECAMGDRGMQLMHANAQRTDALQPPHEYV
PWVTVNGVRIFLALSLTLIVPWSQGWTRQRDQR corresponding to amino acids 1 - 148 of BAC85622; which also corresponds to amino acids 91 - 238 of T59832_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of T59832_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence 1'~ITLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLV of T59832_P7.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832 P7, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFFTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVEACVLDELDMELAFLTIVCMEEFEDMERSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMI-iANAQRTDALQPPHEY'~rPWVTVNG corresponding to amino acids 1 - 212 of Q8WU77; which also corresponds to amino acids 1 - 212 of T59832 P7, and a~second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferablyy at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRIFLALSLTLIVPWSQGWTRQRDQR corresponding to amino acids 213 - 238 of T59832 P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of T59832_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRIFLALSLTLIVPWSQGWTRQRDQR in T59832_P7.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832 P9, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVEACVLDELDMELAFLTIVCMEEFEDMERSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMHANAQRTDALQPPHE corresponding to amino acids 12 - 214 of GILT HUMAN, which also corresponds to amino acids 1 - 203 of T59832 P9, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NPWkIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR corresponding to amino acids 204 - 244 of T59832_P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of T59832_P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NPWKIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR in T59832 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832_P9, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLIrLRGPLKKSNA
PLVNVTLY1'EALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNIiVEACVLDELDMELAFLTIVCMEEFEDMERSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMHANAQRTDALQPPHE corresponding to amino acids 1 - 203 of BAC98466; which also corresponds to amino acids 1 - 203 of T59832_P9, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NPWhIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR corresponding to i2o amino acids 204 - 244 of T59832 P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T59832 P9, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NPWIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR in T59832_P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832_P9, comprising a first amino acid sequence being at least 70%, optionally at least SO%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYILTGNLYLRGPLItItSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLV corresponding to amino acids 1 - 90 of T59832_P9, second amino acid sequence being at least 90 % homologous to MEIL,NVTLVPYGNAQEQNVSGRWEFIkCQHGEEECKFNKVEACVLDELDMELAFLTIVC
MEEFEDMERSLPLCLQLYAPGLSPDTIMECAMGDRGMQLMHANAQRTDALQPPHE
corresponding to amino acids 1 - 113 of BAC85622, which also corresponds to amino acids 91 -203 of T59832_P9, 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 preferablyy at least 95%
homologous to a polypeptide having the sequence NPWKIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR corresponding to amino acids 204 - 244 of T59832_P9, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an -isolated polypeptide encoding for a head of T59832 P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVN5.5.'ILTGNLYLRGPLKILSNA
PLVNVTLf'YEALCGGCRAFLIRELFPTWLLV of T59832 P9.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T5983?_P9, comprising a polypeptide being at least 1~1 70%, optionally at least about SO%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NPWKIR.PSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR in T59832 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832_P9, comprising a first amino acid sequence being at least 90 °,~o homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLI'YEALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECIhFNKVEACVLDELDMELAFLTIVCMEEFEDMERSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMHANAQRTDALQPPHE corresponding to amino acids 1 - 203 of Q8WL177, -which also corresponds to amino acids 1 - 203 of T59832_P9, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NPWKIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR corresponding to amino acids 204 - 244 of T59832_P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T59832 P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NPWKIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR in T59832 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832 P12, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVE corresponding to amino acids 12 - 141 of GILT_HLTMAN, which also corresponds to amino acids 1 - 130 of T59832 P12, and a second amino acid sequence being at least 90 % homologous to CLQLYAPGLSPDTIIvIECAMGDRGMQLMHANAQRTDALQPPHEYVPWVTVNGhPLED
QTQLLTLVCQLYQGKKPDVCPSSTSSLRSVCFK corresponding to amino acids 173 - 261 of GILT HUMAN, which also corresponds to amino acids 131 - 219 of T59832_P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of T59832 P 12, 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 nvo amino acids comprise EC, having a structure as follows: a sequence starting from any of amino acid numbers 130-x to 130; and ending at any of amino acid numbers 131+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832 P12, 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 MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNS.'KTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLV corresponding to amino acids 1 - 90 of T59832 P12, second amino acid sequence being at least 90 % homologous to MEILNVTLVPYGNAQEQNVSGRWEFILCQHGEEEChFNhVE corresponding to amino acids 1 - 40 of BAC85622, which also corresponds to amino acids 91 - 130 of T59832 P12, third amino acid sequence being at least 90 % homologous to CLQLYAPGLSPDTIMECAMGDRGMQLMHANAQRTDALQPPHEYVPW~ITVNG
corresponding to amino acids 72 - 122 of BAC85622, which also corresponds to amino acids 131 - 181 of T59832 P12, and a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence KPLEDQTQLLTLVCQLYQGI~KPDVCPSSTSSLRSVCFIt corresponding to amino acids 182 - 219 of T59832_P12, wherein said first, second, third and fourth amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a head of T59832 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most pref~ai~ly at least about 95% homologous to the sequence MTLSPLLLFLPPLLLLLI~y '~IPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGGdLhFZ,IRELFPT'WLLV of T59832 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for an edge portion of T59832_P12, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EC, having a structure as follows: a sequence starting from any of amino acid numbers 130-x to 130; and ending at any of amino acid numbers 131+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invvention, there is provided an isolated polypeptide encoding for a tail of T59832 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence KPLEDQTQLLTLVCQLYQGKKPDVCPSSTSSLRSVCFK in T59832_P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832 P12, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNI'KTGNLYLRGPLKKSNA
PLVNVTLYI'EALCGGCRAFLIRELFPTWLLVMEIL,NVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVE corresponding to amino acids 1 - 130 of Q8WU77, which also corresponds to amino acids 1 - 130 of T59832_P12, and a second amino acid sequence being at least 90 % ho mologous to CLQLYAPGLSPDTIMECAMGDRGMQLMHANAQRTDALQPPHEYVPWVTVNGKPLED
QTQLLTLVCQLIrQGKKPDVCPSSTSSLRSVCFK corresponding to amino acids 162 - 250 of Q8WU77, _ .which also corresponds to amino acids 131 - 219 of T59832 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for an edge portion of T59832_P12, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EC, having a structure as follows: a sequence starting from any of amino acid numbers 130-x to 130; and ending at any of amino acid numbers 131+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832 P 18, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYK corresponding to amino acids 12 - 55 of GILT_HUMAN, which also corresponds to amino acids 1 - 44 of T59832_P18, and a second amino acid sequence being at least 90 % homologous to CLQLYAPGLSPDTIMECAMGDRGMQLIviHANAQRTDALQPPHEYVPWVTVNGhPLED
QTQLLTLVCQLYQGKKPDVCPSSTSSLR._SVCFK corresponding to amino acids 173 - 261 of GILT HUMAN, which also corresponds to amino acids 45 - 133 of T59832_P18, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for an edge portion of T59832_P18, 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 KC, having a structure as follows: a sequence starting from any of amino acid numbers 44-x to 44; and ending at any of amino acid numbers 45+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832_P1S, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVN-Yh corresponding to amino acids 1 - 44 of Q8WU77, _which also corresponds to amino acids 1 - 44 of T59832_P1S, and a second amino acid sequence being at least 90 % homologous to CLQL1'APGLSPDTIMECAMGDRGMQLMHANAQRTDALQPPHEYVPVWTVNGKPLED
QTQLL,TLVCQLYQGKKPDVCPSSTSSLRSVCFK corresponding to amino acids 162 - 250 of Q8WU77; which also corresponds to amino acids 45 - 133 of T59832 P18, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of T59832 P18, 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 KC, having a structure as follows: a sequence starting from any of amino acid numbers 44-x to 44; and ending at any of amino acid numbers 45+ ((rr2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832_P1S, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVN1'K corresponding to amino acids 1 - 44 of Q8NEI4, which also corresponds to amino acids 1 - 44 of T59832_P18, and a second amino acid sequence being at least 90 % homologous to CLQLYAPGLSPDTIMECAMGDRGMQLMHAN.AQRTDALQPPHEYVPWVTVNGKPLED
QTQLLTLVCQLYQGKKPDVCPSSTSSLRSVCFK corresponding to amino acids 162 - 250 of Q8NEI4; which also corresponds to amino acids 45 - 133 of T59832 P18, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of T59832_P18, 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 Sll amino acids in length, wherein at least t,uo amino acids comprise KC, having a structure as follows: a sequence starting from any of amino acid numbers 44-x to 44; and ending at any of amino acid numbers 45+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HUMGRPSE_P4, comprising a first amino acid sequence being at least 90 % homologous to 12b MRGSELPLVLLALVLCLAPRG)~.~~ ~IPLPAGGGTVLTKMYPRGNHWAVGHLMGKKSTG
ESSSVSERGSLKQQLREYIRWEE~~.,~RNLLGLIEAKENRNHQPPQPKALGNQQPSWDSED
SSNFKDVGSKGK corresponding tc~ amino acids 1 - 127 of GRP_, which also corresponds to amino acids 1 - 127 of HUMGRPSE_P4, and a second amino acid sequence being at least 90 % homologous to GSQREGRNPQLNQQ corresponding to amino acids 148 of GRP_HL~MAN, which also corresponds to amino acids 128 - 141 of HLTMGRPSE_P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of HUMGRPSE_P4, 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 KG, having a structure as follows: a sequence starting from any of amino acid numbers 127-x to 127; and ending at any of amino acid numbers 128 + ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for HLTMGRPSE_P5, comprising a first amino acid sequence being at least 90 % homologous to MRGSELPLVLLALVLCLAPRGRAVPLPAGGGTVLTKMYPRGNHWAVGHLMGKKSTG
ESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSED
SSNFKDVGSKGK corresponding to amino acids 1 - 127 of GRP_H-U1MAN, which also corresponds to amino acids 1 - 127 of HUMGRPSE_P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DSLLQVLNVKEGTPS -corresponding to amino acids 128 - 142 of HUMGRPSE P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of HUMGRPSE_P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DSLLQVLNVKEGTPS in HUMGRPSE P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for AA155578_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MRAPHLHLSAASGARALAKLLPLLMAQLWAAEAALLPQNDTRLDPEAYGAPCARGSQ
PWQVSLFNGLSFHCAGVLVDQSWVLTAAHCGNKPLWARVGDDHLLLLQGEQLRRTT
RSVVI~KYHQGSGPIL,PRRTDEHI)LMLLKLARP corresponding to amino acids 1 - 146 of IkLKA_HUIviAN, which also corresponds to amino acids 1 - 146 of AA
155578_PEA_1 P4, and a second amino acid sequence being at least 90 % homologous to YNKGLTCSSITILSPKECEVFYPGWTNN1~CAGLDRGQDPCQSDSGGPLVCDETLQGIL
SWGVYPCGSAQHPAVYTQICKYMSWINKVIRSN corresponding to amino acids 184 - 276 of KL~ HUMAN, which also correspond s to amino acids 147 - 239 of AA155578 PEA_1 P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for an edge portion of AA155578 PEA_1 P4, 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 tvvo amino acids comprise PY, having a structure as follows: a sequence starting from any of amino acid numbers 146-x to 146; and ending at any of amino acid numbers 147+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for AA155578 PEA_1 P6, comprising a first amino acid sequence being at least 90 % homologous to MR.APHLHLSAASGARALAKLLPLLI\~IAQLW corresponding to amino acids 1 - 29 of KLKA_HUMAN, which also corresponds to amino acids 1 - 29 of.4A155578_PEA_1 P6, and a second amino acid sequence being at least 90 % homologous to VKYNKGLTCSSITILSPKECEVFYPGVVTNNMICAGLDRGQDPCQSDSGGPLVCDETLQ
GILSWGVYPCGSAQHPAVYTQICKYMSWINKVIRSN corresponding to amino acids 182 -276 of KLKA_ITtJNIAN, which also corresponds to amino acids 30 - 124 of i2a AA155575 PEA_1 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of AA15557s PEA_1 P6, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise WV, having a structure as follows: a sequence starting from any of amino acid numbers 29-x to 29; and ending at any of amino acid numbers 30+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for AA155575 PEA_1 P8, comprising a first amino acid sequence being at least 90 % homologous to MRAPHLHLSAASGARALAKLLPLLMAQLW corresponding to amino acids 1 - 29 of KLI~A_HLJMAN, which also corresponds to amino acids 1 - 29 of AA155575 PEA_1 P8, and a second amino acid sequence being at least 70%, optionally at least b0%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GHCGLE corresponding to amino acids 30 - 35 of AA15557g PEA_1 P8, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of AA155575 PEA_1 P8, comprising a polypeptide being at least 70%, optionally at least about RO%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GHCGLE in AA155578 PEA_1 P8.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for AA155578_PEA_I P9, comprising a first amino acid sequence being at least 90 % homologous to MRAPHLHLSAASGARALAKLLPLLMAQLWAAEAALLPQNDTRLDPEAYGAPCARGSQ
PWQVSLFNGLSFHCAGVLVDQSWVLTAAHCGNIL corresponding to amino acids 1 - 90 of KLhA- -HUIvIAN, which also corresponds to amino acids 1 - 90 of AA155578_PEA_1 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for HSENA7S P2, comprising a first amino acid sequence being at least 90 % homologous to MSLLSSRAARVPGPSSSLCALLVLLLLLTQPGPIASAGPAAAVLRELRCVCLQTTQGVHP
KMISNLQVFAIGPQCSKVEVV _corresponding to amino acids 1 - 81 of SZOS_HTJMAN, which also corresponds to amino acids 1 - 81 of HSENA78_P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T94936_PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MMLHSALGLCLLLVTVSSNLAIAIKKEKRPPQTLSRGWGDDITWVQTYEEGLFYAQKS
KKPLMVII-II-ILEDCQYSQALKKVFAQNEEIQEMAQNKFIMLNLMHETTDKNLSPDGQY
VPRIMFVDPSLTVRADIAGR1'SNRLYTYEPRDLPL corresponding to amino acids 1 - 150 of Q8TD06, which also corresponds to amino acids 1 - 150 of T94936_PEA_1 P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for T94936_PEA_1 P3, comprising a first amino acid sequence being at least 90 % homologous to MMLHSALGLCLLLVTVSSNLAIAIKKEKRPPQTLSRGWGDDITWVQTYEEGLFYAQKS
KKPLMVIHI-IL,EDCQYSQALKKVFAQNEEIQEMAQNKFIMLNLMHETTDKNLSPDGQYY
VPRIMFV corresponding to amino acids 1 - 122 of Q8TD06, which also corresponds to amino acids 1 - 122 of T94936_PEA_1 P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence GMYVISFHQIYKISRNQHSCFYF corresponding to amino acids 123 - 145 of T94936_PEA_1 P3, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of T94936_PEA_1 P3, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GMYVISFHQIYhISRNQHSCFYF in T94936_PEA_1 P3.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for 241644 PEA_1 P10, comprising a first amino acid sequence being at least 90 % homologous to MRLLAAALLLLLLALYTARVDGSKCKCSRKGPKIRYSDVKILLEMhPKYPHCEEKn~IVII
TTKSVSRYRGQEHCLHPKLQSTKRFIKWYNAWNEKRR corresponding to amino acids 1 -95 -of SZ14_HUMAN, which also corresponds to amino acids 1 - 95 of Z41644_PEA_1 P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence YAPPLLTFLPTRPSCGSQDGKGPPHQVI corresponding to amino acids 96 - 123 of Z41644_PEA_1 P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of 241644 PEA_1 P10, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence YAPPLLTFLPTRPSCGSQDGKGPPHQVI in 241644 PEA_1 P10.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptidc encoding for 241644 PEA_1 P10, comprising a first amino acid sequence being at least 90 % homologous to MRLLAAALLLLLLALYTARVDGSKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVII
TTKSVSRYRGQEHCLHPKLQSTKRFIKWYNAWNEILRR corresponding to amino acids 13 -107 -of Q9NS21, which also corresponds to amino acids 1 - 95 of Z41644_PEA_1 P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence YAPPLLTFLPTRPSCGSQDGKGPPHQVI corresponding to amino acids 96 - 123 of 241644 PEA_1 P 10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of Z41644_PEA_1 P10, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% b~~!~nologous to the sequence YAPPLLTFLPTRPSCGSQDGhGPPHQVI _in 241644 T'F:.'~_1 P10.
According to preferred embodiments of the present: ijivention, there is provided an isolated chimeric -polypeptide encoding for Z41644_PEA_1 P10, comprising a first amino acid sequence being at least 90 % homologous to MRLLAAALLLLLLALYTARVDGSKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVII
TTKSVSRYRGQEHCLHPKLQSTKRFIKWYNAWNEKRR corresponding to amino acids 13 -107 of AAQ89265, which also corresponds to amino acids 1 - 95 of ~41644_PEA_1 P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence YAPPLLTFLPTRPSCGSQDGKGPPHQVI corresponding'to amino acids 96 - 123 of 241644 PEA_1 P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ _encoding for a tail of 241644 PEA_1 P10, compr7sing 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 YAPPLLTFLPTRPSCGSQDGKGPPHQVI in 241644_PEA_1 P10.
According to preferred embodiments of the present invention, there is provided an isolated 2G oligonucleotide, comprising an amplicon selected from the group consisting of SEQ ID NOs:
891 or 894.
According to preferred embodiments of the present invention, there is provided a primer pair, comprising a pair of isolated oligonucleotides capable of amplifying the above.
Optionally, the pair of isolated oligonucleotides is selected from the group consisting of: SEQ
NOs 889 and 890; or 892 and 893.
According to preferred embodiments of the present invention, there is provided an antibody capable of specifically binding to an epitope of an amino acid sequence as described herein. Optionally, the epitope may comprise a tail, head, or edge portion as described herein.
According to preferred embodiments of the present invention, the antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
According to preferred embodiments of the present invention, there is provided an kit for detecting breast cancer, comprising a kit detecting overexpression of a splice variant as described herein. Optionally, the kit comprises a NAT based technology.
Preferably, the kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein Optionally, the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence as described herein.
Optionally, the kit comprises an antibody as described herein. Preferably, the kit further comprises at least one reagent for performing an ELISA or a Western blot.
According to preferred embodiments of the present invention, there is provided a method for detecting breast cancer, comprising detecting overexpression of a splice variant as described herein.
Optionally detecting overexpression is performed with a NAT-based technology.
Preferably, detecting overexpression is performed with an immunoassay. More preferably, the immunoassay comprises an antibody as described herein.
According to preferred embodiments of the present invention, there is provided a biomarker capable of detecting breast cancer, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
According to preferred embodiments of the present invention, preferably any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology thereto.
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).
All nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate to their isolated form, as isolated polynucleotides (including for all transcripts), oligonucleotides (including for all segments, amplicons and primers), peptides (including for all tails, bridges, insertions or heads, optionally including other antibody epitopes as described herein) and/or polypeptides (including for all proteins). It should be noted that oligonucleotide and polynucleotide, or peptide and polypeptide, may optionally be used interchangeably.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. 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 (Wall:er ed., 1988);
The Glossary of Genetics, SthEd., 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. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
BRIEF DESCRIPTION OF DRAWINGS
Figure I is schematic summary of cancer biomarkers selection engine and the wet validation stages.
Figure 2. Schematic illustration, depicting grouping of transcripts of a give n cluster based on presence or absence of unique sequence regions.
Figure 3 is schematic summary of quantitative real-time PCR analysis.
Figure 4 is schematic presentation of the oligonucleotide based microarray fabrication.
Figure 5 is schematic summary of the oligonucleotide based microarray experimental flow.
Figure 6 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster T10888, demonstrating overexpression in colorectal cancer, a mixture of malignant tumors from different tissues, pancreas carcinoma and gastric carcinoma.
Figure 7 is a histogram showing expression of the CEA6 HLTNLAN
Carcinoembryonic .antigerrrelated cell adhesion molecule 6 (T10888) transcripts, which are detectable by amplicon as depicted in sequence name T10888 juncl l-17, in normal and cancerous breast tissues.
Figure 8 is a histogram showing the expression of CEA6 HUMAN Carcinoembryonic antigen-related cell adhesion molecule 6 T10888 transcripts which are detectable by amplicon as depicted in sequence name T10888juncl 1-17 in different normal tissues.
Figure 9 is a histogram showing Cancer and cell-line vs. awrmal tissue expression for Cluster T39971, demonstrating overexpression in liver cancer, lszng malignant tumors and pancreas carcinoma.
Figure 10 is a histogram showing the expression of of VTNC_HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein) T39971 transcripts, which are detectable by amplicon as depicted in sequence name T39971 junc23-33 in normal and cancerous breast tissues.
Figure 11 is a histogram showing the expression of VTNC_HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein), antisense to SARM1 (T23434), T39971 transcripts, which are detectable by amplicon as depicted in sequence name T39971 junc23-33, in different normal tissues.
Figure 12 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster 221368, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and pancreas carcinoma.
Figure 13 is a histogram showing the expression of SLTL 1 HLTI~IAN -Extracellular sulfatase Sulf 1221368 transcripts, which are detectable by amplicon as depicted in sequence name 221368seg39, in normal and cancerous breast tissues.
Figure 14 is a histogram showing the expression of SLTL 1 HUMAN -Extracellular sulfatase Sulf 1221368 transcripts, which are detectable by amplicon as depicted in sequence name 221368seg39, in different normal tissues.
Figure 15 is a histogram showing the expression of SLTL 1 HUMAN -Extracellular sulfatase Sulf 1 221368 transcripts which are detectable by amplicon as depicted in sequence name 221368junc17-21 in normal and cancerous breast tissues.
Figure 16 is a histogram showing the expression of SUL1 HUMAN - Extracellular sulfatase Sulf 1 221368 transcripts, which are detectable by amplicon as depicted in sequence name 221368junc17-21, in different normal tissues.
Figure 17 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster T59832, demonstrating overexpression in brain malignant tumors, breast malignant tumors, ovarian carcinoma and pancreas carcinoma.
Figure 18 is a histogram showing low over expression observed for cluster T59832, amplicon name: T59832 junc6-25-26, in one experiment earned out with breast cancer samples panel.
Figure 19 is a histogram showing the expression of Gl~_HLJMAN - gastrin-releasing peptide (HUMGRPSE) transcripts, which are detectable by amplicon, as depicted insequence name HUIvIGRPSEjune3-7 in normal and cancerous breast tissues.
Figure 20 is a histogram showing the expression of GI2P-HUMAN -gastrirrreleasing peptide (HCTMGRPSE) transcripts, which are detectable by amplicon, as depicted in sequence name HUMGRPSEjunc3-7, in different normal tissues.
Figure 21 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster AA155578, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and pancreas carcinoma.
Figure 22 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HSENA78, demonstrating overexpression in epithelial malignant tumors and lung malignant tumors.
Figure 23 is a histogram showing the expression ofHomo sapiens breast cancer membrane protein 11 (BCMP11) T94936 transcripts which are detectable by amplicon as depicted in sequence name T94936 segl4 in normal and cancerous Breast tissues.
Figure 24 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster 241644, demonstrating overexpression in lung malignant tumors, breast malignant tumors and pancreas carcinoma.
Figure 25 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster M85491, demonstrating overexpression in epithelial malignant tumors and a mixture of malignant tumors from different tissues.
Figure 26 is a histogram showing the expression ofEphrin type-B receptor 2 precursor (EC 2.7.1.112) (Tyrosine-protein kinase receptor EPH-3) M85491 transcripts which are detectable by amplicon as depicted in sequence name M85491 seg24 in normal and cancerous breast tissues.
Figure 27 is a histogram showing the expression of Ephrin type-B receptor 2 precursor M85491 transcripts, which are detectable by amplicon as depicted in sequence name IvIS5491 seg24, in different normal tissues.
Figure 28 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HSSTROL3, demonstrating overexpression in transitional cell carcinoma, epithelial malignant tumors, a mixture of malignant tumors from different tissues and pancreas carcinoma.
Figure 29A is a histogram showing the expression of Expression of Stromelysin-precursor (EC 3.4.24.-) (lMatrix metalloproteinase-11) (MIvvIP-11) (ST3) SL-3 transcripts which are detectable by amplicon as depicted in sequence name HSSTROL3 seg24 in normal and cancerous breast tissues.
Figure 29B is a histogram showing the expression of Stromelysin-3 precursor (EC
3.4.24.-) (Matrix metalloproteinase-11) (N>IVIy-11) (ST3) (SLr3) HSSTROL3 transcripts, which are detectable by amplicon as depicted in sequence name HSSTROL3 seg24, in different normal tissues.
Figures 30A 30C shows histograms showing over expression of various Stromelysin-3 precursor transcripts in cancerous breast samples relative to the normal samples.
Figure 31 is a histogram showing Cancer and cell-line vs. normal tissue expression far Cluster 875793, demonstrating overexpression in epithelial malignant tumors and a mixture of malignant tumors from different tissues.
Figure 32 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HLJMCA1XIA, demonstrating overexpression in bone malignant tumors, epithelial malignant tumors, a mixture of malignant tumors from different tissues and lung malignant tumors.
Figure 33 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster 820779, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and lung malignant tumors.
Figure 34 is a histogram shoal ng Cancer and cell-line vs. normal tissue expression for Cluster HSS 100PCB, demonstrating overexpression in a mixture of malignant tumors from different tissues.
Figure 35 is a histogram showing Cancer and cell-line vs. nornial tissue expression for Cluster HSCOC4, demonstrating overexpression in brain malignant tumors, a mixture of malignant tumors from different tissues, breast malignant tumors, pancreas carcinoma and prostate cancer.
Figure 36 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HUMTREFAC, demonstrating overexpression in a mixture of malignant 'tumors from different tissues, breast malignant tumors, pancreas carcinoma and prostate cancer.
Figure 37 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HUMOSTRO, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues, lung malignant tumors, breast malignant tumors, ovarian carcinoma and skin malignancies.
Figure 38 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster 811723, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and kidney malignant tumors.
Figure 39 is a histogram showing the expression of of 811723 transcripts which are detectable by amplicon as depicted in sequence name 811723 segl3 in normal and cancerous breast tissues.
Figure 40 is a histogram showing the expression of 811723 transcripts, which are detectable by amplicon as depicted in sequence name RI 1723seg13, in different normal tissues.
Figures 41A and B are histograms showing the expression of 811723 transcripts, which are detectable by amplicon as depicted in sequence name 811723 juncl l-18 in normal and cancerous breast tissues (Figure 41A) or on a panel of normal tissues (Figure 41B).
Figure 42 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster T46984, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues, breast malignant tumors, ovarian carcinoma and pancreas carcinoma.
Figure 43 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HSMUC1A, demonstrating overexpression in a mixture of malignant tumors from different tissues, breast malignant tumors, pancreas carcinoma and prostate cancer.
Figures 44-47 are histograms showing the combined expression of 8 sequences (T10888seg11-17, HUMGRSE junc3-7, HSSTROL3seg24, T94936 Seg 14, 221368 seg39, 221368 juncl7-21 T59832 jun6-25-26 and M85491seg24) in normal and cancerous breast tissues.
Figure 48 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HSU33147, demonstrating overexpression in a mixture of malignant tumors from different tissues.
DESCRIPTION OF PREFERRED EMBODIIVVIENTS
The present invention is of novel markers for breast cancer that are both sensitive and accurate. Furthermore, at least certain of these markers are able to distinguish between different stages of breast cancer, such as 1. Ductal carcinoma (in-situ, invasive) 2.
Lobular carcinoma (is-situ, invasive) 3. inflammatory breast cancer 4. Mutinous carcinoma 5. Tubular carcinoma 6.
Paget's disease of nipple, alone or in combination; or one of the indicative conditions described above.
The markers of the present invention, alone or in combination, can be used for prognosis, prediction, screening, early diagnosis, staging, therapy selection and treatment b monitoring of breast cancer. For example, optionally and preferably, these markers may be used for staging breast 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 then breast. Also, one or more of the markers may optionally be used in combination with one or more other breast cancer markers (other than those described herein).
Biomolecular sequences (amino acid and/or nucleic acid sequences) uncovered using the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease.
These markers are specifically released to the bloodstream under conditions of breast cancer (or one of the above indicative conditions), and/or are otherwise expressed at a much higher level and/or specifically expressed in breast cancer tissue or cells, and/or tissue or cells under one of the above indicative conditions. The measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of breast cancer and/or a condition that it is indicative of a higher risk for breast cancer.
The present invention therefore also relates to diagnostic assays for breast cancer and/or an indicative condition, and methods of use of such markers for detection of breast cancer and/or an indicative condition, optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
According to a preferred embodiment of the present invention, use of the marker optionally and preferably permits a non-cancerous breast disease state to be distinguished from breast cancer and/or an indicative condition. A non limiting example of a non-cancerous breast disease state includes breast fibrosis and/or cysts. According to another preferred embodiment of the present invention, use of the marker optionally and preferably permits an indicative condition to be distinguished from breast cancer.
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.
As used herein a "tail" refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention.
Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (o$en 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.
As used herein a "head" refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein.
As used herein "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 "knov~m 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 "knotvn protein" portion of a variant.
Optionally and preferably, a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant, comprises at least about 10 amino acids, more preferably at least about 20 amino acids, most preferably at least about 30 amino acids, and even more preferably at least about 40 amino acids, in which at least one amino acid is from tl~
tail/head/insertion and at least one amino acid is from the "known protein"
portion of a variant.
Also optionally, the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 1 l, 12, 13...37, 33, 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_Pl (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at East 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 l, nor 50 +
((n-2) - x) (for example) greater than the total sequence length.
In another embodiment, this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention.
Preferably such antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
In another embodiment, this invention provvides an isolated nucleic acid molecule ewcoding for a splice variant according to the present invention, having a nucleotide sequence as 5e~ forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention. In another embodiment, this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.
In another embodiment, this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising:
contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variants) 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 splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.
According to the present invention, the splice variants described herein are norrlimiting examples of markers for diagnosing breast cancer and/or an indicative condition. Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of breast cancer and/or an indicative condition, including a transition from an indicative condition to breast cancer.
According to optional but preferred embodiments of the present invention, any marker according to the present invention may optionally be used alone or combination. Such a comb ination .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, andlor any other mailer. With regard to such a ratio between any marker described herein (or a combination thereof) and a knoGVn marker, more preferably the known marker comprises the "known protein'' as described in greater detail below with regard to each cluster or gene.
According to other preferred 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 breast cancer and/or an indicative condition, such that a biomarker may optionally comprise any of the above.
According to still other preferred 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 fmited 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 thereW th, sequences homologous thereto [e.g., at least 50 %, at least 5~ %, 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 nuc leic 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 ma n 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 irz vivo or in vitro using a DNA dependent DNA
polymerase.
.'~.s used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived d~i~~olated) from a chromosome and thus it represents a contiguous portion of a chromosc~t~ue.
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.
Preferred embodiments of the present invention encompass oligonucleotide probes.
An example of an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
Alternatively, an oligonucleotide probe of the present invention can he 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. ItQ., ed.
(1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland ( 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 preferred 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.
Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other ali~yl phosphonates including 3'-allylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoallylphosphoramidates, 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, mia:ed heteroatom and alkyl or cycloallyl 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 CHI component parts, as disclosed in LT.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 (L~.
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 allyl 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, S-trifluoromethyl and other 5-substituted uracils and cytosines, '~methylguanine and 7 methyladenine, lrazaguanine and &
azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Further bases particularly useful for increasing the binding amity of the oligomeric compounds of the invention include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including ? aminopropyladenine, ~propynyluracil and ~-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, cho1ie acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rao-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: ti,303,374.
It is not necessary for all positions in a given oligonucleotide mo lecule 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.
Preferably, 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., (19891 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 (LT.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 preferably further includes an appropriate selectable marker and/or an origin of replication. Preferably, 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.
Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/ ), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com). Examples of retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif., includingRetro-X
vectors pLNC~ and pLXSN, which permit cloning into multiple cloning sites and the trasgene is transcribed from CMV promoter. Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. 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]]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, A:.~-4:YJ, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes a.t least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present n the viral construct. In addition, such a construct typically includes 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 construe is will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA
synthesis, and a 3' LTR
or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
Hybridization assays Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non-Limiting examples of probes according to the present invention were previously described).
Traditional hybridization assays include PCR, R~1'-PCR, Real-time YC;K, KNase 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 whic h 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 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides 30 long.
lso 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 cond itions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 3ZP
labeled probe, at 65 °C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65°C and whereas moderate hybridization is effected using a hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 3'P
labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.
More generally, hybridization of short nucleic acids (below 200 by in length, e.g. 17-40 by 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 x SSC and 1 SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.S), 1 mM EDTA (pH 7.61, 0.5 %
SDS, 100 pg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 °C below the Tnv final wash solution of 3 M TIvIACI, 0.01 M
sodium phosphate (pH
6.S), 1 mM EDTA (pH 7.6), 0.5 % SDS at I - 1.5 °C below the Tm; (ii) hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.S), 1 mM
EDTA
(pH 7.6), 0.5 °~o SDS, 100 p.g/ml denatured salmon spern~ DNA and 0.1 %
nonfat dried milk, hybridization temperature of 2 - 2.5 °C below the Tm, final wash solution of 3 IvI TMACI, 0.01 M sodium phosphate (pH 6.S), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm, final wash solution of 6 x SSC, and final wash at 22 °C; (iii) hybridization solution of 6 x SSC
and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.S), 1 mNI EDTA (pH
"MAM" for "breast";
"BRAIN" for "brain";
"UTERUS" for "uterus";
"SKIN" for "skin";
10 "KIDNEY" for "kidney";
"MUSCLE" for "muscle";
"ADREN" for "adrenal";
"HEAD" for "head and neck";
"BLADDER" for "bladder";
It should be noted that the terms "segment", "seg" and "node" 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 below. They are also the building blocks that were used to construct complete nucleic acid sequences as described in greater detail below. 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.
As used herein the phrase "breast cancer" refers to cancers of the breast or surrounding tissue, including but not limited to ductal carcinoma (in-situ or invasive), lobular carcinoma (in-situ or invasive), inflammatory breast cancer, mucinous carcinoma, tubular carcinoma, or Paget's disease of the nipple, as well as conditions that are indicative of a higher risk factoz for later development of breast cancer, including but not limited to ductal hyperplasia without atypia and atypical hyperplasia, referred to herein collectively as "indicative conditions".
The term "marker" in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from subjects (patients) having breast cancer (or one of the above indicative conditions) as compared to a comparable sample taken from subjects who do not have breast cancer (or one of the above indicative conditions).
The phxase "differentially present" refers to differences in the quantity of a marker present in a sample taken from patients having breast cancer (or one of the above indicative conditions) as compared to a comparable sample taken from patients who do not have breast cancer (or one of the above indicative 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.
As used herein 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.
As used herein the phrase "diagnosing" refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease andlor prospects of recovery. The term "detecting" may also optionally encompass any of the above.
Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide 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 rer,ncrved from the subject, as described in greater detail below.
As used herein, the term "level" refers to expression levels of RNA andlor 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 biopsylsample 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 andlor amplification and/or a decreased expression, of the variant as opposed to the normal tissues.
A "test amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of breast cancer (or one of the above indicative conditions). A test amount can be either in absolute amount (e.g., microgramlml) or a relative amount (e.g., relative intensity of signals).
A "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a patient with breast cancer (or one of the above indicative conditions) or a person without breast cancer (or one of the above indicative conditions). A
control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
"Detect" refers to identifying the presence, absence or amount of the object to be detected.
A "label" includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means. For example, useful labels include 3zP, 3ss~
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. Itlausner, 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," when referring to a protein or peptide (or other epitope), refers 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 tZVO 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). Typicallyy a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name T10888_PEA_1 T1 T10888_PEA_1T4 T1088SPEA_1 TS
T10888_PEA_1T6 a nucleic acid sequence comprising a sequence in the table below:
SegmentName T10888_PEA_1 node_11 TI0888_PEA_1 12 node_ T10888_PEA_1 17 node_ T10888PEA_1 4 node T 10888_PEA_1 6 node T10888_PEA_1 7 node_ T10888PEA_1 9 node T10888PEA_1 15 node_ According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below amino acid sequence comprising a sequence in the table below:
Proteii,~
Nat~e T 10888_PEA_1P2 T 10888_PEA_P4 T10888_PEA_1PS
5 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name a nucleic acid sequence comprising a sequence in the table below:
Segment Name T39971 node 0 T39971 node 18 T39971 node 21 T399 r~.ode22 i' T39971n:,ode23 T39971~node31 T39971node33 T39971node7 T39971node1 T39971node10 T39971node11 T39971node12 T39971node15 T39971node16 T39971node17 T39971node26 T39971node27 T39971node28 T39971node29 T39971node3 T39971node30 T39971node34 T39971node_35 T39971node36 T39971node4 T39971node5 T39971node8 T39971node9 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below:
Protein ZtTan~ , 39971 P12 , According to preferred embodiments of the present invvention, there is provided an isolated polynucleotide comprising~~a nucleic acid sequence in the table below and/or:
Tran.~cript .
Name I
I
I
a nucleic acid sequence comprising a sequence in the-table below:
Se,gioaerxt Name 221368PEA node Q
221368PEA node 1S
221368PEA node 19 I
221368PEA node 2 221368PEA node 21 I
22.],3,681?EA_1node 33 221368PEA node 36 Z2136S node 37.
PEA
221368PEA node 39 221368PEA node 4 _.
Z21368_PEA_1node 41 221368PEA node 43 Zs 221368PEA_1 node_45 Z21368_PEA_1 node_53 Z21368_PEA_1 node_56 .~213.68~PEA_1 node58 _ .
221368PEA_1 node_66 .:tW..:i. -221368PEA1 node67 Z21368_PEA_1 node_69 Z21368_PEA_1 node_11 Z21368_PEA_1 node_12 221368PEA_1 node_16 Z21368_PEA_1 node_17 Z21368_PEA_1 node_23_ 221368PEA1 node24 Z21368_PEA_1 node_30 221368PEA_1 node_31 221368PEA1 node38 -Z21368PEA1 node47 Z21368_PEA_1 node_49 221368PEA1 node51 Z21368_PEA_1 _61 node Z21368_PEA_I 68 node 221368PEA1 node7 According to preferred embodiments of the present invvention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name 221368 PEA 1~ P2 Z21368_PEA_1 P15 PEA
I
I' l 221368PEAI P:22 221368PEA1 p~23 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name T59832_T8 a nucleic acid sequence comprising a sequence in the table below:
Segment Name , T59832node1 T59832node22 T59832_node_23 T59832node_24 T59832node29 T59832_node39 T59832node7 T59832node10 T59832node11 T59832node12 T59832node14 T59832_node_16 T59832node_19 T59832node2 T59832_node20 2o TS9832node2S
TS9832node26 TS9832node27 TS9832node28 TS9832node3 TS9832node30 TS9832node31 TS9832node32 TS9832node34 TS9832node3S
TS9832node36 TS9832node37 TS9832node3S
TS9832node4 TS9832nodeS
TS9832node._6 TS9832node8 TS9832node9 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name TS9832_PS
TS9832_P18 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transez~pt Name a nucleic acid sequence comprising a sequence in the table below:
Segment ~1'~lame 241644 1 node 0 PEA
241644 1 node_11 PEA_ Z41644_PEA_1 node_12 241644 1 node 15 PEA
241644 1 node 20 PEA
241644 1 node 24 PEA
241644 1_node_1 PEA_ 241644 1 node 10 PEA
241644 1 node_13 PEA_ 241644 1 node 16 PEA
241644 1 node 17 PEA
241644 1 node 19 PEA
Z41644_PEA_1 node_2 241644 1 node 21 PEA
241644 1 node 22 PEA_ 241644 1 node 23 PEA
241644 1 node_25 PEA_ Z41644_PEA_I node 3 241644 1 node 4 PEA
241644 1 node 6 PEA
241644 1 node 9 PEA_ According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript tam HUMGRPSE T4 ~~~
a nucleic acid sequence comprising a sequence in the table below:
Segment Name .
HUMGRPSE node 0 HUMGRPSE node 2 HUMGRPSE node g-_ HUMGRPSE node 3 HUMGRPSE_rbade_'7 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name GRPSE_P5 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Naive AA155578 PEA_1 T13 .4A155578 PEA 1 T8 a nucleic acid sequence comprising a sequence in the table below:
Segtnettt Name AA155575PEA_1 node_11 AA155578PEA 1 node12 AA155578_PEA_1 node_14 AA155578PEA_1 node_19 AA155578PEA_1 node21 AA155578PEA_1 node_23 AA155578PEA_1 node24 AA 155578PEA_1 node25 AA155578PEA_1 node4 AA15557bPEA_1 node_7 AA155578-PEA_1 node_I5 AA155578PEA_1 node_1S
AA155578PEA_1 node22 AA15557RPEA _1 6 node AA 15 PEA_1 node_8 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protei~a Name AA155575PEA_1 P6 AA15S578PEA_1 P8 AA 155578_PEA_1 P9 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
'T'ransc,,ript Name a nucleic acid sequence comprising a sequence in the table below:
S~egrnsnt Name HSENA78 node 0 HSENA7S nude HSENA78 node 6 HSENA7S node 9 HSENA78 node 3 HSENA78 node 4 HSENA78 node a According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below:
Pxotein Name According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcz-i~t Name , 'I T94936_PEA_1 T2 a nucleic acid sequence comprising a sequence in the table below:
Segnnent Name T94936PEA1 nodeI4 T94936_PEA_1 node_16 T9493 PEA_1 _node_2 6_ T94936_PEA_1 node20 T94936PEA1 node23 T94936PEA1 node0 T94936_PEA_ 1 node_11 T94936_PEA_1 node_13 T94936_PEA_1 node_17 T94936PEAI node6 T94936PEA1 node8 T94936PEA1 node9 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name-T94936_PEA_1 P3 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Trattscript Narne M85491_PEA_1 T20 a nucleic acid sequence comprising a sequence in the table below:
Segment Name M85491 PEA_1 node_0 2b node node node M85491PEA_I 24 node M85491_PEA_1 8 node M85491_PEA_1 9 node node MS5491PEA_1 18 node_ node M85491PEA_1 6 node According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name M8549I PEA_1 PI4 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcripx Name a nucleic acid sequence comprising a sequence in the table below:
Segment Name HSSTROL3 node 6 HSSTROL3 node10 HSSTROL3 node13 HSSTROL3 node15 HSSTROL3 node19 HSSTROL3 node21 HSSTROL3 node HSSTROL3 node25 HSSTROL3 node HSSTROL3 node28 HSSTROL3 node29 HSSTROL3 node11 HSSTROL3 node17 HSSTROL3 node_18 HSSTROL3 node20 HSSTROL3 node According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Proteui .Name:
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name 2s a nucleic acid sequence comprising a sequence in the table below:
Segment Names AY180924_PEA_1 node 3 AY 180924 PEA_ 1 node 0 AY180924_PEA_I node 2 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein N~txte ,. , According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name R75793_PEA_1 T5 a nucleic acid sequence comprising a sequence in the table below:
node node node node R75793_PEA_1 4 node 875793PEA_1 5 node R7S793_PEA_1 6 node_ 875793PEA _1 8 node 875793 PEA_1 node_13 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Proteain Name 875793 PEA_1 P2 875793 PEA_1 PS
875793 PEA_1 P6 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
T~anseript I'Jame HLTMC.A 1 XIA_T 16 HUMCAIh'IA T19 HC1NICA1~ T2ll a nucleic acid sequence comprising a sequence in the table below:
Segment-Name HL1MCA 13~IA_node_0 HLiT4CA 1 XIA_node2 HUIVICAI~IA
node 4 node HUIVICA 1 ~'IA 8 node HUMCAIZIA_node 9 HUMCA1~IA node _18 node HUMCAIXIA-node 55 node HUMCAI XIA. 11 node node node node node HUMCA 1 XIA-node_25 node HUMCA1XIA node29 node_ XIA node HjJMCA 1 XIA 35 node node node node 41 node 43 HUMCA1XIA node HUMCA1XIA node HUMCA1XIA node_49 HUMCA1XIA node51 HUMCAIXIA node_S7 node HUMCA1XIA node62 node HUMCA1XIA node66 HUIvICAIXIA 68 node I-IUMCA1XIA_node70 HUMCA 1 XIA-node_72 XIA node 74 I-IUMCA I XIA_node_76 HLTMCA1XIAnode78 HLTMCA node81 HUMCA 1 node83 XIA
node -.
HUMCA1XTA node87 HUMCA1XIA nodeS9 HLTMCA node 1 XIA_ 91 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Prot~iu Name HLTMCA 1 XIA P 17 -__ According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name' a nucleic acid sequence comprising a sequence in the table below:
Segment Na.nn node 820779 2 -_ node node node node node_ 820779node24 820779node27 820779node28 820779node30 820779node3I
820779node32 820779node1 820779node3 820779nodeIO
820779node1l 820779nodeI4 820779node17 820779node19 820779node20 820779node22 820779node23 820779node25 820779node29 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence according to R20779_P2.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name ' ' a nucleic acid sequence comprising a sequence in the table below:
Segnent Name HSS 100PCB_node_3 HSS 100PCB node 4 HSS100PCB node_5 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence according to HSS100PCB
P3.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name HSCOC4_PEA_1 T3 HSCOC4_PEA_1 T4 HSCOC4 PEA_1 T8 HSCOC4 PEA_1 T1?
HSCOC4 PEA_1 T15 HSCOC~ PEA_1 T21 HSCOC4_PEA_1 T25 HSCOC4_PEA_1 T30 HSCOC4_PEA_1 T31 a nucleic acid sequence comprising a sequence in the table below:
Segmenf Name HSCOC4 _PEA_1 1 node_ HSCOC4 _PEA_1 5 node_ HSCOC4 PEA_1 7 node HSCOC4 _PEA_1 30 node HSCOC4 PEA_1 33 node_ node node node node node node node node node PEA node node node node node node node node node node node node node node HSCOC4_PEA_1 2 node_ node HSCOC4_PEA_ 1 10 node_ HSCOC4_PEA_ 1 12 node_ HSCOC4_PEA_1 14 node_ HSCOC4_PEA_1 17 node_ HSCOC4_PEA _1 19 node_ node HSGOC4_PEA_1 22 node node HSCOC4 PEA_1 29 node node node HSCOC4_PEA_ 1 47 node node node node node node node nude node PEA node PEA node node node node node PEA node PEA node HSCOC4_PEA_1 S3 node_ node node node node PEA node node PEA node node node node node node node node node node node node node HSCOC4 _PEA_1 111 node_ According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below protein Name PEA
PEA
HSCOC4 PEA 1 P4?
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name HUIvTTREFAC PEA 2 T4 a nucleic acid sequence comprising a sequence in the table below:
Segment Name HUMTREFAC PEA node 0 HUI~~ITREFACPEA node 9 HUMTREFAC PEA node 2 HLJMTREFAC PEA node 3 HUMTREFAC PEA node 4 HLThrITREFACPEA node 5 HUMTREFAC PEA node 8 According to preferred embodiments of the present invention, there is provided t2x~~
isolated polypeptide comprising an amino acid sequence in the table below Prat~in Name HUMTREFAC_PEA_2 P7 HUMTREFAC_PEA_2_P 8 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name HUMOSTRO PEA_1 PEA_1_T14 ~ HUMOSTRO PEA_1 PEA_1 T16 ~ HUMOSTRO PEA_1 PEA_1 T30 a nucleic acid sequence comprising a sequence in the table below:
'Segnnent .
Natn~
HUMOSTRO PEA_1 1_node0 PEA_ HUMOSTRO PEA_1 1 10 PEA_ node_ HUMOSTRO_ PEA_1 1 16 PEA_ node_ HUNIOSTRO_PEA_1 1 23 PEA_ nods HUMOSTRO PEA_1 1_node_31 PEA_ PEA_ PEA_ node_43 HLTMOSTRO_PEA_ I 1 3 PEA_ node_ HUMOSTRO_PEA_ 1 1 _5 PEA_ node HUMOSTRO_ PEA_1 1_node_7 PEA_ HCTI~~IOSTROPEA_1 1_node8 PEA_ HUMOSTRO_ PEA_1 1_node_15 PEA_ HUMOSTRO PEA_1 1 _17 PEA_ node HLJMOSTRO PEA_1 1 20 PEA_ node PEA_ PEA_ node _ )-ItTMOSTRO_PEA_1 1 22 PEA_ node HLTMOSTRO PEA_1 1 24 PEA_ node_ HUMOSTRO_PEA_ 1 1 26 PEA_ node HUMOSTRO_PEA_ I 1 PEA_ node HUIvI0STR0_PEA_1 1 PEA_ node HLTMOSTRO_PEA_1 1 29 PEA_ node HUMOSTRO PEA_ I 1 30 PEA_ node_ HUMOSTRO PEA_ 1 1 32 PEA_ node HUMOSTRO_PEA_ I I 34 PEA_ node HUMOSTRO PEA_ 1 1 36 PEA_ node HLTMOSTRO PEA_1 1 37 PEA_ node )-IZTMO STRO 1 1 _3 S
PEA_ PEA_ node HLTMOSTRO_PEA_1 _1 35~
PEA rode HUMOSTRO PEA_ 1 1 _40 PEA_ node HUMOSTRO_PEA_ 1 1 PEA_ node HUMOSTRO PEA_ 1 1 PEA_ node_42 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name HUMOSTRO PEA_I PEA_1 P21 HUMOSTRO_PEA_1 PEA_1 P25 HUhZOSTRO PEA_1 PEA_1 P30 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: Rl 1723 PEA_1 TIS, 811723 PEA_1 T17, R11723_PEA_1 T19, 811723 PEA_I T20, R11723_PEA_1 T5, or R11723_PEA_1 T'6.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a mde having a sequence selected from the group consisting of : 811723 PEA_1 node_13, R11723_PEA_1 node_16, 811723 PEA_1 node_19, 811723 PEA_1 node 2, R11723_PEA_1 node 22, R11723_PEA_1 node 31, 5 R11723_PEA_1 node_10, 811723 PEA_1 node_l l, 811723 PEA_1 node_15, 811723 PEA_1 node_18, 811723 PEA_1 node 20,, _811723 PEA_1 node 21, R11723_PEA_1 node_23, 811723 PEA_1 node 24, 811723 PEA_1 node 25, 811723 PEA_1 node_26, 811723 PEA_1 node 27, 811723 PEA_1 node 28, 811723 PEA_1 node 29, 811723 PEA_1 node 3, R11723_PEA_1 node 30, 10 811723 PEA_1 node 4, R11723_PEA_1 node_5, 811723 PEA_1 node 6, 811723 PEA_1 node_7 or 811723 PEA_1 node 8.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of : 811723 PEA_1 P2, _ _811723 PEA_1 P6, 811723 PEA_1 P7, 15 811723 PEA_ 1 P 13, or 811723 PEA_ 1 P 10.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name, PEA
T4698d-PEA_1T3 T46984_PEA_1T12 PEA
PEA_ PEA_ PEA
T46984_PEA_1T23 PEA_ T46984_PEA_1T32 T46984 _1T34 PEA
T46984PEA_1 T40 T46984_PEA_1 T42 T46984PEA_1 T43 T4698~PEA_1 T46 T469S4_PEA_1 T47 T46984PEA_1 T48 T46984PEA_1 T51 T46984PEA_1 T52 T46984_PEA_1 T54 a nucleic acid sequence comprising a sequence in the table below:
Seg~ient Name T46984_PEA_1 2 node_ T46984_PEA_1 4 node T46984_PEA_1 6 node T46984PEA_1 12 node_ T46984_PEA_1 14 node_ T469R4_PEA_1 25 node_ T46934PEA_1 29 node T46984PEA_1 34 node T46984_PEA_1 _46 node T46984PEA_1 47 node T46984PEA_1 52 node T46984PEA_1 65 node T46984PEA_1 69 node T469S4_PEA_1 _75 node T46984PEA_1 86 node T46984_IV'1.;.A_1 9 node_ T469841'1:A1 13 node T46984~'EA_1 19 node_ node T46984_PEA_1 22 node_ T46984_PEA_1 26 node_ T46984_PEA_1 28 node_ T46984PEA_1 31 node T46984_PEA_1 32 node T46984_PEA_1 38 node_ T46984_PEA_1 39 node T46984_PEA_1 40 node T46984_PEA_1 42 node T46984PEA_1 43 node T46984_PEA_1 48 node T46984_PEA_1 49 node T46984_PEA_1 _S0 node T46984_PEA_1 S1 node T46984_PEA_1 _53 node T46984_PEA_1 S4 node T46984_PEA_I SS
node T46984PEA_1 S7 node T46984-PEA_1 60 node T46984-PEA_1 62 node T46984PEA _1 66 node T46984PEA _1 67 node T46984_PEA_ _70 node T46984PEA _1 71 node T46984PEA _1 72 node T46984_PEA_1 73 node T46984_PEA_1 node-74 T46984 1 node_83 PEA_ T46984_PEA_1 node84 T46984 1 node85 PEA
According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein.
Na><axe PEA
PEA
PEA
PEA
T46984 1 P1~
PEA
PEA
PEA
PEA
PEA
PEA
T46984_PEA_1 P3S
PEA
PEA
PEA
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name T116?8 PEA 1 TS
a nucleic acid sequence comprising a sequence in the table belov,~:
Seg~r~.ent I'Jarn~
T11628_PEA_1_node_7 node T11628_PEA_1 16 node_ node node node node rx~de node node node T11628PEA_1 _14 node node node node node node node T11628_PEA_1 29 node node node T11628PEA_1 33 node .~ s T11628 PEA_1_~~~.:o-le 34 T11628_PEA_1 i~oaie 35 T11628_PEA_1 r~~acfe 36 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Frotex~
Name PEA
T11628_PEA_1 PS
PEA
T11628_PEA_1 P10 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name, M78076_PEA_1 M78076_PEA_1 M78076_PEA_1 TS
M78076_PEA_1 M78076_PEA_1 M7807d 1 PEA_ T23 M78076_PEA_1 M78076_PEA_1 M78076_PEA_1 a nucleic acid sequence comprising a sequence in the table below:
Segn~aent Name node node node node node node node node node node node node node M78076_PEA_1 54 node node node M78076_PEA_1 _3 node node node M78076_PEA_1 _12 node node node M78076_PEA_1 30 node M78076_PEA_1 31 node node node node M78076_PEA_1 42 node node M78076_PEA_1 45 node M78076_PEA_1 node_49 M78076 _PEA_1 node_50 M78076 _PEA_1 node_51 M78076 _PEA_1 node_52 M78076 _PEA_1 node53 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name M78076 PEA_1_P3 M78076_PEA_1 P4 M78076_PEA_1 P12 M78076_PEA_1 P14 M78076_PEA_1_P21 M78076_PEA_1 P24 M78076 _PEA_1 P2 M78076 _PEA_1 P25 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Transcript Name HSMUC1A PEA_1 T12 HSMUC 1 A PEA_T26 HSMCTC1A_PEA_1T28 HSMUC1A PEA_1 T29 HSMUC1A_PEA_1 T30 HSMLTC 1 A_PEA_T31 HSMUC1A_PEA_1 T33 HSMUC1A_PEA_1 T34 4a HSMUC1A_ PEA_1 T35 HSMIJC1A_PEA_1 T36 HSIvfUCIA_PEA_1 T40 HSMLTC _PEA_1 T42 HSMUC1A _PEA_1 T43 HSMUC PEA_1 T47 a nucleic acid sequence comprising a sequence in the table below:
Se;rrn~nt Name 1 A node HSMUCIA PEA_1 14 node_ node 1 A_PEA_ node HSMUC1A_ PEA_1 35 node HSMCTC1A PEA_1 38 node HSMUC1A_ PEA_1 3 node 1 A node PEA node HSMUC1A_ PEA_1 6 node HSMUCIA PEA_1 7 node HSMUCIA_PEA_ 1 17 node_ HSMf.JCIA_PEA_1 _18 node HSMUC1A_ PEA_1 20 node HSMUCIA_PEA_ 1 21 node HSMUCIA_ PEA_1 23 node HSMCJC1A_PEA_ 1 26 node HSMUC PEA_1 27 1 A_ node node HSMUC 1 A PEA 1 node 34 HSMUC 1 A PEA 1 node 36 HSMCJC1A_PEA_1 node 37 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below Protein Name HSMLJC1APEA 1 P~5 -. _ HSMUC1A PEA 1 P?9 PEA
_ _ -. _ According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSMLJC 1 A PEA_ 1 P63, comprising a first amino acid sequence being at least 90 % homologous to MTPGTQSPFFLLLLLTVLTWTGSGHASSTPGGEkETSATQRSSV corresponding to amino acids 1 - 45 of MUC 1 HUMAN, which also corresponds to amino acids 1 - 45 of HSMUC1A PEA_1 P63, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence so EEEVSADQVSVGASGVLGSFKEARNAPSFLSWSFSMGPSK corresponding to amino acids 46 - 85 of HSI~fUCIA PEA_1 P63, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSIvfUCIA_PEA_1 P63, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence EEEVSADQVSVGASGVLGSFKEARNAPSFLSWSFSMGPSK in HSMUC1A_PEA_1 P63.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTILHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAKItACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNR1'HVPVVWPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVGDVFELNFMNVKF'SSGYYDFLVEVEGDN
RYIANTVELRVKISTEVGITNVDLSTVDKDQSIAPKTTRVTYPAKAKGTFIADSHQNFAL
FFQLVDVNTGAELTPHQTFVRLHNQKTGQEVVFVAEPDNhNVYKFELDTSERKIEFDS
ASGTYTLY'LIIGDATLKNPILWNV corresponding to amino acids 1 - 498 of RIB2 HUMAN, which also corresponds to amino acids 1 - 498 of T46984_PEA_1 P~, 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 VCA corresponding to amino acids 499 - 501 of T46984_PEA_1 P2, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P3, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAKKACT1'IRSNLDPSNVDSLF1'AAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIS.'HAVAALSGFGLPLASQEALSALTARLSkEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIhEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRY'HVPVVVVPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAhSVASRATVLQKTSFTPVGDVFELNFMNVhF'SSGYYDFLVEVEGDN
RYLANTVELRVILISTEVGITNVDLSTVIaKDQSIAPItTTRVTYPAKAKGTFIADSHQNFAL
FFQLVDVNTGAELTPHQ corresponding to amino acids 1 - 433 of RIB2_HL1MAN, which also corresponds to amino acids 1 - 433 of T46984_PEA_1 P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ICHIWKLIFLP corresponding to amino acids 434 - 444 of T46984_PEA_I P3, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984 PEA_1 P3, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ICHIWKLIFLP in T46984 PEA_1 P3.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P10, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLhASLDRPFTNLESAFIrSIVGLSSL
GAQVPDAKKACT1'IRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAV SEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRYHVPV VVVPEGSASDTHEQ.AILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVGDVFELNFMNVKFSSGYYDFLVEVEGDN
RYIANTVELRVKISTEVGITNVDLSTVDKDQSIAPKTTRVTYPAKAKGTFIADSHQNFAL
FFQLVDVNTGAELTPHQTFVRLHNQKTGQEVVFVAEPDNhNVYKFELDTSERKIEFDS
ASGTYTLYLIIGDATLKNPILVVNV corresponding to amino acids 1 - 49S of RIB2 I-iUNIAN, which also corresponds to amino acids 1 - 498 of T46984 PEA_1 P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence LMDQK corresponding to amino acids 499 - 503 of T46984_PEA_1 P10, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984_PEA_1 P10, comprising a polyypeptide 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 LMDQK
in T46984 PEA 1 P 10.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P1 l, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKA.SLDRPFTNLESAFYSIVGLSSL
GAQVPDAKKACTI'IRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFShKNFESLSEAFSVASAAAVLSHNRYHVPVVVVPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVGDVFELNFMNVKFSSGYYDFLVEVEGDN
RYIANTVELRVKISTEVGITNVDLSTVDILDQSIA.PKTTRVTYPAKAKGTFIADSHQNFAL
FFQLVDVNTGAELTPHQTFVRLHNQKTGQEVVFVAEPDNKNVYKFELDTSERKIEFDS
ASGTYTLYYLIIGDATLKNPILWNVADWThFPEEEAPS'TVL,SQNLFTPKQEIQHLFREPEK
RPPTWSNTFTALILSPLLLLFALWIRIGANVSNFTFAPSTIIFHLGHA.4MLGLMYVYWT
QLNMFQTLKYLAILGSVTFLAGNRMLAQQAVIt.R corresponding to amino acids 1 - 6~8 of RIB2 I-IUIvIAN, which also corresponds to amino acids 1 - 628 of T46984 PEA_1 P11.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P 12, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKASLDRPFTNLESAFIrSIVGLSSL
GAQVPDAKKACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETILDLLLAAVVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTAI,f'V'AAT'YKLMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRYHVI' ~'VWPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVG'f~VFELNFMN corresponding to amino acids 1 - 338 ofRIB2_HZJMAN, which also corresponds to amino acids 1 - 33S of T4698~1 PEA_1 P12, and a second amino acid sequence being at least 70%, optionally at least SO%, preferably at least SS%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence SQDLH corresponding to amino acids 339 -343 of T4698~ PEA_1 P12, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T469S4 PEA_1 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90°~'o and most preferably at least about 95% homologous to the sequence SQDLH in T469S4 PEA 1 P 12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984 PEA_1 P21, 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 M
corresponding to amino acids 1 - 1 of T46984_PEA_1 P21, and a second amino acid sequence being at least 90 % homologous to KACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSSVTQI~.~iAV
AALSGFGLPLASQEALSALTARLSKEE'TVL,ATVQALQTASHLSQQADLRSIVEEIEDLVA
RLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNAIFSKKNFES
LSEAFSVASAAAVLSHNRYHVPVWVPEGSASDTHEQAIL,RLQVTNVLSQPLTQATVKL
EHAhSVASRATVLQILTSFTPVGDVFELNFMNVkFSSGYYYDFLVEVEGDNRYIANTVEL
RVKISTEVGITNVDLSTVDKDQSIAPhTTRVTYPAkAhGTFIADSHQNFALFFQLVDVNT
GAELTPHQTFVRLHNQKTGQEV VFVAEPDNKNVYKFELDTSERKIEFDSASGTYTLY~LII
GDATLhNPILWNVADVVIKFPEEEAPSTVLSQNLFTPKQEIQHLFREPEKRPPTVVSNTF
TAL.u,SPLLLLFAL~~~IRIGANVSNFTFAPSTIIFHLGHAAMLGLMYVI'WTQLNMFQTLK.Y
LAILGSVTFLAGNRMLAQQAVhRTAH corresponding to amino acids 70 - 631 of RIB2_I-ICTMAN, which also corresponds to amino acids 3 - 563 of T46984 PEA_1 P21, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T4698~ PEA_1 P27, comprising a first amino acid 'segue nce being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKI~VERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDA_KKACTI'IRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHL,SQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKIMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRYHVPVVIIVPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVkLEHAhSVASRATVLQhTSFTPVGDVFELNFMNVKFSSG~'~'DFLVEVEGDN
RYIANTVELRVKISTEVGITNVDLSTVDKDQSIAPKTTRVTYPAKAKGTFIADSHQNFA
corresponding to amino acids 1 - 415 of RIB?_HUMAN, which also corresponds to amino acids 1 - 415 of T46984_PEA_1 P27, 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 FGSGLVPMSPTSLLLLARLYFTWDMLLCWDSCMSTGLSSTCSRP corresponding to amino acids 416 - 459 of T46984 PEA_1 P27, wherein said first amino acid sequence and second amino acid sequence axe contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984_PEA_1 P27, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence FGSGLVPMSPTSLLLLARLYFTWDMLLCWDSCMSTGLSSTCSRP in T46984 PEA 1 P27.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1_P32, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTH~.'LTKHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAhHACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQII'~I-IAVAALSGFGLPLASQEALSALTARLSKEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFSkKNFESLSEAFSVASAAAVLSHNR1'HVPVWVPEGSASDTHEQAIL,RLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVGDVFELNFMNVKFSSGYYDFLVEVEGDN
RYIANTVE corresponding to amino acids 1 - 364 of R1B2_HUMAN, which also corresponds 5 to amino acids 1 - 364 of T46984_PEA_1 P32, 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 GQVRWLTPVIPALWEAKAGGSPEVRSSIL,AWPT corresponding to amino acids 365 - 397 of T46984_PEA_I P32, wherein said first amino acid sequence and second amino acid 10 sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T469S4_PEA_1 P32, 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 15 GQVRWLTPVIPALWEAKAGGSPEVRSSILAWPT in T46984 PEA_1 P32.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P34, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKASLDRPFTNLESAFYSIVGLSSL
20 GAQVPDAKKACTI'IRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEALSALTARLSILEETVLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQWQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNRYHVPVVVVPEGSASDTHEQAILRLQVTNVLSQ
PLTQATVKLEHAKSVASRATVLQKTSFTPVG corresponding to amino acids 1 - 329 of 25 RIB2_I-ItJMAN, which also corresponds to amino acids 1 - 329 of T4698~
PEA_1 P34.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P35, comprising a first amino acid sequence being at least 90 % homologous to GAQVPDAKhACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQII'I3AVAALSGFGLPLASQEALSALTARLSKEET'VLATVQALQTASHLSQQADLRSI
VEEIEDLVARLDELGGVI'LQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNA
IFSKKNFESLSEAFSVASAAAVLSHNR.YHVPWVVPEGSASDTHEQAI corresponding to amino acids 1 - 2S7 of RIB? HUMAN, which also corresponds to amino acids 1 -287 of T469S4_PEA_1 P35, 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 GCWPSRQSREQHISSRRKNIEILKTECQEKESRTIHSMR.RIi.MEKKNFI corresponding to amino acids 2S8 - 334 of T469S4_PEA_1_P35, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T469S~ PEA_1 P35, comprising a polypeptide being at least 70°io, optiorally at least about SO%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GCWPSRQSREQHISSRRKMEILKTECQEKESRTIHSMRRI~MEKKNFI in T46984 PEA 1 P35.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P38, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHY'I,TKHDVERLIkASLDRPFTNLESAFYSIVGLSSL
GAQVPDAKKACTYIRSNLDPSIVVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSS
VTQIYHAVAALSGFGLPLASQEAL corresponding to amino acids 1 - 145 of RIB2 HUMAN, which also corresponds to amino acids I - 145 of T4698~ PEA_I P38, and a second amino acid sequence being at least 70%, optionally at least SO%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MDPDWCQCLQLHFCS corresponding to amino acids 146 - 160 of T46984_PEA_1 P38, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984_PEA_1 P3S, 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 MDPDWCQCLQLHFCS in T46984_PEA_1 P3S.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T4698~ PEA_1 P39, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSST~rFLLALTIIASTWALTPTI-IY~LTILHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAI:KACTYIRSNLDPSNVDSLFYAAQASQALSGCEISISNETKDLLLAAV SEDSS
VTQIY'HAVAALSGFGLPLASQEALSALTARLSKEETVLA corresponding to amino acids 1 -160 of RIB2 HTJMAN, which also corresponds to amino acids 1 - 160 of T46984_PEA_1 P39.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T469g4 PEA_1 P45, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTIiY'LTKHDVERLILASLDRPFTNLESAFI'SIVGLSSL
GAQVPDAhKACTI'IRSNLDPSNVDSLFYAAQASQALSGCE corresponding to amino acids 1 - 101 of RIB2_I-ILT(vIAN, which also corresponds to amino acids 1 - 101 of T46984_PEA_1 P45, 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°,~0 homologous to a polypeptide having the sequence NSPGSADSIPPVPAG corresponding to amino acids 102 - 116 of T4698~ PEA_1 P45, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T46984 PEA_1 P45, 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 NSPGSADSIPPVPAG in T46984 PEA 1 P45.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T46984_PEA_1 P46, comprising a first amino acid sequence being at least 90 % homologous to MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKASLDRPFTNLESAFYSIVGLSSL
GAQVPDAh corresponding to amino acids 1 - 69 of RIB2_HUMAN, which also corresponds to amino acids 1 - 69 of T46984 PEA_1 P46, 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 NSPGSADSIFPVPAG corresponding to amino acids 70 - 84 of T46984_PEA_1 P46, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is pnwided an isolated polypeptide encoding for a tail of T46984_PEA_ 1 P46, 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 NSPGSADS1PPVPAG in T46984 PEA 1 P46.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T11628_PEA_1 P2, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MGLSDGEWQLVLNVWGhVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDE
corresponding to amino acids 1 - 55 of T11628 PEA_1 P2, and a second amino acid sequence being at least 90 % homologous to MKASEDLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKII'VKYLEFISECIIQV
LQSKHPGDFGADAQGAMMtALELFRKDMASNYKELGFQG corresponding to amino acids 1 - 99 of QBVW~H6, which also corresponds to amino acids 56 - 154 of T11628_PEA_1 P2, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of T11628_PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFhGHPETLEKFDKFKHLKSEDE of T11628 PEA 1 P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T1162S_PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to I\~IKKASEDLKKHGATVLTALGGIL,KKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQV
LQSKHPGDFGADAQGAMNIiAL,ELFRKDMASNYKELGFQG corresponding to amino acids 56 - 154 of M5.'G_HIIMAN_V 1, which also corresponds to amino acids 1 -99 of T1162S PEA 1 P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T11628_PEA_1 P7, comprising a first amino acid sequence being at least 90 % homologous to MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMK
ASEDLKKHGATVLTALGGILKKKGI-~IEAEIKPLAQSHA.TKHKIPVKYLEFIS~ECIIQVLQ
SKHPGDFGADAQGA.MNK corresponding to amino acids 1 - 134 of IVtYG_HUM.AN V 1, which also corresponds to amino acids 1 - 134 of T11628_PEA_1 P7, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90°~o and most preferably at least 95% homologous to a polypeptide having the sequence G
corresponding to amino acids 135 - 135 of T11628_PEA_1 P7, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T11628_PEA_1 P10, comprising a first amino acid sequence being at least 70°~0, 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 MGLSDGEW QLVLN VWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDE
corresponding to amino acids 1 - 55 of TI 1628_PEA_1 P10, and a second amino acid sequence being at least 90 % homologous to MKASEDLKILHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQV
LQSKHPGDFGADAQGAMNKALELFRKDMASN~'KELGFQG corresponding to amino acids 1 - 99 of Q8WVH6, which also corresponds to amino acids 56 - 154 of T11628_PEA_1 P10, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polygeptide encoding for a head of T11628_PEA_1 PIO, 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 5 MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPET'I,EkFDKFKHLKSEDE of T11628 PEA 1 P10.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076_PEA_I P3, comprising a first amino acid sequence being at least 90 % homologous to CGRLTLHRDLRTGRWEPDPQRSRRCLRDPQRVLEYCRQMYPELQIARVEQATQAIPME
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQGESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGKVTPTPRPTDGV
15 DIYFGMPGEISEHEGFLRAItMDLEERRMRQINEVIvIREWAMA.DNQSKNLPKADRQALN
EHFQSILQTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYLRAEQK.EQRHTLRHYQHVAAVDPEKAQQMRFQVHTHLQVIEERVNQSLGLLD
QNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKD corresponding to amino acids I - 517 of APP1_HUMAN, which also corresponds to amino acids 1 -517 of 20 M78076_PEA_1 P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence GE corresponding to amino acids 51 S - 519 of M78076_PEA_1 P3, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
25 According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M7S076_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
CGRLTLHRDLRTGRWEPDPQRSRRCLRDPQRVLEIfCRQMYPELQIARVEQATQAIPME
30 RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRR_HQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVE1~1CCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGK'~~Tj~TPRPTDGV
DIYFGMPGEISEHEGFLRAI~IvIDLEERI2MRQINEVMREWAMADNQSKNL h'KADRQALN
EHFQSILQTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQl~Li'PQAERVLL
ALRRYLRAEQKEQRHTLRHYQHVAAVDPEKAQQMRFQVHTHLQVIEERVNQSLGLLD
QNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKDD1'PMTLPKG
corresponding to amino acids 1 - 526 of APP 1 ~IUMAN, which also corresponds to amino acids 1 - 526 of M78076_PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence ECLTVNPSLQIPLNP corresponding to amino acids 527 - 541 of M78076_PEA_1 P4, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ECLTVNPSLQIPLNP in M7S076_PEA_1 P4.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076_PEA_1 P12, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGhVTPTPRPTDGV
DIYFGMPGEISEHEGFLRAKN>DLEERRMRQINE~WREWAIViADNQSKNLPKADRQALN
EHFQSII,QTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYI,RAEQKEQRHTLRHYQHVAAVDPEKAQQMRFQVHTHLQVIEERVNQSLGLLD
QNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKDDTPMTLPKG
corresponding to amino acids 1 - 526 of APP1 HUMAN, which also corresponds to amino acids 1 - 536 of M78076_PEA_1 P12, and a second amino acid sequence being at least 70%, b2 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 ECVCSKGFPFPLIGDSEG corresponding to amino acids 527 - 544 ofM78076_PEA_1 P12, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°'0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ECVCSKGFPFPLIGDSEG in M78076 PEA 1 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076_PEA_1 P14, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
CGRLTLHRDLRTGRWBPDPQRSRRCLRDPQRVLEYCRQMYPELQIARVEQATQAIP1~IE
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEf'VCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAWGKVTPTPRPTDGV
DIYFGMPGEISEHEGFLRAI~LEERRIVfRQINEVMREWAMADNQSKNLPKADRQALN
EHFQSILQTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYLRAEQKEQRHTLRHYQHVAAVDPEKAQQMRFQVHTHLQ~~ERVNQSLGLLD
QNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKDDTPMTLPIiGST
EQDAASPEKEKMNPLEQYERKVNASVPRGFPFHSSEIQRDEL corresponding to amino acids 1 - 570 of APP 1 HUMAN, which also corresponds to amino acids 1 - 570 of M78076_PEA_1 P14, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence VRGGTAGYLGEETRGQRPGCDSQSHTGPSKKPSAPSPLPAGTSWDRGVP corresponding to amino acids 571 - 619 of M78076_PEA_1_P14, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P14, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRGGTAGYLGEETRGQRPGCDSQSHTGPSKKPSAPSPLPAGTSWDRGVP in M78076 PEA 1 P14.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076_PEA_1 P21, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
CGRLTLHRDLRTGRWEPDPQRSRRCLRDPQRVLEYCRQMYPELQIARVEQATQAIPME
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSVVPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGILVTPTPRPTDGV
DIYFGMPGEISEHEGFLRAILMDLEERRMRQINEVMREWAMADNQSKNLPKADRQALN
E corresponding to amino acids 1 - 352 of APP1 HUMAN, which also corresponds to amino acids 1 - 352 of M78076_PEA_1 P21, and a second amino acid sequence being at least 90 homologous to AERVLLALRRYLRAEQKEQRHTLRHYQHVAAVDPEILAQQMRFQVHTHLQVIEERVNQ
SLGLLDQNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKDDTPMT
LPKGSTEQDAASPEKEkMNPLEQYERhVNASVPRGFPFHSSEIQRDELAPAGTGVSREA
VSGLLII~~GAGGGSLIVLSMLLLRRhhPYGAISHGVVEVDPMLTLEEQQLRELQRHGYE
NPTYRFLEERP corresponding to amino acids 406 - 650 of APP1 HUMAN, which also corresponds to amino acids 353 - 597 of M78076_PEA_1 P21, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding fox an edge portion of M78076_PEA_1 P21, 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 EA, having a structure as follows: a sequence starting from any of amino acid numbers 353-x to 352; ~,aul ending at any of amino acid numbers 353+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M7807~ PEA_1 P24, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
CGRLTLHRDLRTGRWEPDPQRSRRCLRDPQRVLEYCRQMYPELQIAR.VEQATQAIPME
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGKVTPTPRPTDGV
DIYF'GMPGEISEHEGFLRAICMDLEERRMRQINEVMREWAMADNQSKNLPKADRQALN
EHFQSIL,QTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYLRAEQKEQRHTLRHYQHVAAVDPEIkAQQMRFQVHTHLQVIEERVNQSLGLLD
QNPHLAQELRPQI corresponding to amino acids 1 - 481,of APP1HUMAN, which also corresponds to amino acids 1 - 481 of M78076 PEA_1 P24, 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 RECLLPWLPLQISEGRS corresponding to amino acids 482 - 498 of M78076 PEA_1 P24, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P24, 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 RECLLPWLPLQISEGRS in M78076 PEA_1 P24.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M78076 PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MGPASPAARGLSRRPGQPPLPLLLPLLLLLLRAQPAIGSLAGGSPGAAEAPGSAQVAGL
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQ
EAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSWPPG
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGKVTPTPRPTDGV
DIYFGMPGEISEHEGFLRAI~MDLEERRMRQINEVMREWAMADNQSKNLPKADRQALN
ALRRYLRAEQKEQRHTLRHYQHVAAVDPEKAQQMRFQV corresponding to amino acids 1 - 449 of APP1 HLJIvIAN, which also corresponds to amino acids 1 - 449 of M78076_PEA_1 P2, 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%
10 homologous to a polypeptide having the sequence LTSFQLPNAPLFLRRPRLRLFSCPLDPLSVSWTPSYPLNTASLPLPSLSAQLPDPETWTLT
CCVFDPCFLALGFLLPPPSILCSVVPWIFTAFPRIVFFFFFFLRQVLALSPRQESSVRSWLIAT
STSWVQAIL,LPQPLE corresponding to amino acids 450 - 588 of M78076 PEA_1 P2, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a 15 sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076 PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about S5%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence CCVFDPCFLALGFLLPPPSILCSVPWIFTAFPRNFFFFFFLRQVLALSPRQESSVRSWLIAT
STSVi~VQAILLPQPLE in M78076_PEA_1 P2.
According to preferred embodiments of the present invention, there is provided an 25 isolated chimeric polypeptide encoding for M78076 PEA_1 P25, comprising a first amino acid sequence being at least 90 % homologous to n~IGPASPAARGLSRRPGQPPLP LLLPLLLLLLRAQPAIGSLAGGSPGA.AEAPGSAQVAGL
CGRLTLHRDLRTGRVVEPDPQRSRRCLRDPQRVL,EYCRQMYPELQIARVEQATQAIPME
RWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERIvIDQCESSTRRHQ
SRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAWGKVTPTPRPTDGV
DIYFGIvfPGEISEHEGFLRAI~MDLEERRNiRQINEVIviREWAIVIADNQSKNLPKADRQALN
EHFQSILQTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLL
ALRRYLRAEQILEQRHTLRH~'QHVAAVDPEhAQQMRFQ corresponding to amino acids 1 - 448 of APPIHiCIMAN, which also corresponds to amino acids 1 - 448 of M78076_PEA_1 P25, 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°'0 homologous to a polypeptide having the sequence PQNPNSQPRA.AGSLEVIISHPFVRRLEILISPFQFQNSIPKNSQIVPAASPRGTSSP
corresponding to amino acids 449 - 505 of M78076_PEA_1 P25, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M78076_PEA_1 P25, comprising a polypeptide being at least 70°'0, 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 PQNPNSQPRAAGSLEVIISHPFVRRLEILISPFQFQNSIPfNSQIVPAASPRGTSSP in M78076 PEA 1 P25.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M85491 PEA_1 P13, comprising a first amino acid sequence being at least 90 % homologous to MALRRLGAALLLLPLLAAVEETLMDSTTATAELGWMVHPPSGWEEVSGYDENMNTIR
TYQVCNVFESSQNNWLRTKFIRRRGAHRIHVEIVIILFSVRDCSSIPSVPGSCKETFNLYYY
EADFDSAThTFPNWMENPWVIiVDTIAADESFSQVDLGGRVMICINTEVRSFGPVSRSGF
YLAFQDYGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVAARGSCIANAEEVD
VPIKLYCNGDGEWLVPIGRCMCItAGFEAVENGTVCRGCPSGTFhANQGDEACTHCP1N
SRTTSEGATNCVCRNGYYRADLDPLDMPCTTIPSAPQAVISSVNETSLI~~ILEWTPPRDSG
GREDL~~.'I~1IICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYISDLLAHTQYTFEIQAV
NGVVTDQSPFSPQFASVNITTNQAAPSAVSIIVIIQVSRTVDSITLSWSQPDQPNGVILDI~L
QYYEIk corresponding to amino acids 1 - 476 of EPB2 HU1~~IAN, which also corresponds to amino acids 1 - 476 of M85491 PEA_1 P13, and a second amino acid sequence being at least 70%, optionally at least SO%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VPIGWVLSPSPTSLRAPLPG corresponding to amino acids 477 - 496 of M85491 PEA_1 P13, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M85491 PEA_1 P13, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VPIGWVLSPSPTSLRAPLPG in MS5491 PEA 1 P13.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for M85491 PEA_1 P14, comprising a first amino acid sequence being at least 90 % homologous to MALRFLGAALLLLPLLAAVEETLMDSTTATAELGWI\~IVHPPSGWEEV SGYDENMNTIR
TYQVCNVFESSQNNW~LRTKFIRR.RGAIiRIHVEMKFSVRDCSSIPSVPGSCILETFNLYYY
EADFDSATKTFPNWMENPWVIkVDTIAADESFSQVDLGGRVMK1NTEVRSFGPVSRSGF
Y'LAFQDYGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVVAARGSCIANAEEV'D
VPIKLYCNGDGEWLVPIGRCMCKAGFEAVENGTVCR corresponding to amino acids 1 -270 of EPB2 HLJN1AN, which also corresponds to amino acids 1 - 270 of M85491 PEA_1 P14, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence ERQDLTMLSRLVLNSWPQA~IILPPQPPKVL,EL corresponding to amino acids 271 - 301 of M85491 PEA_1 P14, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of M85491 PEA_1_P14, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ERQDLTMLSRLVLNS~VPQMILPPQPPKVL,EL in M85491 PEA_1 P14.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSSTROL3 P4, comprising a first amino acid sequence Ia;ing at least 90 % homologous to MAPAA~%~n..RSAAARALLPPMLLLLLQPPPLLARALPPDVHLILHAERRGPQPW~LAALPSS
PAPAPA1 QLAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRiZRFP
WQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEGRADIIv>mFARYW corresponding to amino acids 1 - 163 of MM 11 HC>MAN, which also corresponds to amino acids I -163 of HSSTROL3_P4, a bridging amino acid H corresponding to amino acid 164 of HSSTROL3_P4, a second amino acid sequence being at least 90 °'o homologous to GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFYTFRYPLSLSPDDCRGVQHLYGQP~VPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGF~rWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQGAQYWVYDGEKPVLGPAPLTELGLVRFPVHAALVWGPE
KNHIYFFRGRDYWRFHPSTRRVDSPVPRRATDWRGVPSEIDAAFQDADG corresponding to amino acids 165 - 445 of MMI I HLJIVIAN, which also corresponds to amino acids 165 - 445 of HSSTROL3_P4, 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 ALGVRQLVGGGHSSRFSHLVVAGLPHACHRKSGSSSQVLCPEPSALLSVAG
corresponding to amino acids 446 - 496 of HSSTROL3_P4, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSSTROL3 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ALGVRQLVGGGHSSRFSHLVVAGLPHACHRKSGSSSQVLCPEPSALLSVAG in HSSTROL3 P4.
According to preferred embodiments of the present invertion, there is provided an isolated chimeric polypeptide encoding for HSSTROL3_P5, comprising a first amino acid sequence being at least 90 % homologous to MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWHAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRIL,RFP
WQLVQEQVRQTMAEALKVWSDVTPLTFT'EVHEGRADIIVImFARYW corresponding to amino acids 1 - 163 of MM 11 HUMAN, which also corresponds to amino acids 1 -163 of HSSTROL3_P5, a bridging amino acid H corresponding to amino acid 164 of HSSTROL3 P5, a second amino acid sequence being at least 90 % homologous to GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAF1'TFRYPLSLSPDDCRGVQHLI'GQPWPTVTSRTP.ALGPQAG>DTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQ corresponding to amino acids 165 - 358 of MM11 HUMAN, which also corresponds to amino acids 165 - 358 of HSSTROL3_P5, 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 ELGFPSSTGRDESLEHCRCQGLHK corresponding to amino acids 359 - 382 of HSSTROL3_P5, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSSTROL3 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ELGFPSSTGRDESLEHCRCQGLHK in HSSTROL3_P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSSTROL3_P7, comprising a first amino acid sequence being at least 90 % homologous to MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWHAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRILRFP
WQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEGRADIMIDFARYW corresponding to amino acids 1 - 163 of MM 11 HUMAN, which also corresponds to amino acids 1 -163 of HSSTROL3_P7, a bridging amino acid H corresponding to amino acid 164 of HSSTROL3_P7, a second amino acid sequence being at least 90 % homologous to GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGH~TL,G
LQHTTAAhALMSAFYTFRYPLSLSPDDCRGVQHL1'GQPWPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFhAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQG corresponding to amino acids 165 - 359 of MM11 HU~~iA.N, which also corresponds to amino acids 165 - 359 of HSSTROL3_P7, 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 TTGVSTPAPGV corresponding to amino acids 360 - 370 of HSSTROL3_P7, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an 10 isolated polypeptide encoding for a tail of HSSTROL3 P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°'0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence TTGVSTPAPGV in HSSTROL3 P7.
According to preferred embodiments of the present invention, there is provided an 15 isolated chimeric polypeptide encoding for HSSTROL3_P8, comprising a first amino acid sequence being at least 90 % homologous to MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWI-IAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRILRFP
WQLVQEQVRQTMAEALhVWSDVTPLTFTEVHEGRADIMI)7FARYW corresponding to 20 amino acids 1 - 163 of MM11 HLTIVIAN, which also corresponds to amino acids 1 - 163 of HSSTROL3 P8, a bridging amino acid H corresponding to amino acid 164 of HSSTROL3_P8, a second amino acid sequence being at least 90 % homologous to GDDLPFDGPGGILAHAFFPIiTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFYTFRYPLSLSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN
25 EIAPLE corresponding to amino acids 165 - 286 of MM 11 HUI\~IAN, which also corresponds to amino acids 165 - 286 of HSSTROL3 P8, and a third amino acid sequence being at least 70°~0, 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 VRPCLPVPLLLCWPL corresponding to amino acids 287 - 301 of HSSTROL3_P8, wherein 30 said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to lyreferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSSTROL3 P8, comprising a polypeptide being at least 70%, optionally- at least about 80%, preferably at least about 85°./0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRPCLPVPLLLCWPL in HSSTROL3 P8.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSSTROL3_P9, comprising a first amino acid sequence being at least 90 % homologous to IvfAPAAWLRSAAARALLPPMLLLLLQPPPLLAR.ALPPDUHHI_,HAERRGpQpWHAALpSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSA.RNRQK corresponding to amino acids 1 -96 of MM11 HUMAN, which also corresponds to amino acids 1 - 96 of HSSTROL3_P9, a second amino acid sequence being at least 90 % homologous to RILRFPWQLVQEQVRQTMAEALKVW SDVTPLTFTE VHEGRADII'TIDFARYW
corresponding to amino acids 113 - 163 of MM 11 HUMAN, which also corresponds to amino acids 97 - 147 of HSSTROL3_P9, a bridging amino acid H corresponding to amino acid 148 of HSSTROL3 P9, a third amino acid sequence being at least 90 % homologous to GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFhTFRYPLSLSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQG corresponding to amino acids 165 - 359 of 1\w11 1 HUMAN, which also corresponds to amino acids 149 - 343 of HSSTROL3 P9, and a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence TTGVSTPAPGV corresponding to amino acids 344 - 354 of HSSTROL3_P9, wherein said first amino acid sequence, second amino acid sequence, bridging amino acid, third amino acid sequence and fourth amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of HSSTROL3_P9, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about ?0 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 ILR, having a structure as follows: a sequence starting from any of amino acid numbers 96-x to 96; and ending at any of amino acid numbers 97+ ((n-2) - x), in which x varies from 0 to n-?.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSSTR0L3 P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence TTGVSTPAPGV in HSSTROL3 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for AY180924 PEA_1 P3, comprising a first amino acid sequence being at least 90 % homologous to 1\~ILNVSGLFVLLCGLLVSSSAQEVLAGVSSQLLN corresponding to amino acids 1 - 33 of LATH_HUMAN, which also corresponds to amino acids 1 - 33 of AY180924 PEA_1_P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GETVLLWVMQNPEPMPVIkFSLAKYLGHNEHY
corresponding to amino acids 34 - 64 of AY 180924_PEA_1 P3, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of AI'180924_PEA_1 P3, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GETVLLWVMQNPEPMPVkFSLAKYLGI-LNEH1T in AY1809?4_PEA_1 P3.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for 875793 PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MItFLAVLVLLGVSIFLVSAQNPTTAAPADTYPATGPADDEAPDAETTAAATTATTAAPT
TATTAASTTARItDIP corresponding to amino acids 1 - 74 of Q96DR8, which also corresponds to amino acids 1 - 74 of 875793 PEA_1 P2, 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 AP
corresponding to amino acids 75 - 76 of R75793_PEA_1 P?, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HCTI\~iCAI~ P14, comprising a first amino acid sequence being at least 90 % homologous to MEPWSSRWKTICRWLWDFTVTTLALTFLFQAREVRGAAPVDVLKALDFHNSPEGISKTT
GFCTNRKNSKGSDTAl'RVShQAQLSAPThQLFPGGTFPEDFSILFTVItPKKGIQSFLLSIY
NEHGIQQIGVEVGRSPVFLFEDHTGKPAPEDYPLFRTVNIADGKWHRVAISVEIkKTVTM
IVDCKKKTTHPLDRSERAIVDTNGITVFGTRIL,DEEVFEGDIQQFLITGDPKAAYDYCEH
YSPDCDSSAPKAAQAQEPQIDEYAPEDIIEYDYEYGEAEYKEAESVTEGPTVTEETIAQT
EANIVDDFQEYNYGTMESIrQTEAPRHV SGTNEPNPVEEIFTEEYLTGEDYDSQRKNSED
TLY'ENKEIDGRDSDLLVDGDLGEYDFYEYKEYEDKPTSPPNEEFGPGVPAETDITETSIN
GHGAYGEKGQKGEPAVVEPGMLVEGPPGPAGPAGIMGPPGLQGPTGPPGDPGDRGPPG
RPGLPGADGLPGPPGTMLMLPFR1'GGDGSKGPTISAQEAQAQAILQQARIALRGPPGPM
GLTGRPGPVGGPGSSGAhGESGDPGPQGPRGVVQGPPGPTGKPGKRGRPGADGGRGMP
GEPGAI~GDRGFDGLPGLPGDKGHRGERGPQGPPGPPGDDGMRGEDGEIGPRGLPGEAG
PRGLLGPRGTPGAPGQPGMAGVDGPPGPKGNMGPQGEPGPPGQQGNPGPQGLPGPQG
PIGPPGEKGPQGKPGLAGLPGADGPPGHPGILEGQSGEKGALGPPGPQGPIGYPGPRGVIL
GADGVRGLKGSKGEKGEDGFPGFKGDMGLKGDRGEVGQIGPRGEDGPEGPKGRAGPT
GDPGPSGQAGEKGKLGVPGLPGYPGRQGPKGSTGFPGFPGANGEKGARGVAGKPGPR
GQRGPTGPRGSRGARGPTGKPGPKGTSGGDGPPGPPGERGPQGPQGPVGFPGPhGPPGP
PGKDGLPGHPGQRGETGFQGKTGPPGPGGWGPQGPTGETGPIGERGHPGPPGPPGEQG
LPGAAGKEGAKGDPGPQGISGKDGPAGLRGFPGERGLPGAQGAPGLKGGEGPQGPPGP
V corresponding to amino acids 1 - 1056 of CA1B_HUMAN_V5, which also corresponds to amino acids 1 - 1056 of HW~ICAI~iIA_P14, and a second amino acid sequence being at least 70%, optionallyy at least 80%, preferably at least 85%, more preferably at least 90°ro and most preferably at least 95°,~o homologous to a polypeptide having the sequence VSMMIINSQTIMWNYSSSFITLML corresponding to amino acids 1057 - 1081 of HLTMCA1XIA_P14, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HUMCA 1 ~ P 14, comprising a polypeptide being at least 70%, options lly 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 VSI~wIIINSQTI1'SSSFITLML in HCTMCA1.~IA P14.
According to preferred embodiments of the present invent ion, there is provided an isolated chimeric polypeptide encoding for HUMCA1~IA-P15, comprising a first amino acid sequence being at least 90 °,%o homologous to MEPWSSRWKTKRWLWDFTVTTLALTFLFQAREVRGAAPVDVLKALDFHNSPEGISKTT
GFCTNRKNSKGSDTAYRVSKQAQLSAPTKQLFPGGTFPEDFSILFTVKPKKGIQSFLLSIY
NEHGIQQIGVEVGRSPVFLFEDHTGKPAPEDYPLFRTVNIADGKWHRVAISVEKkTVTM
IVDCKKKTTKPLDRSERAIVDTNGITVFGTRILDEEVFEGDIQQFLITGDPKAAYDYCEH
YSPDCDSSAPKAAQAQEPQIDEYAPEDIIEYDYEYGEAEYhEAESVTEGPTVTEETIAQT
EANIVDDFQEYNYGTMESYQTEAPRHVSGTNEPNPVEEIFTEEYLTGEDYDSQRItNSED
TLYENKEIDGRDSDLLVDGDLGEYDFYEYKEYEDhPTSPPNEEFGPGVPAETDITETSIN
GHGAYGEKGQKGEPAVVEPGMLVEGPPGPAGPAGIMGPPGLQGPTGPPGDPGDRGPPG
RPGLPGADGLPGPPGTMLMLPFRYGGDGSKGPTISAQEAQAQAILQQARIALRGPPGPM
GLTGRPGPVGGPGSSGAKGESGDPGPQGPRGVQGPPGPTGKPGKRGRPGADGGRGMP
GEPGAKGDRGFDGLPGLPGDhGHRGERGPQGPPGPPGDDGI'~fRGEDGEIGPRGLPGEAG
PRGLLGPRGTPGAPGQPGIvIAGVDGPPGPKGNMGPQGEPGPPGQQGNPGPQGLPGPQG
PIGPPGEIL corresponding to amino acids 1 - 714 of CA1B hII)MAN, which also corresponds to amino acids 1 - 714 of HLTMCA1~IA P15, 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 MCCNLSFGIL1PLQK corresponding to amino acids 715 - 729 of HUMCA1~A_P15, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding fur a tail of HLTMCA1XIA_P15, comprising a polypeptide being at least 70°./0, optionally at least about SO%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MCCNLSFGILIPLQK in HUMCA 1 ~ P 15.
According to preferred embodiments of the present invention, there is pravided an 5 isolated chimeric polypeptide encoding for HITMCA1~IA P16, comprising a first amino acid sequence being at least 90 % homologous to MEPWSSRWIiTKRWLWDFTVTTLALTFLFQAREVRGAAPVDVLKALDFHNSPEGISKTT
GFCTNRKNSKGSDTAYRVSKQAQLSAPTKQLFPGGTFPEDFSILFTVKPKKGIQSFLLSIY
NEHGIQQIGVEVGRSPVFLFEDHTGKPAPEDYPLFRTVNTADGKWHRVAISVEKKTVTM
10 IVDCK.KKTTKPLDRSERAIVDTNGITVFGTRILDEEVFEGDIQQFLITGDPKAAYDYCEH
YSPDCDSSAPKAAQAQEPQIDEYAPEDIIEYDYEYGEAEI'KEAESVTEGPTVTEETIAQT
EANIVDDFQEYNYGTMESYQTEAP RHVSGTNEPNPVEEIFTEEYLTGEDYDSQRKNSED
TLYENKEIDGRDSDLLVDGDLGEYDF1'EYKEYEDKPTSPPNEEFGPGVPAETDITETSIN
GHGAYGEKGQKGEPAVVEPGMLVEGPPGPAGPAGIMGPPGLQGPTGPPGDPGDRGPPG
GLTGRPGPVGGPGSSGAKGESGDPGPQGPRGVQGPPGPTGKPGKRGRPGADGGRGI\R' GEPGAKGDRGFDGLPGLPGDKGHRGERGPQGPPGPPGDDGMRGEDGETGPRGLPGEA
corresponding to amino acids 1 - 648 of CA1B_HUMAN, which also correspands to amino acids 1 - 64S of HUMCA1XIA P16, a second amina acid sequence being at least 90 20 homologous to GMAGVDGPPGPKGNMGPQGEPGPPGQQGNPGPQGLPGPQGPIGPPGEK
corresponding to amina acids 667 - 714 of CA1B HTJMAN, which also corresponds to amino acids 649 - 696 of HUMCA1XIA-P16, and a third amina 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% homologaus to a polypeptide having the sequence 25 VSFSFSLFYKKVIKFACDICRFVGRHDERKVVKLSLPLY'LIYE carrespanding to amino acids 697 - 738 of HUMCAIs~ P16, wherein said first amino acid sequence, second amino acid sequence and third arnina acid sequence are contiguaus and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of HUMCA1~:IA_P16, comprising a 30 palypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionallyy 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 AG, having a structure as follows: a sequence starting from any of amino acid numbers 64S-x to 648; and ending at any of amino acid numbers 649+ ((rr2) - x), in which x varies from 0 to rr2.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HUMCA1~ P16, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°~0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VSFSFSLFYKKVIKFACDKRFVGRHDERKWKLSLPLYLIYE in HUMCA1~A P16.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HLTMCA1~IA_P17, comprising a first amino acid sequence being at least 90 % homologous to MEPWSSRWKTKRWLWDFTVTTLALTFLFQAREVRGAAPVDVLKALDFHNSPEGISKTT
GFCTNRKNSKGSDTAYRV SKQAQLSAPTKQLFPGGTFPEDFSILFTVKPKKGIQSFLLSIIr NEHGIQQIGVEVGRSPVFLFEDHTGKPAPEDYPLFRTVNIADGKWHRVAISVEKKTVTM
IVDCKKKTTKPLDRSERAIVDTNGITVFGTRILDEEVFEGDIQQFLITGDPKAAYDYCEH
YSPDCDSSAPIkAAQAQEPQIDE corresponding to amino acids I - 260 of CAIB HUMAN, which also corresponds to amino acids 1 - 260 of HUMCA1~ P17, and a second amino acid sequence being at least 70°,~0, 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 VRSTRPEKVFVF'Q corresponding to amino acids 261 - 273 of HUMCAI~ P17, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HUMCA11IA P17, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRSTRPEKVF'VFQ in HUMCA1~ P17.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for R20779_P2, comprising a first amino acid sequence being at least 90 % homologous to MCAERLGQFMTLALVLATFDPARGTDATNPPEGPQDRSSQQKGRLSLQNTAEIQHCLV
NAGDVGCGVFECFENNSCEIRGLHGICMTFLHNAGKFDAQGKSFIKDALKCKAHALRH
RFGCISRKCPAIREMVSQLQRECYLKHDLCAAAQENTRVIVEMII-iF'KDLLLHE
corresponding to amino acids 1 - 169 of STC2_HCTMAN, which also corresponds to amino acids 1 - 169 of R2077Q P2, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence CYKIEITMPKRRKVKLRD
corresponding to amino acids 170 - 187 of R20779_P2, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of R20779_P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°.'0, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence CYKIEITMPKRRKVhLRD in R20779_P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for HSCOC4 PEA_1 P3, comprising a first amino acid sequence being at least 90 °,% homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSWHL,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKhEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIY'GKPVQGV AYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVhVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSH1'~YI'MILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRhILRNVNFQK_AINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPIVISVRRFEQLELRPVLYIVYLDKNLTV corresponding to amino acids 1 -865 of CO~ HUMAN, which also corresponds to amino acids 1 - 865 of HSCOC~
PEA_1 P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence RPHRSLSIQELGEPGPSEGWGG corresponding to amino acids 866 - 887 of HSCOC~ PEA_1 P3, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding fox a tail of HSCOC4 PEA_1 P3, comprising a polypeptide being at least 70°~0, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90°~o and most preferably at least about 95% homologous to the sequence RPHRSLSIQELGEPGPSEGWGG in HSCOC4 PEA_1 P3.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_I P5, comprising a first amino acid sequence being at least 90 % homologous to MRLLWWGLIWASSFFTLSLQKPRLLLFSPSWIiLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLkDAhSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVTtk.kE«~'SSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKI'VL
PNFEVKITPGKPYILTVPGI-IL,DEMQLDIQARl'IYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSkAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSW1'FVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
SRPPRVGDTLNLNLRAVGSGATFSH~'YYMILSRGQIVFMNREPILRTLTSVSVFVDHHLA
PSF1'FVAFYYHGDHPVANSLRVDVQAGAGEGKLELSVDGAKQI'RNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPIs~EKTTRICILRNVNFQKAINEKLGQI'ASPTAKR.CCQDGVTR
LPMhTRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQIL,TLWLPDSLTTWEIFiGLSLSKTKG corresponding to amino acids 1 - 818 of C04_HL1MAN, which also corresponds to amino acids 1 -818 of HSCOC4 PEA_1 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at lca:wk 90% and most preferably at least 95%
homologous to a polypeptide having the sequence L7'~-'TLSGPQVTLLPFPCTPAPCSLCS
corresponding to amino acids 819 - S43 of HSCOC 3~ ~EA_1 P5, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95°ib homologous to the sequence DVTLSGPQVTLLPFPCTPAPCSLCS in HSCOC4 PEA_1 P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P6, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQWKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVfLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQG1NLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQkMRPSTDTITVMV
ENSHGLRVRKKEVI'MPSSIFQDDFVIPDISEPGTWILISARFSDGLESNSSTQFEVILK~'VL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKILTFFR
LTSWYFVSSPFSLDLSKTKRF-ILVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYZ'MILSRGQIVFMNREPHRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVVQAGACEGKLELSVDGAKQYRNGESVKL,HLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEA1~~1SYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPhwIRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALELLQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAIHREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASR1'LDKTEQWSTLPPETKDHAVDLIQKG corresponding to amino acids 1 - 1052 of C04 HUMAN, which also corresponds to amino acids 1 -1052 of sa HSCOC4 PEA_1 P6, 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°.~0 homologous to a polypeptide having the sequence SGCKGKQEGGQERTVTGRWTAQEATEGKKGGP corresponding to amino acids 1053 -1084 of HSCOC~ PEA_1 P6, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SGCKGKQEGGQERTVTGRWTAQEATEGIVKGGP in HSCOC4 PEA_1 P6.
Accarding to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P1?, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQIiPRLLLFSPSWHLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
ENSHGLRVRKICEVI'MPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
GLESQTKLVNGQSHISLSKAEFQDALEItLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKThRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHY-YYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAI~~INSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKICEZNVNFQKAINEKLGQYASPTAh:RCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRILKSRDILGQAGLQRALEII,QEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTIS.GLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLI'NYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGS.ARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAI13REEL
VY~ELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTIviIYLAPTLAASRI'LDKTEQWSTLPPEThDHAVDLIQKG1'MRIQQFRIi ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISK.ASS
FLGEKASAGLLGAH.AAAITAYALTLTKAPADLRGVAHI~NLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKV corresponding to amino acids 1 - 1380 of C04 HLTM.~ V1, which also corresponds to amino acids 1 - 1380 ofHSCOC4 PEA_1 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence RAREGVGPGTGGGEGVE corresponding to amino acids 1381 - 1397 of HSCOC4 PEA_1 P12, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence R.AREGVGPGTGGGEGVE in HSCOC~ PEA_1 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1~,P15, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHL,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRK~EVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMILSRGQIVFIvINREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGShSHKPLNIViGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRhkRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPItwIRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRD)LGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQIL,TLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDhNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAI~-IREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTI~I~APTLAASRY'I,DKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEIkASAGLLGAHA AAITAYALTLTKAPADLRGVAHNNLIvIAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAI'WIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQ corresponding to amino acids 1 - 1359 of C04_HUMAN_V 1, which also corresponds to amino acids 1 - 1359 of HSCOC4_PEA_1 P15, 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 VNHSLVNHSLAWVARTPGPRGQARSRPQPPTRGIPAALLPGVFGGRLTSWLRDLEL
corresponding to amino acids 1360 - 1415 of HSCOC4_PEA_1 P15, wherein said first amino ?0 acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4_PEA_1 P15, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VNHSLVNHSLAWVARTPGPRGQARSRPQPPTRG1PAALLPGVFGGRLTSWLRDLEL in HSCOC4 PEA 1 P15.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P16, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSGGLHQLLRGPEVQLVAHSPWLK
a3 DSLSRTTNTQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRICKEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVIL.ITPGKPYIL,TVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYT~IILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHK.PLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKILRNVNFQKAINEKLGQYY ASPTAILRCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQIL,TLW LPDSLTTVVEIHGLSLShTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAII-IR.EEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRIt ADGSYYAAWLSRDSSTWLTAFVLKVL,SLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTILAPADLRGVAI-INNLMAMIAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IR.GLEEELQFSLGSKINVItVGGNSKGTLKVLRT~'NVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDYEYDELPAKDDPDAPLQPVTPLQLFEGRRNRRR.REAPK corresponding to amino acids 1 - 1457 of C04_HLTMAN_V 1, which also corresponds to amino acids 1 - 1457 of HSCOC4_PEA_1_P16, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence AERQGGAVWHGHRGRHPPEWIPRPAC corresponding to amino acids 1458 - 1483 of HSCOC4 PEA_1 P16, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P16, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90°,/° and most preferably at least about 95%
homologous to the sequence AERQGGAVWHGHRGRHPPEWIPRPAC in HSCOC4 PEA_1 P16.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P20, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLG~TPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRIiKEVYMPSSTFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYIL,TVPGHLDEMQLDIQARI'IYGKPVQGVAYVRFGLLDEDGIS.KTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYY~~SRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAG ACEGILLELSVDGAKQYRNGESVhLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEANINSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRI::RLSCPKEKTTRkKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEII,QEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTIS'_GLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAIHREEL
V1'ELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTVvrL,TAFVLKVL,SLAQEQVGGSPEKLQETSN'VLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAI-IAAAITAYALTLTKAPADLRGVAHNNLMAIvIAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQ corresponding to amino acids 1 - 1303 of C04 HLJMAN_V 1, which also e5 corresponds to amino acids 1 - 1303 of HSCOC4_PEA_1 P20, 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 VGAVPGLWRGWVVL,RPRACLSPGSTSLGHGDCPGCPVCLLDCLPHH corresponding to amino acids 1304 - 1349 of HSCOC4 PEA_1 P20, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P20, 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 VGAVPGLWRGWVULRPRACLSPGSTSLGHGDCPGCPVCLLDCLPHI-i in HSCOC4 PEA 1 P20.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P9, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIVVASSFFTLSLQKPRLLLFSPSVVHI,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRI~EV~'MPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDiQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSVVYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHCrDHPVANSLRVDVQAGACEGKLELSVDGAKQ1'RNGESVItLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNS1'DLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPIviMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVL1'NYLDhNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGA114R.EEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRI'LDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSI\~IQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTIiAPADLRGVAHNNLMAMAQETGDNL~'WGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDI'EYDELPAKDDPDAPLQPVTPLQLFEGRF~NRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
1'FDSV corresponding to amino acids 1 - 1529 of CO~ HLTMAN_V 1, which also corresponds to amino acids 1 - 1529 of HSCOC4 PEA_1 P9, and a second amino acid sequence being at least 70%, optionally at least SO%, preferably at least SS%, more. preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SGER
corresponding to amino acids 1530 - 1533 of HSCOC~ PEA_1 P9, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P9, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SGER in HSCOC4 PEA 1 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P22, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSWHL,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPILVDFTLSSERDFALLSLQVFLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKhEV~~'SSIFQDDFVIPDISEPGTVvThISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKhTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRL1'VAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
s7 EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGCy't'C'rrLSLERPD
SRPPRVGDTLNLNLRAVGSGATFSHYY1'MiLSRGQIVFMNREPKRTLTSV;i'~FVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESV~;..LHLETDS
LALVALGALDTALYAAGSIkSHKPLNMGKVFEAMNSYDLGCGPGGGDSAL,QVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPIviNIRSCEQRAARV QQPDCREPFLSCCQFAESLRKhSRDKGQAGLQRALEIL,QEEDLID
EDDIPVRSFFPENWLWRVETV'DRFQII,TLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYN'~'LDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVI,QIEKEGAIHREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMTYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGS1'AAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITA~'ALTLTKAPADLRGVAHNNLMAMAQETGDNL1TWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDYEI'DELPAKDDPDAPLQPVTPLQLFEGRF~NRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
YFDSVPTSRECVGFEAVQEVPVGLVQPASATLYDYZ'1VPERRCSVFYGAPSKSRLLATLC
SAEVCQCAEGKCPRQRRALERGLQDEDGYRMKFACYYPRVEYGFQVKVLREDSRAAF
RLFETKITQVLHF corresponding to amino acids 1 - 1653 of C04 HUMAN V1, which also corresponds to amino acids 1 - 1653 of HSCOC4_PEA_1 P22, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SMKQTGEAGRAGGRQGG corresponding to amino acids 1654 - 1670 of HSCOC~ PEA_1 P22, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1_P22, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about 85%, more preferably ss at least about ~0% and most preferably at least about 95% homologous to the sequence SMKQTGEAGRAGGRQGG in HSCOC4 PEA_1 P22.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC~ PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologots to MRLLWGL1WASSFFTLSLQILPRLLLFSPSV~~HI,GVPLSVGVQLQDVPRGQVVKGSVF'LR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTIT~VMV
ENSHGLRVRKKEVYIviPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSFFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYY~YMILSRGQIVFMNREPKRTLTSVSVF'VDHHLA
PSFYFVAFYYI-iGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKICRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEIL,QEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLY~NYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAA.AVSLKVVARGSFEFPVGDAVShVLQIEKEGAIHREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSY~VRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYL,APTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISkASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVAHNNLMAMAQETGDNLYWGSV
TGSQSNAV SPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDI~~NTTCQDLQIEVTVKGHVE
YTMEANEDY~EDYEYDELPAKDDPDAPLQPVTPLQLFEGRRNRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
YFDSVPTSRECVGFEAVQEVPVGLVQPASATLYDY~~NPERRCSVFYGAPShSRLLATLC
SAEVCQCAEGKCPRQRRALERGLQDEDGYRMKFACYYPRVEYG corresponding to amino acids 1 - 1626 of C04_HIJMAN_V 1, which also corresponds to amino acids 1 - 1626 of HSCOC4_PEA_1 P23, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90°.~o and most preferably at least 95% homologous to a polypeptide having the sequence QSSHRGPGLTLPRGPAVLVSLGVACSSYRSCTQPVCSDTNFLPSQPQSNSPFPLLLTPS
corresponding to amino acids 1627 - 1685 of HSCOC~ PEA_1 P23, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4_PEA_1 P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence QSSHRGPGLTLPRGPAVLVSLGVACSSYRSCTQPVCSDTNFLPSQPQSNSPFPLLLTPS in HSCOC4 PEA 1 P23.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P24, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQV~~ICGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLIkDAhSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKKEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKK1'VL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEIvIEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVhVSA'f~jSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSH1'YY~MILSRGQIVFMNREPKRTLTSVSVFVDHHI,A
PSFYFVAFY~f'HGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQRAARVQQPDCR.EPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATP ~Ii~L, RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGI,!~.Q
QVLVPAGSARPVAFSVVPTAAAAVSLKWARGSFEFPVGDAVSKVLQIEKEGAIFiR~Ei, VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
ADGSY AAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVA~INNLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDI'~EYDELPAkDDPDAPLQPVTPLQLFEGRFSJRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
1'FDS corresponding to amino acids I - 1528 of C04 HUl~ V1, which also corresponds to 15 amino acids 1 - 1528 of HSCOC4 PEA_1 P24, 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 SADVLCFTGHQVR.ADSWPPCVLLILSASVLRGSALASVAPWSGVCRTRMATG
corresponding to amino acids 1529 - 1579 of HSCOC4_PEA_1 P24, wherein said first amino 20 acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P24, 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 25 SADVLCFTGHQVRADSWPCVLLKSASVLRGSALASVAPWSGVCRTRMATG in HSCOC4 PEA 1 P24.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P25, comprising a first amino acid sequence being at least 90 % homologous to NPSRNNVPCSPKWFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTIVIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKKEVYMPSSIFQDDFVIPDISEPGTWhISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTkLUNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAlIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMIL,SRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGIhLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKKRNVNFQKAIIVEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQR.AARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLm EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRF'EQLELRPVLYNYLDhNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAffiREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMII'LAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLILVLSLAQEQVGGSPEKL.QETSNWLLSQQQADGSFQ
DPCPVLDRSIVIQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVAHNNLMAMAQETGDNLYWGS V
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
1'TI~~ANEDY'EDY'EYDELPAKDDPDAPLQPVTPLQLFEGRFS~IItRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
YFDSVPTSRECVGFEAVQEVPVGLVQPASATLYDYYNPERRCSVFYGAPSKSRLLATLC
SAEVCQCAEG corresponding to amino acids 1 - 1593 of C04_HUMAN_V1, which also corresponds to amino acids 1 - 1593 of HSCOC4_PEA_1 P25, 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 ETEGLGRGSGGGMAGAPPTLSDGFPNFREVPSPASRPGAGSAGRGWLQDEVCLLLPPC
GVRLPG corresponding to amino acids 1594 - 1657 of HSCOC4 PEA_1 P25, wwherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is pro~~ided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P35, 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 ETEGLGRGSGGGMAGAPPTLSDGFPNFREVPSPASRPGAGSAGRGWLQDEVCLLLPPC
GVRLPG in HSCOC4 PEA 1 P25.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P?6, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPS~~GVPLSVGVQ LQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
ENSHGLRVRKKEV~'MPSSIFQDDFVIPDISEPGTWhISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTI~.LVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAA.AIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSH1'1'~c'IvIILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNS1'DLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPh:EKTTRKICRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMNIRSCEQR.AARVQQPDCREPFLSCCQFAESLRhhSRDKGQAGLQRALEILQEEDLII7 EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSWPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGA)TIREEL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAVhL.SRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISIiASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVAI-INNLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDYEYDELPAKDDPDAPLQPVTPLQLFEGR.RNRRRREAPKVVEEQESRV
YFDSVPTSRECVGFEAVQEVPVGLVQPASATLYDYYNPERRCSVFYGAPSKSRLLATLC
SAEVCQCAEG corresponding to amino acids 1 - 1593 of C04_HLTIVI~_V1, which also corresponds to amino acids 1 - 1593 of HSCOC4_PEA_1 P26, 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 ETEGLGRGSGGGMAGAPPTLSDGFPNFREVPSPASRPGAGSAGRGWLQDEVCLLLPPC
GVRSVFPPRPWPDPPSGTGCFGLSGCSLLLLQVMHAACLL corresponding to amino acids 1594 - 1691 of HSCOC4_PEA_1 P26, wherein said first amino acid sequence and second I S amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P26, 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 ETEGL.GRGSGGGMAGAPPTLSDGFPNFREVPSPASRPGAGSAGRGWLQDEVCLLLPPC
GVRSVFPPRPWPDPPSGTGCFGLSGCSLLLLQVMHAACLL in HSCOC4 PEA_1 P26.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSCOC4_PEA_1 P30, comprising a first amino acid sequence being at least 90 % homologous to I~~LLWGLIWASSFFTLSLQKPRLLLFSPSWHLGVPLSVGVQLQDVPRGQVVICGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQG1NLLFSSRRGHLFLQTDQPIYNPGQRVR1'RVFALDQKMRPSTDTITVMV
ENSHGLRVRKKEVYn~IPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVILKY'VL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLShAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEIvIEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQF:( _rIjGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGD T I,NLNLRAVGSGATFSHYYYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFI'FVAF ;''~.c iiGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRICI~E~NVNFQKAINEILLGQYASPTAKRCCQDGVTR
EDDIPVRSFFPENWLWRVETVDRFQIL,TLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGA)HREEEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSY'VRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLTKAPADLRGVAHNNLMAMAQETGDNLl'WGS
corresponding to amino acids 1 - 1232 of C04 HLIMAN_V3, which also corresponds to amino acids 1 - 1232 of HSCC~C4 PEA_1 P30, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90°ro and most preferably at least 95% homologous to a polypeptide having the sequence RNPVRLLQPRAQMFCVLRGTK corresponding to amino acids 1233 - 1253 of HSCOC4 PEA_1 P30, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC~ PEA_1 P30, 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 RNPVRLLQPRAQIvfFCVLRGTK in HSCOC4 PEA_1 P30.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HSC(aC4 PEA_1 P38, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTIVIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQIhMRPSTDTITVMV
ENSHGLRVRKKEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVhITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEI'~EAE
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHY~1'MILSRGQNFMIVREPKRTLTSVSVFVDHHLA
PSFYFVAFY1'HGDHPVANSLRVDVQAGACEGkLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTAL1'AAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
10 LAFSDGDQWTL.SRKRLSCPKEKTTRKKRNVNFQK.AINEKLGQYASPTAKRCCQDGVTR
LPMMRSCEQR.AARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQIL,TLWLPDSLTTWEIHGLSLSKTKG corresponding to amino acids 1 - 818 of CO4 HUMAN, which also corresponds to amino acids 1 -818 of HSCOC4 PEA_1 P38, and a second amino acid sequence being at least 70%, optionally at 15 least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DVTLSGPQVTLL.PFPCTPAPCSLCS
corresponding to amino acids 819 - 843 of HSCOC~ PEA_1_P38, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an 20 isolated polypeptide encoding for a tail of HSCOC4 PEA_1 P3S, comprising a polypeptide being at least 70%, optionally at least about SO°'/°, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DVTLSGPQVTLLPFPCTPAPCSLCS in HSCOC4 PEA_1 P38.
According to preferred embodiments of the present invention, there is provided an 25 isolated chimeric polypeptide encoding for HSCOC4_PEA_1_P39, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQILPRLLLFSPSV~~,GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTT'NIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
30 ENSHGLRVRKKEVI'1VIPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQ corresponding to amino acids 1 - 387 of C04_ _ _HUMAN, which also corresponds to amino acids 1 - 387 of HSCOC4 PEA_1 P39, 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 VSSRGEG corresponding to amino acids 388 - 394 of HSCOC4 PEA_I P39, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4_PEA_1 P39, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VSSRGEG in HSCOC4_PEA_1 P39.
According to preferred embodiments of the present invention, there is provided an IS isolated chimeric _polypeptide encoding for HSCOC4_PEA_1 P40, comprising a first amino acid sequence being at least 90 % homologous to MRLLVvjGLIWASSFFTLSLQKPRLLLFSPSVVHL,GVPLSVGVQLQDVPRGQV~~kGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRhKEVI'MPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVKKY
corresponding to amino acids 1 - 236 ofC04_HUMAN, which also corresponds to amino acids I - 236 of HSCOC4_PEA_1 P40, 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 AGEWTEPHFPLKGRVPGRPGEAEYGHY corresponding to amino acids 237 - 263 of HSCOC4_PEA_1 P40, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of HSCOC4_PEA_I P40, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% anc~. a~aost preferably at least about 95% homologous to the sequence AGEWTEPHFPLKGRevfGRPGEAEYGHY in HSCOC4 PEA 1 P40.
According to preferred embodiments of the present invvention, there is provided an isolated chimeric polypeptide encoding for HSCOC4 PEA_1 P41, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQIkPRLLLFSPSWHI.GVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAItSCGLHQLLRGPEVQLVAHSPWLK
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRhhEVYMPSSIFQDDFVIPDISEPGTWKISARFSDGLESNSSTQFEVIiKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVA1'VRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVKVSATVSSPGSVP
EVQDIQQNTDGSGQVSIPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWTLSRKRLSCPKEKTTRKICRNVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMNIRSCEQRAARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEILQEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVLYNYLDKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSWPTAAAAVSLKVVARGSFEFPVGDAVSKVLQIEKEGAIHREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKG1'MRIQQFRK
ADGSYAAWLSRDSSTWLTAFVL,KVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAI3AA.AITAYALTLTKAPADLRGVAFINNLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
LRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVZDMKNTTCQDLQIEVTVKGHVE
1'TMEANEDYEDYEYDELPAKDDPDAPLQPVTPLQLFEGR.FSJRRRREAPKVVEEQESRV
HYTVCIWRNGKVGLSGMAIADVTLLSGFHALRADLEKLTSLSDRYVSHFETEGPHVLL
YFDSV _corresponding to amino acids 1 - 1529 of C04 HLTMAN_V 1, which also corresponds to amino acids 1 - 1529 of HSCOC4 PEA_1 P41, 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 SGER
corresponding to amino acids 1530 - 1533 of HSCOC4 PEA_1 P41, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of HSCOC4 PEA_1 P41, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and mast preferably at least about 95% homologous to the sequence SGER in HSCOC4 PEA 1 P41.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for HSCOC4 PEA_1_P42, comprising a first amino acid sequence being at least 90 % homologous to MRLLWGLIWASSFFTLSLQKPRLLLFSPSVVHLGVPLSVGVQLQDVPRGQVVKGSVFLR
NPSRNNVPCSPKVDFTLSSERDFALLSLQVPLKDAKSCGL,HQLLRGPEVQLVAHSPVrL,K
DSLSRTTNIQGINLLFSSRRGHLFLQTDQPIYNPGQRVRYRVFALDQKMRPSTDTITVMV
ENSHGLRVRKKEVYMPSSIFQDDFVIPDISEPGTWILISARFSDGLESNSSTQFEVILKYVL
PNFEVKITPGKPYILTVPGHLDEMQLDIQARYIYGKPVQGVAYVRFGLLDEDGKKTFFR
GLESQTKLVNGQSHISLSKAEFQDALEKLNMGITDLQGLRLYVAAAIIESPGGEMEEAE
LTSWYFVSSPFSLDLSKTKRHLVPGAPFLLQALVREMSGSPASGIPVhVSATVSSPGSVP
EVQDIQQNTDGSGQVSTPIIIPQTISELQLSVSAGSPHPAIARLTVAAPPSGGPGFLSIERPD
SRPPRVGDTLNLNLRAVGSGATFSHYYYMILSRGQIVFMNREPKRTLTSVSVFVDHHLA
PSFYFVAFYYHGDHPVANSLRVDVQAGACEGKLELSVDGAKQYRNGESVKLHLETDS
LALVALGALDTALYAAGSKSHKPLNMGKVFEAMNSYDLGCGPGGGDSALQVFQAAG
LAFSDGDQWWTLSRKRLSCPKEKTTRKI~E~NVNFQKAINEKLGQYASPTAKRCCQDGVTR
LPMI\~IRSCEQR.AARVQQPDCREPFLSCCQFAESLRKKSRDKGQAGLQRALEIL,QEEDLID
EDDIPVRSFFPENWLWRVETVDRFQILTLWLPDSLTTWEIHGLSLSKTKGLCVATPVQL
RVFREFHLHLRLPMSVRRFEQLELRPVL1'NYL,DKNLTVSVHVSPVEGLCLAGGGGLAQ
QVLVPAGSARPVAFSVVPTAAAAVSLKVVWARGSFEFPVGDAVSKVLQIEKEGA)T REIREEL
VYELNPLDHRGRTLEIPGNSDPNMIPDGDFNSYVRVTASDPLDTLGSEGALSPGGVASL
LRLPRGCGEQTMIYLAPTLAASRYLDKTEQWSTLPPETKDHAVDLIQKGYMRIQQFRK
ADGSYAAWLSRDSSTWLTAFVLKVLSLAQEQVGGSPEKLQETSNWLLSQQQADGSFQ
DPCPVLDRSMQGGLVGNDETVALTAFVTIALHHGLAVFQDEGAEPLKQRVEASISKASS
FLGEKASAGLLGAHAAAITAYALTLThAPADLRGVAHNNLMAMAQETGDNLYWGSV
TGSQSNAVSPTPAPRNPSDPMPQAPALWIETTAYALLHLLLHEGKAEMADQAAAWLTR
QGSFQGGFRSTQDTVIALDALSAYWIASHTTEERGLNVTLSSTGRNGFKSHALQLNNRQ
IRGLEEELQFSLGSKINVKVGGNSKGTLKVLRTYNVLDMKNTTCQDLQIEVTVKGHVE
YTMEANEDYEDYEYDELPAKDDPDAPLQPVTPLQLFEGRTS~RRRREAPKVVEEQESRV
HYTVCIW -corresponding to amino acids 1 - 1473 of C04_HUMAN_V 1, which also corresponds to amino acids 1 - 1473 of HSCOC4_PEA_1 P42, 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 WAPGAALGQGREGRTQAGAGLLEPAQAEPGRQLTRLHR corresponding to amino acids 1474 - 1511 of HSCOC4 PEA_1 P42, a third amino acid sequence being at least 90 homologous to RNGKVGLSGMAIADVTLLSGFHALRADLEK corresponding to amino acids 1474 - 1503 of C04_HLTMAN_V 1, which also corresponds to amino acids 1512 -1541 of HSCOC4 PEA_1 P42, and a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence VWSATQGNPLCPRY corresponding to amino acids 1542 - 1555 of HSCOC4-PEA_1 P42, wherein said first amino acid sequence, second amino acid sequence, third amino acid sequence and fourth amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ -encoding for an edge portion of HSCOC4_PEA_1 P42, 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 encoding for WAPGAALGQGREGRTQAGAGLLEPAQAEPGRQLTRLHR, corresponding to HSCOC4 PEA_1 P42.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of HSCOC~ PEA_1 F42, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about 85%, more preferably at least about 90% and most pref:~~-~hly at least about 95% homologous to the sequence VWSATQGNPLCPRY in HSCOC4i, PEA_1 P4?.
According to preferred eml~o~liments of the present invention, there is provided an isolated chimeric _ _ _polypeptide encoding for HLTMTREFAC PEA 2 P8, comprising a first amino acid sequence being at least 90 % homologous to MAARALCMLGLVLALLSSSSAEEYVGL corresponding to amino acids 1 - 27 of TFF3 HUI\~L~1N, which also corresponds to amino acids 1 - 27 of HUMTREFAC_PEA
2 P8, 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 WkVHLPKGEGFSSG corresponding to amino acids ~8 of HUMTREFAC_PEA_2 P8, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ _encoding for a tail of HLTIvITREFAC PEA 2 P8, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence WKVHLPKGEGFSSG in HLTMTREFAC_PEA 2 P8.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _ _polypeptide encoding for HLTMOSTRO PEA_1 PEA_1 P21, comprising a first amino acid sequence being at least 90 % homologous to MRIAVICFCLLGITCAIPVILQADSGSSEEKQLYNKYPDAVATWLNPDPSQKQNLLAPQ
corresponding to amino acids 1 - 58 of OSTP_I-IIJMAN, which also corresponds to amino acids 1 - 58 of HUMOSTRO PEA_1 PEA_1 P?1, 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 VFLNFS
corresponding to amino acids 59 - 64 of HUMOSTRO PEA_1 PEA_1 P21, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ _encoding for a tail of HUMOSTRO_PEA_1 FEA_1 P21, comprising a polypeptide being at least 70%, optionallyy at least about 80%, preferably at least about S5%, 1oi more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VFLNFS in HLTMOSTRO PEA_1 PEA_1 P21.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HLTMOSTRO PEA_1 PEA_1 P25, comprising a first amino acid sequence being at least 90 % homologous to MRIAVICFCLLGITCAIPVILQADSGSSEEhQ corresponding to amino acids 1 - 31 of OSTP_HTJMAN, which also corresponds to amino acids 1 - 31 of HUMOSTRO _PEA_1 PEA_1 P25, 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 H
corresponding to amino acids 32 - 32 of HLTMOSTRO _PEA_1 PEA_1 P25, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric - - -polypeptide encoding for HUMOSTRO PEA_1 PEA_1 P30, comprising a first amino acid sequence being at least 90 % homologous to NIRIAVICFCLLGITCAIPVKQADSGSSEEKQ corresponding to amino acids 1 - 31 of OSTP_HUMAN, which also corresponds to amino acids 1 - 31 of IiUMOSTRO_PEA_1 PEA_1 P30, 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 VSIFYVFI
corresponding to amino acids 32 - 39 of HUMOSTRO PEA_1 PEA_1 P30, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide - - -encoding for a tail of HLTMOSTRO PEA_1 PEA_1 P30, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VSIFYVFI _ _ -in IiUMOSTRO PEA_1 PEA_1 P30.
According to preferred embodiments of the present invention, there is provided an isolated chimeric - .polypeptide encoding for T10888_PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWIiEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVYPELPhPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
WWVNGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLY
GPDVPTISPSItANYRPGENLNLSCHAASNPPAQYSWF1NGTFQQSTQELFIPNITVNNSGS
YMCQAHNSATGLNRTTVTMITVS co~esponding to amino acids 1 - 319 of CEA6_ -HUMAN, which also corresponds to amino acids 1 - 319 of T10888_PEA_1 P2, 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 DWTRP -corresponding to amino acids 320 - 324 of T10888_PEA_1 P2, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ _encoding for a tail of T10888 PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DWTRP in T1088S_PEA_1 P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for T10888 PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPFSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYIiGERVDGNSLI~~GYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDILDAVAFTCEPEVQNTTYI, WWVNGQSLPVSPRI,QLSNGNMTLTLLSVhRNDAGSYECEIQNPASANRSDPVTLNVL
corresponding to amino acids 1 - 234 of CEA6_HUMAN, which also corresponds to amino acids 1 - 234 of T10888_PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence LLLSSQLWPPSASRLECWPGWL corresponding to amino acids 235 - 256 of T10888_PEA_1 P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T10888_PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LLLSSQLWPPSASRLECWPGWL in T10888_PEA_1 P4.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T10888_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYKGERVDGNSLIVGI'~~IGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFI-IVYPELPIiPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
WW-VNGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNP ASANRSDPVTLNVL
corresponding to amino acids 1 - 234 of Q 13774, which also corresponds to amino acids 1 - 234 of T10888_PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence LLLSSQLWPPSASRLECWPGWL
corresponding to amino acids 235 - 256 of T10888_PEA_1 P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T10888_PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LLLSSQLWPPSASRLECWPGVhL, in T10888_PEA_1 P4.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T108SS PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGhEVLLLAHNLP
QNRIGYS WYKGERVDGNSLIVGYV IGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTY'L
GQSLPVSPRLQLSNGNMTLTLLSVhRNDAGSYECEIQNPASANRSDPVVTLNVLY
GPDVPTISPSK:ANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGS
YMCQAHNSATGLNRTTVTMITVSG corresponding to amino acids 1 - 320 of CEA6_ _ _HUMAN, which also corresponds to amino acids 1 - 320 of T10888 PEA_1 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, mare preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence KWIHEALASHFQVESGSQRRARItICFSFPTCVQGAHANPhFSPEPSQFTSADSFPLVFLFF
VVFCFLISHV _ _corresponding to amino acids 321 - 390 of T10888 PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of T10888_PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence hWIHEALASHFQVESGSQRRARI~FSFFTCVQGAHANPKFSPEPSQFTSADSFPLVFLFF
VVFCFLISHV in T10888 PEA_I P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T10888_PEA_1 P6, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVP'WItEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGILEVLLLA
HNLPQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIY'PNASLLIQNVTQ
NDTGFYTLQVIK.SDLVNEEATGQFHVY
corresponding to amino acids 1 - 141 of CEA6 HUMAN, which also corresponds to amino acids 1 - 141 of T10888_PEA_1 P6, 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 REYFHMTSGCWGSVLLPTYGIVRPGLCLWPSLHYILYQGLDI
corresponding to amino acids 142 - 183 of T10888_PEA_1 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T10888_PEA_1 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 9>".s;, homologous to the sequence REYFHMTSGCWGSVLLPTYGIVRPGLCLWPSLH~'u''LYQGLDI in T10888 PEA_1 P6.
According to preferred embodiments of the pre,acnt invention, there is provided an isolated chimeric polypeptide encoding for T39971 P6, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLIL,ALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQY~WRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKG corresponding to amino acids 1 - 276 of VTNC_HLTMAN, which also corresponds to amino acids 1 - 276 of T39971 P6, 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 TQGVVGD corresponding to amino acids 277 - 283 of T39971 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T39971 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologois to the sequence TQGWGD
in T39971 P6.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T39971 P9, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDE1'TVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLIiNGSLFAFR
GQYCI'ELDEKAVRFGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQ1'WRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEI'QFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRT corresponding to amino acids 1 - 325 of VTNC_HUMAN, which also corresponds to amino acids 1 - 325 of T39971 P9, and a second amino acid sequence being at least 90 % homologous to SGMAPRPSLAKKQRFRHIZNRI~GYRSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGA
NNYDDYRMDWLVPATCEPIQSVFFFSGDK1'YRVNLRTRRVDTVDPPYPRSIAQYWLGC
PAPGHL corresponding to amino acids 357 - 478 of VTNC_HUMAN, which also corresponds to amino acids 326 - 447 of T39971 P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of T39971 P9, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise TS, having a structure as follows: a sequence starting from any of amino acid numbers 325-x to 325; and ending at any of amino acid numbers 326 + ((n-2) - x), in which x vanes from 0 to t~-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T39971 P11, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTI~~'EDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHS1'SGRERVYFFKGKQYWEYQFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDLFELLFWGRTS corresponding to amino acids 1 - 326 of VTNC_ -HUMAN, which also corresponds to amino acids 1 - 326 of T39971 PI 1, and a second amino acid sequence being at least 90 % homologous to DKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL corresponding to amino acids 442 - 478 of VTNC'_HLJIvIAN, which also corresponds to amino acids 327 - 363 of T39971 P11,, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for an edge portion of T39971 PI 1, 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 SD, having a structure as follows: a sequence starting from any of amino acid numbers 326-x to 326; and ending at any of amino acid numbers 327 + ((n-2) - x), in which x varies from 0 to rr2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T39971 P1 l, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNA': VHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASkPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQI'WRFEDGV
LDPDYPRIVISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTS corresponding to amino acids 1 - 326 of Q9BSH7, -which also corresponds to amino acids 1 - 326 of T39971 P11, and a second amino acid sequence being at least 90 % homologous to DKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL corresponding to amino acids 442 - 478 of Q9BSH7,, -which also corresponds to amino acids 327 - 363 ofT39971 P11, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of T39971 P11, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise SD, having a structure as follows: a sequence starting from any of amino acid numbers 326-x to 326; and ending at any of amino acid numbers 327 + ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T39971 P12, comprising a first amino acid sequence being at least 90 % homologous to 1\~IAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
1os KPQVTRGDVFTMPEDEI'TVYDDGEEKNNATVHEQVGC?~:1's I.TSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELC'S~~KPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFK corresponding to amino acids 1 - 223 of VTNC_HTJMAN, which also corresponds to amino acids 1 -223 of T39971 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence VPGAVGQGRKHLGRV corresponding to amino acids 224 - 233 of T39971 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T39971 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VPGAVGQGRKHLGRV in T39971 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T39971 P12, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLIL,ALLAWVALADQESChGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCY~ELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFK corresponding to amino acids 1 - 223 of Q9BSH7, _which also corresponds to amino acids 1 - 223 of T39971 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VPGAVGQGRKHLGRV corresponding to amino acids 238 of T39971 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T39971 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VPGAVGQGRKHLGRV in T39971 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for 221366 PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIPvfNIILVLTDDQDVELGSL
QVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKY~r'><~TNNENCSSPSW
QAMFIEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYYIPPGWREWLGLIKNSRFYNYTVCR
NGIKEKHGFDYAKDYFTDLITNESINYFKMSKRW'PHRPVNEVIVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYNYAPNMDKIiWIMQYTGPMLPIHMEFTMLQRKRLQTLMSVDD
SVERLYNMLVETGELENTYIIYTADHGYHIGQFGLVKGKSMP~'DFDIRVPFFIRGPSVEP
GSIVPQIVLNIDLAPTILDIAGLDTPPDVDGKSVLKLLDPEKPGNRFRTNKKAKIWRDTFL
VERGKFLRKKEESSKNIQQSNHLPKYERVKELCQQARYQTACEQPGQKWQCIEDTSGK
LRIHKCKGPSDLLTVRQSTRNLYARGF HDKDKECSCRESGYRASRSQRKSQRQFLRNQ
GTPKYKPRFV~-iTRQTRSLSVEFEGEIYDINLEEEEELQVLQPRNIAkRHDEGHKGPRDLQ
ASSGGNRGRMLADSSNAVGPPTTVRVTHKCFILPNDSIHCERELYQSARAWKDHKAYI
DKEIEALQDKIKNLREVRGHLKRRILPEECSCSKQSYYNKEKGVIkKQEKLKSHLHPFKE
AAQEVDSKLQLFKENNRRRICKERKEKRRQRKGEECSLPGLTCFTHDNNIiWQTAPFWN
corresponding to amino acids 1 - 761 of SUL1 HUMAN, which also corresponds to amino acids 1 - 761 of 221368 PEA_1 P2, 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 PHKYSAHGRTRHFESATRTTNGAQKLSRI corresponding to amino acids 762 - 790 of Z21368_PEA_1 P2, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of 221368 PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence PHKI'SAHGRTRHFESATRTTNGAQKLSRI in 221368 PEA_1 P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ -polypeptide encoding for 221368 PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIIL,VLTDDQDVEL
corresponding to amino acids 1 - 57 of Q7Z2W2, which also corresponds to amino acids 1 - 57 of 221368 PEA_1 P5, second bridging amino acid sequence comprising A, and a third amino acid sequence being at least 90 % homologous to FFGKYLNEYNGSYIPPGWREWLGLIKNSRFYNIrTVCRNGIKEKHGFDYAKDYFTDLITN
ES1NYFKMSKRIvfYPHRPVMIvIVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNM
DKHWIMQYYTGPMLPIHMEFTNILQRKRLQTLMSVDDSWRLYNMLVETGELENTYIIYT
ADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTILDIAGLDT
PPDVDGKSVLkLLDPEKPGNRFRTNKKAKIWRDTFLVERGKFLRKKEESSKNIQQSNHL
PKYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRNLY
ARGFHDIkDKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRF~~I-ITRQTRSLSVEFE
GEIYDINLEEEEELQVLQPRNIAKItHDEGHhGPRDLQASSGGNRGRMLADSSNAVGPPT
TVRVTHKCFILPNDSIHCERELYQSARAWKDHKAYIDKEIEALQDK)kNLREVRGHLKR
RKPEECSCSKQSYYNKEKGVKKQEKLKSHLHPFKEAAQEVDSKLQLFKErTNRRRIiICER
KEKRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNE
THNFLFCEFATGFLEYFDMNTDPYQLTNTVHTVERGIL,NQLHVQLMELRSCQGYILQCN
PRPKNLDVGNKDGGSYDLHRGQLWDGWEG corresponding to amino acids 139 - 571 of Q7Z2W2; which also corresponds to amino acids 59 - 791 of Z2136S PEA_1 P5, wherein said first, second arid third amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ -encoding for an edge portion of 221368 PEA_1 P5, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least three amino acids comprise LAF having a structure as follows (numbering according to Z2136g PEA_1 PS): a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 59 + ((n-2) - x), in which x varies from 0 to n-2.
m According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for 221368_PEA_1 P5, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MKI'SCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIII,VLTDDQDVELAFF
GKYLNBYNGSY1PPGWREWLGLIKNSRFYNYTVCRNG)KF~HGFDYAKDYFTDLITNES
INYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNMDK
HWIMQI'TGPMLPIHIviEF1 NILQRKRLQTLMSVDDSVERLYNNiZVETGELENTYIIYTAD
HGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTILDIAGLDTPP
DVDGKSVLKLLDPEKPGNRFRTNKKAhiWRDTFLVERGKFLRKKEESSKNIQQSNHLP
KYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRNLYA
RGFHDKDKECSCRESG1'RASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEFEGE
RVTHKCFILPNDSIHCERELYQSARAWItDHKAI'IDKELEAL,QDKI~NLREVRGHLKRRK
PEECSCSKQSYYNKEKGVKKQEKLKSHLHPFKEAAQEVDSKLQLFKENNR7f~RItKERhE
KRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNETH
NFLFCEFATGFLEYFDMNTDPYQLT'NTVHTVERGILNQLHVQLME corresponding to amino acids 1 - 751 of Z21368_PEA_1 P5, and a second amino acid sequence being at least 90 % homologous to LRSCQGI'ICQCNPRPKNLDVGNILDGGSYDLHRGQLWDGWEG
corresponding to amino acids 1 - 40 of AAH12997, which also corresponds to amino acids 752 -791 of 221368_PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a head of 221368 PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELAFF
GKYLNEYNGSYIPPGWREWLGLIKNSRFYNYTVCRNGIKEKHGFDYAKDYFTDLIT'NES
IN1'FKMSKRMYPHRFVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNMDK
HWIMQl'TGPMLPII~-iMEFTNILQRKRLQTLMSVDDSVERLI'NMI,VETGELENT'YIIYTAD
HGYHIGQFGLVKGKSIvIPI'DFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTILDIAGLDTPP
DVDGKSVLKLLDPEKPGNRFRTNItKAKIWRDTFLVERGKFLRKKEESSKNIQQSNHLP
KYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRII~hCKGPSDLLTVRQSTRNLYA
RGFHDKDKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEFEGE
I1'DINLEEEEELQVLQPRNL4ICRHDEGHKGPRDLQASSGGNRGRMLADSSNAVGPPTTV
RVTHKCFILPNDSIHCERELYQSARAWKDHKAY'mKEIEALQDKIKNLREVRGHLKRRK
PEECSCSKQSYYNKEKGVKKQEKLKSHLHPFKEAAQEVDSKLQLFKENNRRRKICERKE
ILRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNETH
NFLFCEFATGFLEYFDMNTDPYQLTNT'VHTVERGILNQLHVQLME of Z21368_PEA_1_P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric - -polypeptide encoding for Z2136S PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERhNIRPNIIL,VLTDDQDVEL
corresponding to amino acids 1 - 57 of SL1L 1 HTJMAN, which also corresponds to amino acids 1 - 57 of X21365 PEA_1 P5, and a second amino acid sequence being at least 90 homologous to AFFGKYLNEYNGS1'IPPGWREWLGLIKNSRFYNYTVCRNGIKEhHGFDYAKDYFTDLIT
NESINYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPN
MDhHWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDDSVERLYNIviLVETGELENTYII
YTADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQNLNIDLAPTILDIAGL
DTPPDVDGILSVLKLLDPEKPGNRFRTNKKAhIWRDTFLVERGKFLRKKEESSKNIQQSN
HLPKYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRN
LYARGFHDKDKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVE
FEGEIYDINLEEEEELQVLQPRNL~1KRI-IDEGHKGPRDLQASSGGNRGRMLADSSNAVGP
PTTVRVTHKCFILPNDSIHCERELYQSARAWKDHKAYIDKEIEALQD 'I~NLREVRGHL
KRRKPEECSCSKQSY1'NKEKGVhKQEKLKSHI,HPFKEAAQEVDSKLQLFKENNRRRK
ILERKEKRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRT
VNETHNFLFCEFATGFLEI'FDMNTDPYQLTNTVHTVERGIL,NQLHVQLMELRSCQGYK
QCNPRPKNLDVGNKDGGSYDLHRGQLWDGWEG corresponding to amino acids 138 - 871 of SLTL1 HUT~IAN, which also corresponds to amino acids 58 - 791 of Z2136S
PEr~~._,1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential .order.
According to preferred embodiments of the present invention, there is providc~i an isolated chimeric -polypeptide encoding for an edge portion of Z21368_PEA_1 P5, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more.
preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise LA, having a structure as follows: a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 58 + ((n-2) - x), in which x varies from 0 to n-?.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for Z2136s PEA_1 P15, comprising a first amino acid sequence being at least 90 °~o homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIiLVLTDDQDVELGSL
QVMNkTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYV'I~IHIVVYTNNENCSSPSW
QAMTIEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYIPPGWREWLGLIKNSRFIrNI'TVCR
NGIKEKHGFDYAKDYFTDLITNESINYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYNYAPNMDKHWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDD
SVERLYNNIL,VETGELENTYIIYTADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEP
GSNPQIVT,NIDLAPTILDIAGLDTPPDVDGKSVLKLLDPEKPGNRFRTNKKAkIWRDTFL
VERG corresponding to amino acids 1 - 416 of SL1L1 HCTMAN, which also corresponds to amino acids 1 - 416 of Z2136s PEA_1 P15.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for 221368 PEA_1 P16, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCST'VRSPRFRGRIQQERhNIRPNIILVLTDDQDVELGSL
QVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYYVI~VHNVYYTNNENCSSPSW
QAIVgiEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYIPPGWREWLGLIKNSRFYNYTVCR
NGIKEKHGFDYAKD1'FTDLITNESINYFIiMSKRMYPHRPVMMVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYNYAPNMDKHWIIvIQYYTGPMLPIHI\~FTNILQRKRLQTLMSVDD
SVERLYNNILJVETGELENTYIIYTADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEP
GSIVPQIVLNIDLAPTILDIAGLDTPPDVDGKSVLKLLDPEKPGNR corresponding to amino acids 1 - 397 of SLTL1 HUMAN, which also corresponds to amino acids 1 - 397 of 221368 PEA_1 P16, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence CVIVPPLSQPQIH corresponding to amino acids 398 - 410 of 221368 PEA_1 P16, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of Z21368_PEA_1 P 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 CVIVPPLSQPQIH in Z21368_PEA_1 P16.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for Z21368_PEA_ 1 P22; comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVI,TDDQDVELGSL
QVNINKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYVI3fNHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRTAFFGKYLNEYNGSZ'IPPGVvrREWLGLIItNSRFYNYTVCR
NGIKEhHGFDYAK corresponding to amino acids 1 - 188 of SUL1 HUMAN, which also corresponds to amino acids 1 - 1S8 of Z21368_PEA_1 P22, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ARYDGDQPRCAPRPRGLSPTVF corresponding to amino acids 189 - 210 of Z21368_PEA_1 P22, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of 221368 PEA_1 P22, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ARYDGDQPRCAPRPRGLSPTVF in Z21368_PEA_1 P22.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for Z21368_PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologous to MILYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERIiNIRPNIILVLTDDQDVELGSL
QVMNKTRK~EHGGATFINAFVTTPMCCPSRSSIvIL,TGKYV~-1NHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRT corresponding to amino acids 1 - 137 of Q7Z2W2, which also corresponds to amino acids I - 137 of Z21368_PEA_1 P23, 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 GLLHRLNH _ _corresponding to amino acids 138 - 145 of 221368 PEA_1 P23, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of Z2136S PEA_I P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GLLHRLNH in Z21368_PEA_1 P23.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for Z21368_PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVL,GTELLGSLCSTVRSPRFRGRIQQERhNIRPNIILVLTDDQDVELGSL
QVIvINKTRKIMEHGGATF1NAFVTTPMCCPSRSSMLTGKY~VHNHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRT corresponding to amino acids 1 - 137 of SLTL IHiUMAN, which also corresponds to amino acids 1 - 137 of Z21368_PEA_1 P23, 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 GLLHRLNH _corresponding to amino acids 138 - 145 of Z21368_PEA_1 P23, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of 221368 PEA_1 P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GLLHRLNH in 221368 PEA_I P23.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832_P5, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYK corresponding to amino acids 12 - 55 of GILT_1-IUMAN, which also corresponds to amino acids 1 - 44 of T59832 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VGTATGRAGWREQAPCRGTRLLLSPQTSQGKTRAPRGRCPCRVPGhTLFSSRRCGHTP
SVPFRFRIPHLRGAAASTRLVPPKGSI''lSAYCVLLGQELGSPFVAQGTSS.AAGQGPPACIL, AATLDAFIPARAGLACLWDLLGRCPRG corresponding to amino acids 45 - 189 of T59832_P5, wherein said first and second amino acid~sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T5983? P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VGTATCTRAGWREQAPCRGTRLLLSPQTSQGKTRAPRGRCPCRVPGKTLFSSRRCGHTP
SVPFRFRIPHLRGAAASTRLVPPKGSMSAYCVLLGQELGSPFVAQGTSSAAGQGPPACIL
AATLDAFIPARAGLACLWDLLGRCPRG in T59832 P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832 P7, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLVI~~ILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVEAGVLDELDMELAFLTIVCMEEFEDI~~RSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMIiANAQRTDALQPPHEYVPWVTVNG corresponding to amino acids 12 - 223 of GILT HUMAN, which also corresponds to amino acids 1 - 212 of T59832_P7, 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 VRIFLALSLTLNPWSQGWTRQRDQR corresponding to amino acids 213 - 238 of T59832 P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T59832_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRIFLALSLTLIVPWSQGWTRQRDQR in T59832 P7.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59833 P7, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPV~NYhTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFItC
QHGEEECKFNKVEACVLDELDMELAFLTIVCI\~EFEDMERSLPLCLQLYAPGLSPDTII~i ECAMGDRGMQLMHANAQRTDALQPPHEYVPWVTVNG corresponding to amino acids 1 - 212 of BAC98466, which also corresponds to amino acids 1 - 212 of T59832_P7, 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 VRIFLALSLTLIVPWSQGWTRQRDQR corresponding to amino acids 213 - 238 of T59832_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T59832_P7, comprising a polypeptide being at least 70%, optionallyy 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 VRIFLALSLTLIVPWSQGWTRQRDQR in T59832_P7.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832_P7, comprising a first amino acid sequence being at least 70°.~0, optionally at least 80%, preferably at least 85%, more preferablyy at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ms MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKILSNA
PLVN VTLYYEALCGGCRAFLIRELFPTWLLV corresponding to amino acids 1 - 90 of T59832_P7, and a second amino acid sequence being at least 90 % homologous to MEILNVTLVPYGNAQEQNVSGRWEFhCQHGEEECKFNKVEACVLDELDMELAFLTIVC
MEEFEDMERSLPLCLQLYAPGLSPDT)IvvIECAMGDRGMQLMHANAQRTDALQPPHEYV
PWVTVNGVRIFLALSLTLIVPWSQGWTRQRDQR corresponding to amino acids 1 - 148 of BAC85622; which also corresponds to amino acids 91 - 238 of T59832_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of T59832_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence 1'~ITLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLV of T59832_P7.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832 P7, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFFTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVEACVLDELDMELAFLTIVCMEEFEDMERSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMI-iANAQRTDALQPPHEY'~rPWVTVNG corresponding to amino acids 1 - 212 of Q8WU77; which also corresponds to amino acids 1 - 212 of T59832 P7, and a~second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferablyy at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRIFLALSLTLIVPWSQGWTRQRDQR corresponding to amino acids 213 - 238 of T59832 P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of T59832_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRIFLALSLTLIVPWSQGWTRQRDQR in T59832_P7.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832 P9, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVEACVLDELDMELAFLTIVCMEEFEDMERSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMHANAQRTDALQPPHE corresponding to amino acids 12 - 214 of GILT HUMAN, which also corresponds to amino acids 1 - 203 of T59832 P9, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NPWkIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR corresponding to amino acids 204 - 244 of T59832_P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of T59832_P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NPWKIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR in T59832 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832_P9, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLIrLRGPLKKSNA
PLVNVTLY1'EALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNIiVEACVLDELDMELAFLTIVCMEEFEDMERSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMHANAQRTDALQPPHE corresponding to amino acids 1 - 203 of BAC98466; which also corresponds to amino acids 1 - 203 of T59832_P9, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NPWhIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR corresponding to i2o amino acids 204 - 244 of T59832 P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T59832 P9, comprising a polypeptide being at least 70%, optionally at least about SO%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NPWIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR in T59832_P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832_P9, comprising a first amino acid sequence being at least 70%, optionally at least SO%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYILTGNLYLRGPLItItSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLV corresponding to amino acids 1 - 90 of T59832_P9, second amino acid sequence being at least 90 % homologous to MEIL,NVTLVPYGNAQEQNVSGRWEFIkCQHGEEECKFNKVEACVLDELDMELAFLTIVC
MEEFEDMERSLPLCLQLYAPGLSPDTIMECAMGDRGMQLMHANAQRTDALQPPHE
corresponding to amino acids 1 - 113 of BAC85622, which also corresponds to amino acids 91 -203 of T59832_P9, 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 preferablyy at least 95%
homologous to a polypeptide having the sequence NPWKIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR corresponding to amino acids 204 - 244 of T59832_P9, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an -isolated polypeptide encoding for a head of T59832 P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVN5.5.'ILTGNLYLRGPLKILSNA
PLVNVTLf'YEALCGGCRAFLIRELFPTWLLV of T59832 P9.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T5983?_P9, comprising a polypeptide being at least 1~1 70%, optionally at least about SO%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NPWKIR.PSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR in T59832 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832_P9, comprising a first amino acid sequence being at least 90 °,~o homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLI'YEALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECIhFNKVEACVLDELDMELAFLTIVCMEEFEDMERSLPLCLQLYAPGLSPDTIM
ECAMGDRGMQLMHANAQRTDALQPPHE corresponding to amino acids 1 - 203 of Q8WL177, -which also corresponds to amino acids 1 - 203 of T59832_P9, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NPWKIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR corresponding to amino acids 204 - 244 of T59832_P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of T59832 P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NPWKIRPSSLPLSASCTRARSRMSALPQPAPSGVFASSDGR in T59832 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832 P12, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLVMEILNVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVE corresponding to amino acids 12 - 141 of GILT_HLTMAN, which also corresponds to amino acids 1 - 130 of T59832 P12, and a second amino acid sequence being at least 90 % homologous to CLQLYAPGLSPDTIIvIECAMGDRGMQLMHANAQRTDALQPPHEYVPWVTVNGhPLED
QTQLLTLVCQLYQGKKPDVCPSSTSSLRSVCFK corresponding to amino acids 173 - 261 of GILT HUMAN, which also corresponds to amino acids 131 - 219 of T59832_P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of T59832 P 12, 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 nvo amino acids comprise EC, having a structure as follows: a sequence starting from any of amino acid numbers 130-x to 130; and ending at any of amino acid numbers 131+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832 P12, 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 MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNS.'KTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGCRAFLIRELFPTWLLV corresponding to amino acids 1 - 90 of T59832 P12, second amino acid sequence being at least 90 % homologous to MEILNVTLVPYGNAQEQNVSGRWEFILCQHGEEEChFNhVE corresponding to amino acids 1 - 40 of BAC85622, which also corresponds to amino acids 91 - 130 of T59832 P12, third amino acid sequence being at least 90 % homologous to CLQLYAPGLSPDTIMECAMGDRGMQLMHANAQRTDALQPPHEYVPW~ITVNG
corresponding to amino acids 72 - 122 of BAC85622, which also corresponds to amino acids 131 - 181 of T59832 P12, and a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence KPLEDQTQLLTLVCQLYQGI~KPDVCPSSTSSLRSVCFIt corresponding to amino acids 182 - 219 of T59832_P12, wherein said first, second, third and fourth amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a head of T59832 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most pref~ai~ly at least about 95% homologous to the sequence MTLSPLLLFLPPLLLLLI~y '~IPTAAVQASPLQALDFFGNGPPVNYKTGNLYLRGPLKKSNA
PLVNVTLYYEALCGGGdLhFZ,IRELFPT'WLLV of T59832 P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for an edge portion of T59832_P12, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EC, having a structure as follows: a sequence starting from any of amino acid numbers 130-x to 130; and ending at any of amino acid numbers 131+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invvention, there is provided an isolated polypeptide encoding for a tail of T59832 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence KPLEDQTQLLTLVCQLYQGKKPDVCPSSTSSLRSVCFK in T59832_P12.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832 P12, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNI'KTGNLYLRGPLKKSNA
PLVNVTLYI'EALCGGCRAFLIRELFPTWLLVMEIL,NVTLVPYGNAQEQNVSGRWEFKC
QHGEEECKFNKVE corresponding to amino acids 1 - 130 of Q8WU77, which also corresponds to amino acids 1 - 130 of T59832_P12, and a second amino acid sequence being at least 90 % ho mologous to CLQLYAPGLSPDTIMECAMGDRGMQLMHANAQRTDALQPPHEYVPWVTVNGKPLED
QTQLLTLVCQLIrQGKKPDVCPSSTSSLRSVCFK corresponding to amino acids 162 - 250 of Q8WU77, _ .which also corresponds to amino acids 131 - 219 of T59832 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for an edge portion of T59832_P12, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EC, having a structure as follows: a sequence starting from any of amino acid numbers 130-x to 130; and ending at any of amino acid numbers 131+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832 P 18, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVNYK corresponding to amino acids 12 - 55 of GILT_HUMAN, which also corresponds to amino acids 1 - 44 of T59832_P18, and a second amino acid sequence being at least 90 % homologous to CLQLYAPGLSPDTIMECAMGDRGMQLIviHANAQRTDALQPPHEYVPWVTVNGhPLED
QTQLLTLVCQLYQGKKPDVCPSSTSSLR._SVCFK corresponding to amino acids 173 - 261 of GILT HUMAN, which also corresponds to amino acids 45 - 133 of T59832_P18, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for an edge portion of T59832_P18, 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 KC, having a structure as follows: a sequence starting from any of amino acid numbers 44-x to 44; and ending at any of amino acid numbers 45+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T59832_P1S, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVN-Yh corresponding to amino acids 1 - 44 of Q8WU77, _which also corresponds to amino acids 1 - 44 of T59832_P1S, and a second amino acid sequence being at least 90 % homologous to CLQL1'APGLSPDTIMECAMGDRGMQLMHANAQRTDALQPPHEYVPVWTVNGKPLED
QTQLL,TLVCQLYQGKKPDVCPSSTSSLRSVCFK corresponding to amino acids 162 - 250 of Q8WU77; which also corresponds to amino acids 45 - 133 of T59832 P18, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of T59832 P18, 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 KC, having a structure as follows: a sequence starting from any of amino acid numbers 44-x to 44; and ending at any of amino acid numbers 45+ ((rr2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T59832_P1S, comprising a first amino acid sequence being at least 90 % homologous to MTLSPLLLFLPPLLLLLDVPTAAVQASPLQALDFFGNGPPVN1'K corresponding to amino acids 1 - 44 of Q8NEI4, which also corresponds to amino acids 1 - 44 of T59832_P18, and a second amino acid sequence being at least 90 % homologous to CLQLYAPGLSPDTIMECAMGDRGMQLMHAN.AQRTDALQPPHEYVPWVTVNGKPLED
QTQLLTLVCQLYQGKKPDVCPSSTSSLRSVCFK corresponding to amino acids 162 - 250 of Q8NEI4; which also corresponds to amino acids 45 - 133 of T59832 P18, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of T59832_P18, 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 Sll amino acids in length, wherein at least t,uo amino acids comprise KC, having a structure as follows: a sequence starting from any of amino acid numbers 44-x to 44; and ending at any of amino acid numbers 45+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HUMGRPSE_P4, comprising a first amino acid sequence being at least 90 % homologous to 12b MRGSELPLVLLALVLCLAPRG)~.~~ ~IPLPAGGGTVLTKMYPRGNHWAVGHLMGKKSTG
ESSSVSERGSLKQQLREYIRWEE~~.,~RNLLGLIEAKENRNHQPPQPKALGNQQPSWDSED
SSNFKDVGSKGK corresponding tc~ amino acids 1 - 127 of GRP_, which also corresponds to amino acids 1 - 127 of HUMGRPSE_P4, and a second amino acid sequence being at least 90 % homologous to GSQREGRNPQLNQQ corresponding to amino acids 148 of GRP_HL~MAN, which also corresponds to amino acids 128 - 141 of HLTMGRPSE_P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of HUMGRPSE_P4, 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 KG, having a structure as follows: a sequence starting from any of amino acid numbers 127-x to 127; and ending at any of amino acid numbers 128 + ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for HLTMGRPSE_P5, comprising a first amino acid sequence being at least 90 % homologous to MRGSELPLVLLALVLCLAPRGRAVPLPAGGGTVLTKMYPRGNHWAVGHLMGKKSTG
ESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSED
SSNFKDVGSKGK corresponding to amino acids 1 - 127 of GRP_H-U1MAN, which also corresponds to amino acids 1 - 127 of HUMGRPSE_P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DSLLQVLNVKEGTPS -corresponding to amino acids 128 - 142 of HUMGRPSE P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of HUMGRPSE_P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DSLLQVLNVKEGTPS in HUMGRPSE P5.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for AA155578_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MRAPHLHLSAASGARALAKLLPLLMAQLWAAEAALLPQNDTRLDPEAYGAPCARGSQ
PWQVSLFNGLSFHCAGVLVDQSWVLTAAHCGNKPLWARVGDDHLLLLQGEQLRRTT
RSVVI~KYHQGSGPIL,PRRTDEHI)LMLLKLARP corresponding to amino acids 1 - 146 of IkLKA_HUIviAN, which also corresponds to amino acids 1 - 146 of AA
155578_PEA_1 P4, and a second amino acid sequence being at least 90 % homologous to YNKGLTCSSITILSPKECEVFYPGWTNN1~CAGLDRGQDPCQSDSGGPLVCDETLQGIL
SWGVYPCGSAQHPAVYTQICKYMSWINKVIRSN corresponding to amino acids 184 - 276 of KL~ HUMAN, which also correspond s to amino acids 147 - 239 of AA155578 PEA_1 P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for an edge portion of AA155578 PEA_1 P4, 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 tvvo amino acids comprise PY, having a structure as follows: a sequence starting from any of amino acid numbers 146-x to 146; and ending at any of amino acid numbers 147+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for AA155578 PEA_1 P6, comprising a first amino acid sequence being at least 90 % homologous to MR.APHLHLSAASGARALAKLLPLLI\~IAQLW corresponding to amino acids 1 - 29 of KLKA_HUMAN, which also corresponds to amino acids 1 - 29 of.4A155578_PEA_1 P6, and a second amino acid sequence being at least 90 % homologous to VKYNKGLTCSSITILSPKECEVFYPGVVTNNMICAGLDRGQDPCQSDSGGPLVCDETLQ
GILSWGVYPCGSAQHPAVYTQICKYMSWINKVIRSN corresponding to amino acids 182 -276 of KLKA_ITtJNIAN, which also corresponds to amino acids 30 - 124 of i2a AA155575 PEA_1 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for an edge portion of AA15557s PEA_1 P6, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise WV, having a structure as follows: a sequence starting from any of amino acid numbers 29-x to 29; and ending at any of amino acid numbers 30+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for AA155575 PEA_1 P8, comprising a first amino acid sequence being at least 90 % homologous to MRAPHLHLSAASGARALAKLLPLLMAQLW corresponding to amino acids 1 - 29 of KLI~A_HLJMAN, which also corresponds to amino acids 1 - 29 of AA155575 PEA_1 P8, and a second amino acid sequence being at least 70%, optionally at least b0%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GHCGLE corresponding to amino acids 30 - 35 of AA15557g PEA_1 P8, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of AA155575 PEA_1 P8, comprising a polypeptide being at least 70%, optionally at least about RO%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GHCGLE in AA155578 PEA_1 P8.
According to preferred embodiments of the present invention, there is provided an isolated chimeric -polypeptide encoding for AA155578_PEA_I P9, comprising a first amino acid sequence being at least 90 % homologous to MRAPHLHLSAASGARALAKLLPLLMAQLWAAEAALLPQNDTRLDPEAYGAPCARGSQ
PWQVSLFNGLSFHCAGVLVDQSWVLTAAHCGNIL corresponding to amino acids 1 - 90 of KLhA- -HUIvIAN, which also corresponds to amino acids 1 - 90 of AA155578_PEA_1 P9.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for HSENA7S P2, comprising a first amino acid sequence being at least 90 % homologous to MSLLSSRAARVPGPSSSLCALLVLLLLLTQPGPIASAGPAAAVLRELRCVCLQTTQGVHP
KMISNLQVFAIGPQCSKVEVV _corresponding to amino acids 1 - 81 of SZOS_HTJMAN, which also corresponds to amino acids 1 - 81 of HSENA78_P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptide encoding for T94936_PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MMLHSALGLCLLLVTVSSNLAIAIKKEKRPPQTLSRGWGDDITWVQTYEEGLFYAQKS
KKPLMVII-II-ILEDCQYSQALKKVFAQNEEIQEMAQNKFIMLNLMHETTDKNLSPDGQY
VPRIMFVDPSLTVRADIAGR1'SNRLYTYEPRDLPL corresponding to amino acids 1 - 150 of Q8TD06, which also corresponds to amino acids 1 - 150 of T94936_PEA_1 P2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _ _polypeptide encoding for T94936_PEA_1 P3, comprising a first amino acid sequence being at least 90 % homologous to MMLHSALGLCLLLVTVSSNLAIAIKKEKRPPQTLSRGWGDDITWVQTYEEGLFYAQKS
KKPLMVIHI-IL,EDCQYSQALKKVFAQNEEIQEMAQNKFIMLNLMHETTDKNLSPDGQYY
VPRIMFV corresponding to amino acids 1 - 122 of Q8TD06, which also corresponds to amino acids 1 - 122 of T94936_PEA_1 P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence GMYVISFHQIYKISRNQHSCFYF corresponding to amino acids 123 - 145 of T94936_PEA_1 P3, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of T94936_PEA_1 P3, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GMYVISFHQIYhISRNQHSCFYF in T94936_PEA_1 P3.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for 241644 PEA_1 P10, comprising a first amino acid sequence being at least 90 % homologous to MRLLAAALLLLLLALYTARVDGSKCKCSRKGPKIRYSDVKILLEMhPKYPHCEEKn~IVII
TTKSVSRYRGQEHCLHPKLQSTKRFIKWYNAWNEKRR corresponding to amino acids 1 -95 -of SZ14_HUMAN, which also corresponds to amino acids 1 - 95 of Z41644_PEA_1 P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence YAPPLLTFLPTRPSCGSQDGKGPPHQVI corresponding to amino acids 96 - 123 of Z41644_PEA_1 P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of 241644 PEA_1 P10, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence YAPPLLTFLPTRPSCGSQDGKGPPHQVI in 241644 PEA_1 P10.
According to preferred embodiments of the present invention, there is provided an isolated chimeric _polypeptidc encoding for 241644 PEA_1 P10, comprising a first amino acid sequence being at least 90 % homologous to MRLLAAALLLLLLALYTARVDGSKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVII
TTKSVSRYRGQEHCLHPKLQSTKRFIKWYNAWNEILRR corresponding to amino acids 13 -107 -of Q9NS21, which also corresponds to amino acids 1 - 95 of Z41644_PEA_1 P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence YAPPLLTFLPTRPSCGSQDGKGPPHQVI corresponding to amino acids 96 - 123 of 241644 PEA_1 P 10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _encoding for a tail of Z41644_PEA_1 P10, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% b~~!~nologous to the sequence YAPPLLTFLPTRPSCGSQDGhGPPHQVI _in 241644 T'F:.'~_1 P10.
According to preferred embodiments of the present: ijivention, there is provided an isolated chimeric -polypeptide encoding for Z41644_PEA_1 P10, comprising a first amino acid sequence being at least 90 % homologous to MRLLAAALLLLLLALYTARVDGSKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVII
TTKSVSRYRGQEHCLHPKLQSTKRFIKWYNAWNEKRR corresponding to amino acids 13 -107 of AAQ89265, which also corresponds to amino acids 1 - 95 of ~41644_PEA_1 P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence YAPPLLTFLPTRPSCGSQDGKGPPHQVI corresponding'to amino acids 96 - 123 of 241644 PEA_1 P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide _ _encoding for a tail of 241644 PEA_1 P10, compr7sing 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 YAPPLLTFLPTRPSCGSQDGKGPPHQVI in 241644_PEA_1 P10.
According to preferred embodiments of the present invention, there is provided an isolated 2G oligonucleotide, comprising an amplicon selected from the group consisting of SEQ ID NOs:
891 or 894.
According to preferred embodiments of the present invention, there is provided a primer pair, comprising a pair of isolated oligonucleotides capable of amplifying the above.
Optionally, the pair of isolated oligonucleotides is selected from the group consisting of: SEQ
NOs 889 and 890; or 892 and 893.
According to preferred embodiments of the present invention, there is provided an antibody capable of specifically binding to an epitope of an amino acid sequence as described herein. Optionally, the epitope may comprise a tail, head, or edge portion as described herein.
According to preferred embodiments of the present invention, the antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
According to preferred embodiments of the present invention, there is provided an kit for detecting breast cancer, comprising a kit detecting overexpression of a splice variant as described herein. Optionally, the kit comprises a NAT based technology.
Preferably, the kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein Optionally, the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence as described herein.
Optionally, the kit comprises an antibody as described herein. Preferably, the kit further comprises at least one reagent for performing an ELISA or a Western blot.
According to preferred embodiments of the present invention, there is provided a method for detecting breast cancer, comprising detecting overexpression of a splice variant as described herein.
Optionally detecting overexpression is performed with a NAT-based technology.
Preferably, detecting overexpression is performed with an immunoassay. More preferably, the immunoassay comprises an antibody as described herein.
According to preferred embodiments of the present invention, there is provided a biomarker capable of detecting breast cancer, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
According to preferred embodiments of the present invention, preferably any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology thereto.
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).
All nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate to their isolated form, as isolated polynucleotides (including for all transcripts), oligonucleotides (including for all segments, amplicons and primers), peptides (including for all tails, bridges, insertions or heads, optionally including other antibody epitopes as described herein) and/or polypeptides (including for all proteins). It should be noted that oligonucleotide and polynucleotide, or peptide and polypeptide, may optionally be used interchangeably.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. 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 (Wall:er ed., 1988);
The Glossary of Genetics, SthEd., 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. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
BRIEF DESCRIPTION OF DRAWINGS
Figure I is schematic summary of cancer biomarkers selection engine and the wet validation stages.
Figure 2. Schematic illustration, depicting grouping of transcripts of a give n cluster based on presence or absence of unique sequence regions.
Figure 3 is schematic summary of quantitative real-time PCR analysis.
Figure 4 is schematic presentation of the oligonucleotide based microarray fabrication.
Figure 5 is schematic summary of the oligonucleotide based microarray experimental flow.
Figure 6 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster T10888, demonstrating overexpression in colorectal cancer, a mixture of malignant tumors from different tissues, pancreas carcinoma and gastric carcinoma.
Figure 7 is a histogram showing expression of the CEA6 HLTNLAN
Carcinoembryonic .antigerrrelated cell adhesion molecule 6 (T10888) transcripts, which are detectable by amplicon as depicted in sequence name T10888 juncl l-17, in normal and cancerous breast tissues.
Figure 8 is a histogram showing the expression of CEA6 HUMAN Carcinoembryonic antigen-related cell adhesion molecule 6 T10888 transcripts which are detectable by amplicon as depicted in sequence name T10888juncl 1-17 in different normal tissues.
Figure 9 is a histogram showing Cancer and cell-line vs. awrmal tissue expression for Cluster T39971, demonstrating overexpression in liver cancer, lszng malignant tumors and pancreas carcinoma.
Figure 10 is a histogram showing the expression of of VTNC_HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein) T39971 transcripts, which are detectable by amplicon as depicted in sequence name T39971 junc23-33 in normal and cancerous breast tissues.
Figure 11 is a histogram showing the expression of VTNC_HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein), antisense to SARM1 (T23434), T39971 transcripts, which are detectable by amplicon as depicted in sequence name T39971 junc23-33, in different normal tissues.
Figure 12 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster 221368, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and pancreas carcinoma.
Figure 13 is a histogram showing the expression of SLTL 1 HLTI~IAN -Extracellular sulfatase Sulf 1221368 transcripts, which are detectable by amplicon as depicted in sequence name 221368seg39, in normal and cancerous breast tissues.
Figure 14 is a histogram showing the expression of SLTL 1 HUMAN -Extracellular sulfatase Sulf 1221368 transcripts, which are detectable by amplicon as depicted in sequence name 221368seg39, in different normal tissues.
Figure 15 is a histogram showing the expression of SLTL 1 HUMAN -Extracellular sulfatase Sulf 1 221368 transcripts which are detectable by amplicon as depicted in sequence name 221368junc17-21 in normal and cancerous breast tissues.
Figure 16 is a histogram showing the expression of SUL1 HUMAN - Extracellular sulfatase Sulf 1 221368 transcripts, which are detectable by amplicon as depicted in sequence name 221368junc17-21, in different normal tissues.
Figure 17 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster T59832, demonstrating overexpression in brain malignant tumors, breast malignant tumors, ovarian carcinoma and pancreas carcinoma.
Figure 18 is a histogram showing low over expression observed for cluster T59832, amplicon name: T59832 junc6-25-26, in one experiment earned out with breast cancer samples panel.
Figure 19 is a histogram showing the expression of Gl~_HLJMAN - gastrin-releasing peptide (HUMGRPSE) transcripts, which are detectable by amplicon, as depicted insequence name HUIvIGRPSEjune3-7 in normal and cancerous breast tissues.
Figure 20 is a histogram showing the expression of GI2P-HUMAN -gastrirrreleasing peptide (HCTMGRPSE) transcripts, which are detectable by amplicon, as depicted in sequence name HUMGRPSEjunc3-7, in different normal tissues.
Figure 21 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster AA155578, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and pancreas carcinoma.
Figure 22 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HSENA78, demonstrating overexpression in epithelial malignant tumors and lung malignant tumors.
Figure 23 is a histogram showing the expression ofHomo sapiens breast cancer membrane protein 11 (BCMP11) T94936 transcripts which are detectable by amplicon as depicted in sequence name T94936 segl4 in normal and cancerous Breast tissues.
Figure 24 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster 241644, demonstrating overexpression in lung malignant tumors, breast malignant tumors and pancreas carcinoma.
Figure 25 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster M85491, demonstrating overexpression in epithelial malignant tumors and a mixture of malignant tumors from different tissues.
Figure 26 is a histogram showing the expression ofEphrin type-B receptor 2 precursor (EC 2.7.1.112) (Tyrosine-protein kinase receptor EPH-3) M85491 transcripts which are detectable by amplicon as depicted in sequence name M85491 seg24 in normal and cancerous breast tissues.
Figure 27 is a histogram showing the expression of Ephrin type-B receptor 2 precursor M85491 transcripts, which are detectable by amplicon as depicted in sequence name IvIS5491 seg24, in different normal tissues.
Figure 28 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HSSTROL3, demonstrating overexpression in transitional cell carcinoma, epithelial malignant tumors, a mixture of malignant tumors from different tissues and pancreas carcinoma.
Figure 29A is a histogram showing the expression of Expression of Stromelysin-precursor (EC 3.4.24.-) (lMatrix metalloproteinase-11) (MIvvIP-11) (ST3) SL-3 transcripts which are detectable by amplicon as depicted in sequence name HSSTROL3 seg24 in normal and cancerous breast tissues.
Figure 29B is a histogram showing the expression of Stromelysin-3 precursor (EC
3.4.24.-) (Matrix metalloproteinase-11) (N>IVIy-11) (ST3) (SLr3) HSSTROL3 transcripts, which are detectable by amplicon as depicted in sequence name HSSTROL3 seg24, in different normal tissues.
Figures 30A 30C shows histograms showing over expression of various Stromelysin-3 precursor transcripts in cancerous breast samples relative to the normal samples.
Figure 31 is a histogram showing Cancer and cell-line vs. normal tissue expression far Cluster 875793, demonstrating overexpression in epithelial malignant tumors and a mixture of malignant tumors from different tissues.
Figure 32 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HLJMCA1XIA, demonstrating overexpression in bone malignant tumors, epithelial malignant tumors, a mixture of malignant tumors from different tissues and lung malignant tumors.
Figure 33 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster 820779, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and lung malignant tumors.
Figure 34 is a histogram shoal ng Cancer and cell-line vs. normal tissue expression for Cluster HSS 100PCB, demonstrating overexpression in a mixture of malignant tumors from different tissues.
Figure 35 is a histogram showing Cancer and cell-line vs. nornial tissue expression for Cluster HSCOC4, demonstrating overexpression in brain malignant tumors, a mixture of malignant tumors from different tissues, breast malignant tumors, pancreas carcinoma and prostate cancer.
Figure 36 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HUMTREFAC, demonstrating overexpression in a mixture of malignant 'tumors from different tissues, breast malignant tumors, pancreas carcinoma and prostate cancer.
Figure 37 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HUMOSTRO, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues, lung malignant tumors, breast malignant tumors, ovarian carcinoma and skin malignancies.
Figure 38 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster 811723, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and kidney malignant tumors.
Figure 39 is a histogram showing the expression of of 811723 transcripts which are detectable by amplicon as depicted in sequence name 811723 segl3 in normal and cancerous breast tissues.
Figure 40 is a histogram showing the expression of 811723 transcripts, which are detectable by amplicon as depicted in sequence name RI 1723seg13, in different normal tissues.
Figures 41A and B are histograms showing the expression of 811723 transcripts, which are detectable by amplicon as depicted in sequence name 811723 juncl l-18 in normal and cancerous breast tissues (Figure 41A) or on a panel of normal tissues (Figure 41B).
Figure 42 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster T46984, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues, breast malignant tumors, ovarian carcinoma and pancreas carcinoma.
Figure 43 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HSMUC1A, demonstrating overexpression in a mixture of malignant tumors from different tissues, breast malignant tumors, pancreas carcinoma and prostate cancer.
Figures 44-47 are histograms showing the combined expression of 8 sequences (T10888seg11-17, HUMGRSE junc3-7, HSSTROL3seg24, T94936 Seg 14, 221368 seg39, 221368 juncl7-21 T59832 jun6-25-26 and M85491seg24) in normal and cancerous breast tissues.
Figure 48 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HSU33147, demonstrating overexpression in a mixture of malignant tumors from different tissues.
DESCRIPTION OF PREFERRED EMBODIIVVIENTS
The present invention is of novel markers for breast cancer that are both sensitive and accurate. Furthermore, at least certain of these markers are able to distinguish between different stages of breast cancer, such as 1. Ductal carcinoma (in-situ, invasive) 2.
Lobular carcinoma (is-situ, invasive) 3. inflammatory breast cancer 4. Mutinous carcinoma 5. Tubular carcinoma 6.
Paget's disease of nipple, alone or in combination; or one of the indicative conditions described above.
The markers of the present invention, alone or in combination, can be used for prognosis, prediction, screening, early diagnosis, staging, therapy selection and treatment b monitoring of breast cancer. For example, optionally and preferably, these markers may be used for staging breast 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 then breast. Also, one or more of the markers may optionally be used in combination with one or more other breast cancer markers (other than those described herein).
Biomolecular sequences (amino acid and/or nucleic acid sequences) uncovered using the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease.
These markers are specifically released to the bloodstream under conditions of breast cancer (or one of the above indicative conditions), and/or are otherwise expressed at a much higher level and/or specifically expressed in breast cancer tissue or cells, and/or tissue or cells under one of the above indicative conditions. The measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of breast cancer and/or a condition that it is indicative of a higher risk for breast cancer.
The present invention therefore also relates to diagnostic assays for breast cancer and/or an indicative condition, and methods of use of such markers for detection of breast cancer and/or an indicative condition, optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
According to a preferred embodiment of the present invention, use of the marker optionally and preferably permits a non-cancerous breast disease state to be distinguished from breast cancer and/or an indicative condition. A non limiting example of a non-cancerous breast disease state includes breast fibrosis and/or cysts. According to another preferred embodiment of the present invention, use of the marker optionally and preferably permits an indicative condition to be distinguished from breast cancer.
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.
As used herein a "tail" refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention.
Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (o$en 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.
As used herein a "head" refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein.
As used herein "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 "knov~m 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 "knotvn protein" portion of a variant.
Optionally and preferably, a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant, comprises at least about 10 amino acids, more preferably at least about 20 amino acids, most preferably at least about 30 amino acids, and even more preferably at least about 40 amino acids, in which at least one amino acid is from tl~
tail/head/insertion and at least one amino acid is from the "known protein"
portion of a variant.
Also optionally, the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 1 l, 12, 13...37, 33, 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_Pl (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at East 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 l, nor 50 +
((n-2) - x) (for example) greater than the total sequence length.
In another embodiment, this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention.
Preferably such antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
In another embodiment, this invention provvides an isolated nucleic acid molecule ewcoding for a splice variant according to the present invention, having a nucleotide sequence as 5e~ forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention. In another embodiment, this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.
In another embodiment, this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising:
contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variants) 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 splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.
According to the present invention, the splice variants described herein are norrlimiting examples of markers for diagnosing breast cancer and/or an indicative condition. Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of breast cancer and/or an indicative condition, including a transition from an indicative condition to breast cancer.
According to optional but preferred embodiments of the present invention, any marker according to the present invention may optionally be used alone or combination. Such a comb ination .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, andlor any other mailer. With regard to such a ratio between any marker described herein (or a combination thereof) and a knoGVn marker, more preferably the known marker comprises the "known protein'' as described in greater detail below with regard to each cluster or gene.
According to other preferred 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 breast cancer and/or an indicative condition, such that a biomarker may optionally comprise any of the above.
According to still other preferred 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 fmited 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 thereW th, sequences homologous thereto [e.g., at least 50 %, at least 5~ %, 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 nuc leic 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 ma n 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 irz vivo or in vitro using a DNA dependent DNA
polymerase.
.'~.s used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived d~i~~olated) from a chromosome and thus it represents a contiguous portion of a chromosc~t~ue.
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.
Preferred embodiments of the present invention encompass oligonucleotide probes.
An example of an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
Alternatively, an oligonucleotide probe of the present invention can he 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. ItQ., ed.
(1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland ( 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 preferred 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.
Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other ali~yl phosphonates including 3'-allylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoallylphosphoramidates, 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, mia:ed heteroatom and alkyl or cycloallyl 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 CHI component parts, as disclosed in LT.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 (L~.
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 allyl 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, S-trifluoromethyl and other 5-substituted uracils and cytosines, '~methylguanine and 7 methyladenine, lrazaguanine and &
azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Further bases particularly useful for increasing the binding amity of the oligomeric compounds of the invention include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including ? aminopropyladenine, ~propynyluracil and ~-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, cho1ie acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rao-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: ti,303,374.
It is not necessary for all positions in a given oligonucleotide mo lecule 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.
Preferably, 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., (19891 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 (LT.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 preferably further includes an appropriate selectable marker and/or an origin of replication. Preferably, 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.
Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/ ), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com). Examples of retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif., includingRetro-X
vectors pLNC~ and pLXSN, which permit cloning into multiple cloning sites and the trasgene is transcribed from CMV promoter. Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. 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]]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, A:.~-4:YJ, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes a.t least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present n the viral construct. In addition, such a construct typically includes 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 construe is will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA
synthesis, and a 3' LTR
or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
Hybridization assays Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non-Limiting examples of probes according to the present invention were previously described).
Traditional hybridization assays include PCR, R~1'-PCR, Real-time YC;K, KNase 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 whic h 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 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides 30 long.
lso 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 cond itions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 3ZP
labeled probe, at 65 °C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65°C and whereas moderate hybridization is effected using a hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 3'P
labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.
More generally, hybridization of short nucleic acids (below 200 by in length, e.g. 17-40 by 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 x SSC and 1 SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.S), 1 mM EDTA (pH 7.61, 0.5 %
SDS, 100 pg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 °C below the Tnv final wash solution of 3 M TIvIACI, 0.01 M
sodium phosphate (pH
6.S), 1 mM EDTA (pH 7.6), 0.5 % SDS at I - 1.5 °C below the Tm; (ii) hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.S), 1 mM
EDTA
(pH 7.6), 0.5 °~o SDS, 100 p.g/ml denatured salmon spern~ DNA and 0.1 %
nonfat dried milk, hybridization temperature of 2 - 2.5 °C below the Tm, final wash solution of 3 IvI TMACI, 0.01 M sodium phosphate (pH 6.S), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm, final wash solution of 6 x SSC, and final wash at 22 °C; (iii) hybridization solution of 6 x SSC
and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.S), 1 mNI EDTA (pH
7.6), 0.5 SDS, 100 p.g/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 he 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, CyS, Cy5.5, Cy7, FluorX
(Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, CalifJ 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 hyb ridization 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, ~~C, 32P, and 35S.
3U 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 vau:,~ty 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 earned 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
(Kwon et al.; 1989, Proc. NatI. 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. Uther 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 preferred 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 preferred embodiment, the amplification of the differentially expressed nucleic acids is earned 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 Biologyy, 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 u~ 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 lresent 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 Claain Reaction (PCR): The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,653,195 and 4,683,202 to Mullis and IVlullis 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 respectivve strands of the double-stranded target sequence to the DNA mixture containing the desired tart 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 Chairs 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, tvvo~ 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. W09001069 A 1 ( 1990).
However, because the four oligonuc leotides used in this assay can pair to form two short ligatable iss fragments, there is the potential for the generation of target independent background signal. The use of LCR for mutant screening is limited to the exanunation of specific nucleic acid positions.
Self-Sustained Synthetic Reaction (3SRlNASBA): The self sustained sequence replication reaction (3SR) is a transcriptionbased 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 (Q(3) Replicase: In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Q~i 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 Q~3 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 nonspecific hybridization of th° 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+~'~n =y, where "~" 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 polymerise 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 e~ciency to 85 °io, 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 SS % 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 polymerise chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate fo r 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 DN.A
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 misma tches 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 preferred 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, the re 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).
C3~cling 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.
Br-anelzed 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 nonspecific binding is similarly increased.
The detection of at least one sequence change according to various preferred 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), Suigle-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 wwithin 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 comb inations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. The presence of specific sequences witlun 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.
Restr-ictiora fragrnent 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
manipulatior~.
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 oligonaceleotide .(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 metlbd is based on the differences in the melting temperature of short DNA fragn,e:raia differing by a single nucleotide. Stringent hybridization and washing conditions can differeartiate bet<veen mutant and wild-type alleles. The ASO approach applied to PCR products also ha . 'been extensively utilized by various researchers to detect and characterize point mutations ui 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.
Derzatur~irtglTenrperattsre Gradient Gel Electr-oplror-esis (DGGElTGGE): 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 n 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-&0) 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 Confortnatiott Polyntotphism (SSCP): Another common method, called "Single-Strand Conformation Polymorphism" (SSCP) was developed by Hayashi, Sel<ya 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 W I1 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 infra-molecular interactions can form and not be disturbed during tl~
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 fingetpr~intirtg (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 presently preferred 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, Q~3-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 occurnng 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) andlor .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 wwell 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 BIastP
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. Finally, the present invention also encompasses fragments of the abovve 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.
Homology/identity of nucleic acid sequences is preferably determined by using BIastN 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.
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. Such modifications include, but are not limited to N
terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CPL?-S=O, O=C-NH, CPL?-O, CH2-CH?, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are wwell known in the art and are specified. Further Mails in this respect are provided hereinunder.
Peptide bonds ECO-NH-) within the peptide may be substituted, for example, by N-methylated bonds ~N(CH3)-CO-), ester bonds ~C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (-CO-CH2-), oc-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioarnide bonds (-CS-NH-), olefinic double bonds ( CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives ( N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom.
These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic norr natural acid such as Phenylglycine, TIC., naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
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 occurnng amino acids; those amino acids often modifted .post-translationally irr 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.
Table I non-cc~rrventiorral or modified arnino acids which can be used with the present irrvention.
Table 1 Non-conventional Code Non-conventional Code amino amino acid acid a,-aminobutyric Abu L-N-methylalanine Nmala acid a-amino-a-methylbutyr.3teMgabu L-N-methylarginine Nmarg aminocyclopropane-Cpro IrN-methylasparagineNmasn Carboxylate L-N-methylaspartic Nmasp acid aminoisobutyric Aib LrN-methylcysteine Nmcys acid aminonorbornyl- Norb LrN-methylglutamineNmgin Carboxylate IrN-methylglutamic Nmglu acid Cyclohexylalanine Chexa L~-N-methylhistidineNmhis CyclopentylalanineCpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg IrN-methyllysine Nmlys D-aspartic acid Dasp LrN-methylmethionineNmmet D-cysteine Dcys L,-N-methylnorleucineNmnle D-glutamine Dgln L-N-methylnorvalineNmnva D-glutamic acid Dglu 1rN-methylornithineNmorn D-histidine Dhis LrN-methylphenylalanineNmphe D-isoleucine Dile IrN-methylproline Nmpro D-leucine Dleu IrN-methylserine Nmser D-lysine Dlys L-N-methylthreonineNmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn IrN-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycineNmetg D-serine Dser IrN-methyl-t-butylglycineNmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp Irnorvaline Nva D-tyrosine Dtyr a-methyl-aminoisobutyrateMaib D-valine Dval a-methyl-y-aminobutyrateMgabu D-a-methylalanine Dmala a-methylcyclohexylalanineMchexa D-a-methylarginineDmarg a-methylcyclopentylalanineMcpen D-a-methylasparagineDmasn a-methyl-a-napthylalanineManap D-a-methylaspartateDmasp a- methylpenicillamineMpen D-~:-methylcysteineDmcys N-(4-aminobutyl)glycineNglu , D-~~c.-methylglutamineDmgln N-(2-aminoethyl)glycineNaeg D-c~ -methylhistidineDmhis N-(3-aminopropyl)glycineNorn D-a-.methylisoleucineDmile N- amino-a-methylbutyrateNmaabu D-a-methylleucineDmleu a-napthylalanine Anap D-a-methyllysine Dmlys N-benzylglycine Nphe D-a-methylmethionineDmmet N-(2-carbamylethyl)glycineNgln D-a-methylornithineDmorn N-(carbamylmethyl)glycineNasn D-a-methylphenylalanineDmphe N-(2-carboxyethyl)glycineNglu D-a-methylprolineDmpro N-(carboxymethyl)glycineNasp .
D-a-methylserine Dmser N-cyclobutylglycineNcbut D-a-methylthreonineDmthr N-cycloheptylglycineNchep D-a-methyltryptophanDmtrp N-cyclohexylglycineNchex D-a-methyltyrosineDmty N-cyclodecylglycineNcdec D-a-methylvaline Dmval N-cyclododeclglycineNcdod D-a-methylalnine Dnmala N-cyclooctylglycineNcoct D-a-methylarginineDnmarg N-cyclopropylglycineNcpro D-a-methylasparagineDnmasn N-cycloundecylglycineNcund D-a-methylasparatateDnmasp N-(2,2-diphenylethyl)glycineNbhm ~ D-a-methylcysteineDnmcys N-(3,3- Nbhe diphenylpropyl)glycine D-N-methylleucineDnmleu N-(3-indolylyethyl)Nhtrp glycine D-N-methyllysine Dnmlys N-methyl-'y-aminobutyrateNmgabu N- Nmchexa D-N-methylmethionineDnmmet methylcyclohexylalanine D-N-methylornithineDnmorn N-methylcyclopentylalanineNmcpen N-methylglycine Nala D-N-methylphenyylalanineDnmphe N-methylaminoisobutyrateNmaib D-N-methylproline Dnmpro N-(1-methylpropyl)glycineNile D-N-methylserine Dnmser N-(2-methylpropyl)glycineNile D-N-methylserine Dnmser N-(2-methylpropyl)glycineNleu D-N-methylthreonineDnmthr I
D-N-methyltryptophanDnmtrp N-(1-methylethyl)glycineNva D-N-methyltyrosineDnmtyr N-methyla-napthylalanineNmanap D-N-methylvaline Dnmval N-methylpenicillamineNmpen y-aminobutyric Gabu N-(p-hydroxyphenyl)glycineNhtyr acid Lrt-butylglycine Tbug N-(thiomethyl)glycineNcys L-ethylglycine Etg penicillamine Pen L-homophenylalanineHphe L-a-methylalanine Mala Lr-a-methylarginineMarg L-a-methylasparagineMasn L-a-methylaspartateMasp L-a-methyl-t-butylglycineMtbug L-a-methylcysteineMcys L-methylethylglycineMetg L-a-methylglutamineMgln lra-methylglutamateMglu L-a-methylhisti:dineMhis Ira-methylhomo Mhphe phenylalanine L-a-methylisoleucineMile N-(~-methylthioethyl)glycineNmet D-N-methylglutamineDnmgln N-(3- Narg guanidinopropyl)glycine D-N-methylglutamateDnmglu N-(1-hydroxyethyl)glycineNthr D-N-methylhistidineDnmhis N-(hydroxyethyl)glycineNser D-N-methylisoleucineDnmile N-(imidazolylethyl)glycineNhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycineNhtrp D-N-methyllysine Dnmlys N-methyl-y-aminobutyrateNmgabu N- Nmchexa D-N-methylmethionineDnmmet methylcyclohexylalanine D-N-methylornithineDnmorn N-methylcyclopentylalanineNmcpen N-methylglycine Nala D-N-methylphenylalanineDnmphe N-methylaminoisobutyrateNmaib D-N-methylproline Dnmpro N-( 1-methylpropyl)glycineNile D-N-methylserine Dnmser N-(2-methylpropyl)glycineNleu D-N-methylthreonineDnmthr D-N-methyltryptophanDnmtrp N-(1-methylethyl)glycineNval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanineNmanap D-N-methylvaline Dnmval N-methylpenicillamineNmpen y-aminobutyric acidGabu N-(p-hydroxyphenyl)glycineNhtyr butylglycine Tbug N-(thiomethyl)glycineNcys Irt- .
Lrethylglycine Etg penicillamine Pen IrhomophenylalanineHphe Lra-methylalanine Mala Ira-methylarginine Marg L-a-methylasparagineIVl;asn L,-a-methylaspartateMasp Lra-methyl-t-butylglycineMtbug Lra-methylcysteine Mcys L-methylethylglycineMetg Lr-a-methylglutamineMgln Ira-methylglutamateMglu L-a-me.thylhistidineNlhis L-a- Mhphe methylhomophenylalanine L-a-methylisoleucineMile N-(2-methylthioethyl)glycineNmet L-a-methylleucine Mleu L-a-methyllysine Mlys L-a-methylmethionineMmet L-a-methylnorleucineMule L-a-methylnorvalineMnva Lra-methylornithineMorn Lra-methylphenylalanineMphe L-a-methylproline Mpro L-a-methylserine mser L-a-methylthreonineMthr L-a-methylvaline Mtrp L-a-methyltyrosine Mtyr L-a-methylleucine Mval L-N- Nmhphe Nnbhm methylhomophenylalanine (2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl) N-(N- .
carbamylmethyl-glycineNnbhm carbamylmethyl(1)glycineNnbhe 1-carboxy-1-(2,2-diphenylNmbc ethylamino)cyclopropane Table 1 Cont.
Sine; lk~e peptides of the present invention are preferably utilized in diagnostics which require the lrehvtides to be in soluble form, the peptides of the present invention preferably include one o~~ more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere v~rith 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 relativelyy 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 far 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 inununoglobulin 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, CHI, 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, I X88, incorporated herein by reference).
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 SS 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 pepsin 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 mbar et al. [Proc. Naf1 Acad. Sci. USA 69:2659-62 ( 19720].
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.
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 uiterest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
Humanized forms of non-human (e.g., mur-ine) 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) wwhich contain minimal sequence derived from norrhuman immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in v~hich all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323 329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These norrhuman amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S.
Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR
residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage .display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in uwhich the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. .Pat. Nos. 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).
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 s~f the present invention, an immunoassay can be used to qualitatively or quantitatively dei:~ca and analyze markers in a sample. This method comprises:
providing an antibody that specifi,;ahy 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 imrnunosorbent 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. LTsually 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 iced 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 incubarion conditions described above. The amount of an antibod~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.
Preferably used are antibodies which specifically interact with the polypeptides of the present invention and not with wild type proteins or other isoforms thereof, for example. Such antibodies are directed, for example, to the unique sequence portions of the polypeptide variants of the present invention, including but not limited to bridges, heads, tails and insertions described in greater detail below. Preferred embodiments of antibodies according to the present invention are described in greater detail with regard to the section entitled "Antibodies".
Radio-irnnzz~noassay (RIA): In one version, this method invvolves 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 11"5) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precip itated 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 unknov~m amount of substrate is added in varying amounts. ')he decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
Ey~nze lir7kecl irn~fzmaosorhertt assay (ELIS4): 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 enzyyme 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.
l~restern 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).
S 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.
InunmZahistaclaernical analysis: This method involves detection of a substrate ira 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 (FAGS): This method involves detection of a substrate ira 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 (SPELT). 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, SPELT
can optionally be used with two labels simultaneously. SPELT has some other advantages as well, for example with regard to cost and the types of labels that can be used. For example, LTS
Patent No.
6,696,686 describes the use of SPELT for detection of breast cancer, and is hereby incorporated by reference as if fully set forth herein.
i7s Display Libraries According to still another aspect of the present invention there is provided a display library comprising a plurality of display ~;hicles (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 AC, 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 LB et al. "Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high amities" Biochemistry 1995 Nov 28;34(47):15430-5; Davies EL 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 RT al. "Current trends in molecular recognition and bioseparation" J
Chromatogr A 1995 Jul 14;707(1):3-22; Deng SJ et al. "Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries"
Proc Natl Acad Sci U S A 1995 I\~Iay 23;92(11):4992-6; and Deng SJ 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.
The following sections relate to Candidate Marker Examp les (first section) and to Experimental Data for these Marker Examples (second section).
CANDIDATE MARKER E~-1MPLES SECTION
This Section relates to Examples of sequences according to the present invention, including illustrative methods of selection thereof.
Description of the methodology undertaken to uncover the biomolecular sequences of the present invention Human ESTs and cDNAs were obtained from GenBanl: versions 136 (June 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 Oct 2003); Ref~eq sequences from December 2003; and from LifeSeq library of Incyte Corp (Wilmington, DE, USA; ESTs only). With regard to GenBank sequences, the human EST sequences from the EST (GBEST) section and the human mRNA sequences from the primate (GBPRI) section were used; also the human nucleotide RefSeq mRNA sequences were used (see for example www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html and for a reference to the EST section, see wunv.nebi.nlm.nih.gov/dbEST/; a general reference to dbEST, the EST
database in GenBank, may be found in Boguski et al, Nat Genet. 1993 Aug;4(4):332-3; all of which are hereby incorporated by reference as if fully set forth herein).
Novel splice variants were predicted using the LEADS clustering and assembly system as described in Sorek, R., Ast, G. & Graur, D. Alu-containing exons are alternatively spliced.
Genome Res 12, 1060-7 (2002); US patent No: 6,625,545; and U.S. Pat. Appl. No.
10/426,002, published as L1S20040101876 on May 27 2004; all of which are hereby incorporated by reference as if fully set forth herein. Briet7y, 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 (particularlyy of spliced sequences), chromosomal information, alignments, and additional information such as SNPs, .gene or~ology 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 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 Figure 1.
Identification of differentially expressed gene pr-odatcts - 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 over expressed in cancer is described hereinbelow.
Dry analysis Library annotation - EST libraries are manually classified according to:
(i) Tissue origin (ii) Biological source - Examples of frequently used biological sources for construction of EST libraries include cancer cell-lines; normal tissues;
cancer tissues; fetal tissues; and others such as normal cell lines and pools of normal cell-lines, cancer cell-lines and combinations thereof. A
specific description of abbreviations used below with regard to these tissues/cell lines etc is given above.
?0 (iii) Protocol of library construction - various methods are known in the art for library construction including normalized library construction;
non-normalized library construction; subtracted libraries; ORESTES and others. It will be appreciated that at times the protocol of library construction is not indicated.
The following rules were followed:
EST libraries originating from identical biological samples are 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 deviation., ~.bove the average for all libraries being analyzed, this library was tagged as being contasn~~nated and was eliminated from further consideration in the below analysis (see also Sorek, h.. & 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.
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 our belief 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 cumber 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 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 Rvalues were computed both for library and weighted clone counts to check that the counts are statistically significant.
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 ? 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 (S 1 ).
(ii) Supporting transcripts of group TB (S~).
(iii) Supporting transcripts from both groups (S3).
Library and clones number scores described above were given to S 1 group.
Fisher Exact Test P-values were used to check if S 1 is significantly enriched by cancer EST clones compared to S2; and S 1 is significantly enriched by cancer EST clones compared to cluster background (S 1+S2+S3).
Identification of unique sequence regions and division of the group of transcripts accordingly is illustrated in Figure 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 preferably not considered for determining differential expression between variants; Region 2: speciEc to Transcript 1;
Region 3: specific to Transcripts 2+3; Region 4: specific to Transcript 3; Region 5: specific to Transcripts 1 and 2;
Region 6: specific to Transcript 1.
E?~AMPLE 5 Identification of cancer specific splice variants of genes over expressed in cancer A search for EST supported (no mRNA) regions for genes of:
(i) known cancer markers (ii) Genes shown to be over-expressed in cancer in published micro-array experiments.
Reliable EST supported-regions were defined as supported by minimum of one of the following:
(i) 3 spliced ESTs; or (ii) 2 spliced ESTs from 2 libraries;
(iii) 10 unspliced ESTs from 2 libraries, or (iv) 3libraries.
Actual Marker Examples The following examples relate to specific actual marker examples.
EXPERIMENTAL EXAMPLES SECTION
This Section relates to Examples describing experiments involving these sequences, and illustrative, non-limiting examples of methods, assays and uses thereof. The materials and experimental procedures are explained first, as all experiments used them as a basis for the work that was performed.
The markers of the present invention were tested with regard to their expression in various cancerous and non-cancerous tissue samples. A description of the samples used in the panel is provided in Table 1 below. A description of the samples used in the normal tissue panel is provided in Table 2 below. Tests were then performed as described in the "Materials and Experimental Procedures" section below.
Table 1: Tissue samples in testing panel sample sex/
rename Lot no sourcepathologygrade age TNM stage Invasive1 Lobular 52-B-ILG A605360 BiochainCarcinoma F/60 51-B-IDC A605361 BiochainIDC 1 F/79 T2NOM stage NOM
7-A-IDC 7263T ABS IDG 2 F/43 0 stage T2NOM stage 13-A-IDC IA0133T IABS IIDC I 2 IF/63 IT2N1a ~ f~~lX
_ _.
~ -~
14-A-IDC A0135T ABS DC 2 F/37 ?N2M
G2 I ~ h 'r3N1Mstage 16-A-IDC 49040200327ABS DC 2 NA x 17-A-IDC 49040200367ABS DC 2-3 NA x 43-B-IDC A609183 BiochainDC 2 F140 44-B-IDG A609198 BiochainDG 2 F/77 45-B-IDC A609181 BiochainDC 2 F/58 48-B-IDG A609222 BiochainDG 2 F/44 49-B-IDC A609223 BiochainIDC 2 F/54 50-B-IDG A609224 BiochainIDG 2 F/69 53-B-IDC A605151 BiochainIDC 2 F/44 54-B-IDC A605353 BiochainIDC 2 F/41 55-B-IDC A609179 BiochainIDG 2 F/42 61-B-IDC A610029 BiochainIDC 2 F/46 62-B-IDC A609194 BiochainIDG 2 F/51 47-B-IDG A609221 BiochainIDC 2 46-B-CarciA609177 BiochainCarcinoma2 F/48 T2NOMstage 27-A-IDC 49070200727ABS IDC 3 NA x T1cN0 42-A-IDG 60050200317ABS IDC 3 NA Mx 31-GG-IDG CG-154 IchilovIDC NA
32-A-Muc 171167 I ABS MucinousI I F/54 IT2NOM(stage I
Carci carcinoma 0 2A
Normal matched 35-A-N 7238N ABS o 6T F/60 M6 t Normal matched 36-A-N 7263N ABS to 7T F/43 Normal matched 39-A-N 7259N ABS to 15T F159 Normal matched 40-A-N 1432N ABS to 12T F/46 Normal matched 41-A-N 7249N ABS to 26T F/60 Normal 56-B-N A609235 BiochainPM F/59 Normal 57-B-N A609233 BiochainPM F/34 Normal 58-B-N A609232 BiochainPM F/65 Normal 59-B-N A607155 BiochainPM F/35 Normal 60-B-N A609234 BiochainPM FI36 63-Am-N 26486 AmbionNormal F/43 PS
Normal 64-Am-N 23036 AmbionPM F/57 65-Am-N 131410 IAmbionNormal I I F/63 ( zaa PM
Normal 66-Am-N 36678 AmbionPM F/45 073P0106020 Normal 67-Am-N 6A AmbionPM F/64 Table 2: Tissue samples in normal panel:
Lot no. ource issue Pathology Sex/Age 1-Am-Colon (C71 071P10B mbion Colon PM F/43 ) 2-B-Colon (C69) A411078 BiochainColon PM-Pool M&F
of 10 3-CI-Colon (C70)1110101 ClontechColon PM-Pool M&F
of 3 4-Am-Small Intestine091P0201mbion Small IntestinePM M/75 5-B-Small IntestineA501158 BiochainSmall IntestinePM M/63 6-B-Rectum A605138 BiochainRectum PM M/25 7-B-Rectum A610297 BiochainRectum PM M/24 8-B-Rectum A610298 BiochainRectum PM M/27 9-Am-Stomach 110P04A mbion Stomach PM M/16 10-B-Stomach A501159 BiochainStomach PM M/24 11-B-Esophagus A603814 BiochainEsophagus PM M/26 12-B-Esophagus A603813 BiochainEsophagus PM M141 13-Am-Pancreas 071P25C mbion Pancreas PM M/25 14-CG-Pancreas CG-255-2IchilovPancreas PM M/75 15-B-Lung A409363 BiochainLung PM F126 16-Am-Lung (L93)111P0103mbion Lung PM F161 17-B-Lung (L92) A503204 BiochainLung PM M/28 18-Am-Ovary (047)061P43A mbion Ovary PM F/16 19-B-Ovary (048)A504087 BiochainOvary PM F/51 20-B-Ovary (046)A504086 BiochainOvary PM F/41 21-Am-Cervix IOlPOlOlAmbion Cervix PM F/40 22-B-Cervix A408211 BiochainCervix PM i =~''~~fi 23-B-Cervix A504089 BiochainCervix PM-Pool Ni~i of 5 24-B-Uterus A411074 BiochainUterus PM-Pool M~~xF
of 10 25-B-Uterus A409248 BiochainUterus PM F/43 26-B-Uterus A504090 BiochainUterus PM-Pool M&F
of 5 27-B-Bladder A501157 BiochainBladder PM M/29 28-Am-Bladder 071P02C mbion Bladder PM M/20 29-B-Bladder A504088 BiochainBladder PM-Pool M&F
of 5 30-Am-Placenta 021P33A mbion Placenta PB F/33 31-B-Placenta A410165 BiochainPlacenta PB F/26 32-B-Placenta A411073 BiochainPlacenta PB-Pool M&F
of 5 33-B-Breast (B59)A607155 BiochainBreast PM F/36 34-Am-Breast 26486 mbion Breast PM F/43 (B63) 35-Am-Breast 23036 mbion Breast PM F/57 (B64) 36-CI-Prostate 1070317 ClontechProstate PB-Pool M&F
(P53) of 47 37-Am-Prostate 061P04A mbion Prostate PM M/47 (P42) 38-Am-Prostate 25955 mbion Prostate PM M/62 (P59) 39-Am-Testis 111P0104Ambion estis PM M/25 40-B-Testis A411147 Biochainestis PM MI74 41-CI-Testis 1110320 Clontechestis PB-Pool M&F
of 45 42-CG-Adrenal CG-184-101chilovdrenal PM F/81 43-B-Adrenal A610374 Biochaindrenal PM F/83 44-B-Heart A411077 BiochainHeart PB-Pool M&F
of 5 45-GG-Heart CG-255-9IchilovHeart PM M/75 46-CG-Heart CG-227-1IchilovHeart PM F/36 47-Am-Liver 081P0101mbion Liver PM M/64 48-CG-Liver CG-93-3 IchilovLiver PM F/19 49-CG-Liver CG-124-4IchilovLiver PM F/34 50-CI-BM 1110932 GlontechBone MarrowPM-Pool M&F
of 8 51-CGEN-Blood WBC#5 GLEN Blood M
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 he 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, CyS, Cy5.5, Cy7, FluorX
(Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, CalifJ 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 hyb ridization 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, ~~C, 32P, and 35S.
3U 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 vau:,~ty 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 earned 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
(Kwon et al.; 1989, Proc. NatI. 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. Uther 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 preferred 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 preferred embodiment, the amplification of the differentially expressed nucleic acids is earned 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 Biologyy, 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 u~ 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 lresent 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 Claain Reaction (PCR): The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,653,195 and 4,683,202 to Mullis and IVlullis 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 respectivve strands of the double-stranded target sequence to the DNA mixture containing the desired tart 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 Chairs 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, tvvo~ 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. W09001069 A 1 ( 1990).
However, because the four oligonuc leotides used in this assay can pair to form two short ligatable iss fragments, there is the potential for the generation of target independent background signal. The use of LCR for mutant screening is limited to the exanunation of specific nucleic acid positions.
Self-Sustained Synthetic Reaction (3SRlNASBA): The self sustained sequence replication reaction (3SR) is a transcriptionbased 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 (Q(3) Replicase: In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Q~i 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 Q~3 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 nonspecific hybridization of th° 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+~'~n =y, where "~" 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 polymerise 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 e~ciency to 85 °io, 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 SS % 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 polymerise chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate fo r 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 DN.A
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 misma tches 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 preferred 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, the re 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).
C3~cling 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.
Br-anelzed 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 nonspecific binding is similarly increased.
The detection of at least one sequence change according to various preferred 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), Suigle-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 wwithin 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 comb inations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. The presence of specific sequences witlun 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.
Restr-ictiora fragrnent 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
manipulatior~.
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 oligonaceleotide .(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 metlbd is based on the differences in the melting temperature of short DNA fragn,e:raia differing by a single nucleotide. Stringent hybridization and washing conditions can differeartiate bet<veen mutant and wild-type alleles. The ASO approach applied to PCR products also ha . 'been extensively utilized by various researchers to detect and characterize point mutations ui 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.
Derzatur~irtglTenrperattsre Gradient Gel Electr-oplror-esis (DGGElTGGE): 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 n 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-&0) 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 Confortnatiott Polyntotphism (SSCP): Another common method, called "Single-Strand Conformation Polymorphism" (SSCP) was developed by Hayashi, Sel<ya 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 W I1 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 infra-molecular interactions can form and not be disturbed during tl~
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 fingetpr~intirtg (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 presently preferred 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, Q~3-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 occurnng 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) andlor .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 wwell 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 BIastP
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. Finally, the present invention also encompasses fragments of the abovve 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.
Homology/identity of nucleic acid sequences is preferably determined by using BIastN 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.
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. Such modifications include, but are not limited to N
terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CPL?-S=O, O=C-NH, CPL?-O, CH2-CH?, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are wwell known in the art and are specified. Further Mails in this respect are provided hereinunder.
Peptide bonds ECO-NH-) within the peptide may be substituted, for example, by N-methylated bonds ~N(CH3)-CO-), ester bonds ~C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (-CO-CH2-), oc-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioarnide bonds (-CS-NH-), olefinic double bonds ( CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives ( N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom.
These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic norr natural acid such as Phenylglycine, TIC., naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
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 occurnng amino acids; those amino acids often modifted .post-translationally irr 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.
Table I non-cc~rrventiorral or modified arnino acids which can be used with the present irrvention.
Table 1 Non-conventional Code Non-conventional Code amino amino acid acid a,-aminobutyric Abu L-N-methylalanine Nmala acid a-amino-a-methylbutyr.3teMgabu L-N-methylarginine Nmarg aminocyclopropane-Cpro IrN-methylasparagineNmasn Carboxylate L-N-methylaspartic Nmasp acid aminoisobutyric Aib LrN-methylcysteine Nmcys acid aminonorbornyl- Norb LrN-methylglutamineNmgin Carboxylate IrN-methylglutamic Nmglu acid Cyclohexylalanine Chexa L~-N-methylhistidineNmhis CyclopentylalanineCpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg IrN-methyllysine Nmlys D-aspartic acid Dasp LrN-methylmethionineNmmet D-cysteine Dcys L,-N-methylnorleucineNmnle D-glutamine Dgln L-N-methylnorvalineNmnva D-glutamic acid Dglu 1rN-methylornithineNmorn D-histidine Dhis LrN-methylphenylalanineNmphe D-isoleucine Dile IrN-methylproline Nmpro D-leucine Dleu IrN-methylserine Nmser D-lysine Dlys L-N-methylthreonineNmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn IrN-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycineNmetg D-serine Dser IrN-methyl-t-butylglycineNmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp Irnorvaline Nva D-tyrosine Dtyr a-methyl-aminoisobutyrateMaib D-valine Dval a-methyl-y-aminobutyrateMgabu D-a-methylalanine Dmala a-methylcyclohexylalanineMchexa D-a-methylarginineDmarg a-methylcyclopentylalanineMcpen D-a-methylasparagineDmasn a-methyl-a-napthylalanineManap D-a-methylaspartateDmasp a- methylpenicillamineMpen D-~:-methylcysteineDmcys N-(4-aminobutyl)glycineNglu , D-~~c.-methylglutamineDmgln N-(2-aminoethyl)glycineNaeg D-c~ -methylhistidineDmhis N-(3-aminopropyl)glycineNorn D-a-.methylisoleucineDmile N- amino-a-methylbutyrateNmaabu D-a-methylleucineDmleu a-napthylalanine Anap D-a-methyllysine Dmlys N-benzylglycine Nphe D-a-methylmethionineDmmet N-(2-carbamylethyl)glycineNgln D-a-methylornithineDmorn N-(carbamylmethyl)glycineNasn D-a-methylphenylalanineDmphe N-(2-carboxyethyl)glycineNglu D-a-methylprolineDmpro N-(carboxymethyl)glycineNasp .
D-a-methylserine Dmser N-cyclobutylglycineNcbut D-a-methylthreonineDmthr N-cycloheptylglycineNchep D-a-methyltryptophanDmtrp N-cyclohexylglycineNchex D-a-methyltyrosineDmty N-cyclodecylglycineNcdec D-a-methylvaline Dmval N-cyclododeclglycineNcdod D-a-methylalnine Dnmala N-cyclooctylglycineNcoct D-a-methylarginineDnmarg N-cyclopropylglycineNcpro D-a-methylasparagineDnmasn N-cycloundecylglycineNcund D-a-methylasparatateDnmasp N-(2,2-diphenylethyl)glycineNbhm ~ D-a-methylcysteineDnmcys N-(3,3- Nbhe diphenylpropyl)glycine D-N-methylleucineDnmleu N-(3-indolylyethyl)Nhtrp glycine D-N-methyllysine Dnmlys N-methyl-'y-aminobutyrateNmgabu N- Nmchexa D-N-methylmethionineDnmmet methylcyclohexylalanine D-N-methylornithineDnmorn N-methylcyclopentylalanineNmcpen N-methylglycine Nala D-N-methylphenyylalanineDnmphe N-methylaminoisobutyrateNmaib D-N-methylproline Dnmpro N-(1-methylpropyl)glycineNile D-N-methylserine Dnmser N-(2-methylpropyl)glycineNile D-N-methylserine Dnmser N-(2-methylpropyl)glycineNleu D-N-methylthreonineDnmthr I
D-N-methyltryptophanDnmtrp N-(1-methylethyl)glycineNva D-N-methyltyrosineDnmtyr N-methyla-napthylalanineNmanap D-N-methylvaline Dnmval N-methylpenicillamineNmpen y-aminobutyric Gabu N-(p-hydroxyphenyl)glycineNhtyr acid Lrt-butylglycine Tbug N-(thiomethyl)glycineNcys L-ethylglycine Etg penicillamine Pen L-homophenylalanineHphe L-a-methylalanine Mala Lr-a-methylarginineMarg L-a-methylasparagineMasn L-a-methylaspartateMasp L-a-methyl-t-butylglycineMtbug L-a-methylcysteineMcys L-methylethylglycineMetg L-a-methylglutamineMgln lra-methylglutamateMglu L-a-methylhisti:dineMhis Ira-methylhomo Mhphe phenylalanine L-a-methylisoleucineMile N-(~-methylthioethyl)glycineNmet D-N-methylglutamineDnmgln N-(3- Narg guanidinopropyl)glycine D-N-methylglutamateDnmglu N-(1-hydroxyethyl)glycineNthr D-N-methylhistidineDnmhis N-(hydroxyethyl)glycineNser D-N-methylisoleucineDnmile N-(imidazolylethyl)glycineNhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycineNhtrp D-N-methyllysine Dnmlys N-methyl-y-aminobutyrateNmgabu N- Nmchexa D-N-methylmethionineDnmmet methylcyclohexylalanine D-N-methylornithineDnmorn N-methylcyclopentylalanineNmcpen N-methylglycine Nala D-N-methylphenylalanineDnmphe N-methylaminoisobutyrateNmaib D-N-methylproline Dnmpro N-( 1-methylpropyl)glycineNile D-N-methylserine Dnmser N-(2-methylpropyl)glycineNleu D-N-methylthreonineDnmthr D-N-methyltryptophanDnmtrp N-(1-methylethyl)glycineNval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanineNmanap D-N-methylvaline Dnmval N-methylpenicillamineNmpen y-aminobutyric acidGabu N-(p-hydroxyphenyl)glycineNhtyr butylglycine Tbug N-(thiomethyl)glycineNcys Irt- .
Lrethylglycine Etg penicillamine Pen IrhomophenylalanineHphe Lra-methylalanine Mala Ira-methylarginine Marg L-a-methylasparagineIVl;asn L,-a-methylaspartateMasp Lra-methyl-t-butylglycineMtbug Lra-methylcysteine Mcys L-methylethylglycineMetg Lr-a-methylglutamineMgln Ira-methylglutamateMglu L-a-me.thylhistidineNlhis L-a- Mhphe methylhomophenylalanine L-a-methylisoleucineMile N-(2-methylthioethyl)glycineNmet L-a-methylleucine Mleu L-a-methyllysine Mlys L-a-methylmethionineMmet L-a-methylnorleucineMule L-a-methylnorvalineMnva Lra-methylornithineMorn Lra-methylphenylalanineMphe L-a-methylproline Mpro L-a-methylserine mser L-a-methylthreonineMthr L-a-methylvaline Mtrp L-a-methyltyrosine Mtyr L-a-methylleucine Mval L-N- Nmhphe Nnbhm methylhomophenylalanine (2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl) N-(N- .
carbamylmethyl-glycineNnbhm carbamylmethyl(1)glycineNnbhe 1-carboxy-1-(2,2-diphenylNmbc ethylamino)cyclopropane Table 1 Cont.
Sine; lk~e peptides of the present invention are preferably utilized in diagnostics which require the lrehvtides to be in soluble form, the peptides of the present invention preferably include one o~~ more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere v~rith 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 relativelyy 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 far 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 inununoglobulin 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, CHI, 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, I X88, incorporated herein by reference).
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 SS 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 pepsin 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 mbar et al. [Proc. Naf1 Acad. Sci. USA 69:2659-62 ( 19720].
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.
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 uiterest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
Humanized forms of non-human (e.g., mur-ine) 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) wwhich contain minimal sequence derived from norrhuman immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in v~hich all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323 329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These norrhuman amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S.
Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR
residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage .display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in uwhich the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. .Pat. Nos. 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).
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 s~f the present invention, an immunoassay can be used to qualitatively or quantitatively dei:~ca and analyze markers in a sample. This method comprises:
providing an antibody that specifi,;ahy 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 imrnunosorbent 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. LTsually 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 iced 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 incubarion conditions described above. The amount of an antibod~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.
Preferably used are antibodies which specifically interact with the polypeptides of the present invention and not with wild type proteins or other isoforms thereof, for example. Such antibodies are directed, for example, to the unique sequence portions of the polypeptide variants of the present invention, including but not limited to bridges, heads, tails and insertions described in greater detail below. Preferred embodiments of antibodies according to the present invention are described in greater detail with regard to the section entitled "Antibodies".
Radio-irnnzz~noassay (RIA): In one version, this method invvolves 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 11"5) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precip itated 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 unknov~m amount of substrate is added in varying amounts. ')he decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
Ey~nze lir7kecl irn~fzmaosorhertt assay (ELIS4): 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 enzyyme 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.
l~restern 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).
S 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.
InunmZahistaclaernical analysis: This method involves detection of a substrate ira 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 (FAGS): This method involves detection of a substrate ira 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 (SPELT). 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, SPELT
can optionally be used with two labels simultaneously. SPELT has some other advantages as well, for example with regard to cost and the types of labels that can be used. For example, LTS
Patent No.
6,696,686 describes the use of SPELT for detection of breast cancer, and is hereby incorporated by reference as if fully set forth herein.
i7s Display Libraries According to still another aspect of the present invention there is provided a display library comprising a plurality of display ~;hicles (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 AC, 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 LB et al. "Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high amities" Biochemistry 1995 Nov 28;34(47):15430-5; Davies EL 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 RT al. "Current trends in molecular recognition and bioseparation" J
Chromatogr A 1995 Jul 14;707(1):3-22; Deng SJ et al. "Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries"
Proc Natl Acad Sci U S A 1995 I\~Iay 23;92(11):4992-6; and Deng SJ 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.
The following sections relate to Candidate Marker Examp les (first section) and to Experimental Data for these Marker Examples (second section).
CANDIDATE MARKER E~-1MPLES SECTION
This Section relates to Examples of sequences according to the present invention, including illustrative methods of selection thereof.
Description of the methodology undertaken to uncover the biomolecular sequences of the present invention Human ESTs and cDNAs were obtained from GenBanl: versions 136 (June 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 Oct 2003); Ref~eq sequences from December 2003; and from LifeSeq library of Incyte Corp (Wilmington, DE, USA; ESTs only). With regard to GenBank sequences, the human EST sequences from the EST (GBEST) section and the human mRNA sequences from the primate (GBPRI) section were used; also the human nucleotide RefSeq mRNA sequences were used (see for example www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html and for a reference to the EST section, see wunv.nebi.nlm.nih.gov/dbEST/; a general reference to dbEST, the EST
database in GenBank, may be found in Boguski et al, Nat Genet. 1993 Aug;4(4):332-3; all of which are hereby incorporated by reference as if fully set forth herein).
Novel splice variants were predicted using the LEADS clustering and assembly system as described in Sorek, R., Ast, G. & Graur, D. Alu-containing exons are alternatively spliced.
Genome Res 12, 1060-7 (2002); US patent No: 6,625,545; and U.S. Pat. Appl. No.
10/426,002, published as L1S20040101876 on May 27 2004; all of which are hereby incorporated by reference as if fully set forth herein. Briet7y, 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 (particularlyy of spliced sequences), chromosomal information, alignments, and additional information such as SNPs, .gene or~ology 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 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 Figure 1.
Identification of differentially expressed gene pr-odatcts - 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 over expressed in cancer is described hereinbelow.
Dry analysis Library annotation - EST libraries are manually classified according to:
(i) Tissue origin (ii) Biological source - Examples of frequently used biological sources for construction of EST libraries include cancer cell-lines; normal tissues;
cancer tissues; fetal tissues; and others such as normal cell lines and pools of normal cell-lines, cancer cell-lines and combinations thereof. A
specific description of abbreviations used below with regard to these tissues/cell lines etc is given above.
?0 (iii) Protocol of library construction - various methods are known in the art for library construction including normalized library construction;
non-normalized library construction; subtracted libraries; ORESTES and others. It will be appreciated that at times the protocol of library construction is not indicated.
The following rules were followed:
EST libraries originating from identical biological samples are 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 deviation., ~.bove the average for all libraries being analyzed, this library was tagged as being contasn~~nated and was eliminated from further consideration in the below analysis (see also Sorek, h.. & 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.
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 our belief 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 cumber 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 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 Rvalues were computed both for library and weighted clone counts to check that the counts are statistically significant.
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 ? 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 (S 1 ).
(ii) Supporting transcripts of group TB (S~).
(iii) Supporting transcripts from both groups (S3).
Library and clones number scores described above were given to S 1 group.
Fisher Exact Test P-values were used to check if S 1 is significantly enriched by cancer EST clones compared to S2; and S 1 is significantly enriched by cancer EST clones compared to cluster background (S 1+S2+S3).
Identification of unique sequence regions and division of the group of transcripts accordingly is illustrated in Figure 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 preferably not considered for determining differential expression between variants; Region 2: speciEc to Transcript 1;
Region 3: specific to Transcripts 2+3; Region 4: specific to Transcript 3; Region 5: specific to Transcripts 1 and 2;
Region 6: specific to Transcript 1.
E?~AMPLE 5 Identification of cancer specific splice variants of genes over expressed in cancer A search for EST supported (no mRNA) regions for genes of:
(i) known cancer markers (ii) Genes shown to be over-expressed in cancer in published micro-array experiments.
Reliable EST supported-regions were defined as supported by minimum of one of the following:
(i) 3 spliced ESTs; or (ii) 2 spliced ESTs from 2 libraries;
(iii) 10 unspliced ESTs from 2 libraries, or (iv) 3libraries.
Actual Marker Examples The following examples relate to specific actual marker examples.
EXPERIMENTAL EXAMPLES SECTION
This Section relates to Examples describing experiments involving these sequences, and illustrative, non-limiting examples of methods, assays and uses thereof. The materials and experimental procedures are explained first, as all experiments used them as a basis for the work that was performed.
The markers of the present invention were tested with regard to their expression in various cancerous and non-cancerous tissue samples. A description of the samples used in the panel is provided in Table 1 below. A description of the samples used in the normal tissue panel is provided in Table 2 below. Tests were then performed as described in the "Materials and Experimental Procedures" section below.
Table 1: Tissue samples in testing panel sample sex/
rename Lot no sourcepathologygrade age TNM stage Invasive1 Lobular 52-B-ILG A605360 BiochainCarcinoma F/60 51-B-IDC A605361 BiochainIDC 1 F/79 T2NOM stage NOM
7-A-IDC 7263T ABS IDG 2 F/43 0 stage T2NOM stage 13-A-IDC IA0133T IABS IIDC I 2 IF/63 IT2N1a ~ f~~lX
_ _.
~ -~
14-A-IDC A0135T ABS DC 2 F/37 ?N2M
G2 I ~ h 'r3N1Mstage 16-A-IDC 49040200327ABS DC 2 NA x 17-A-IDC 49040200367ABS DC 2-3 NA x 43-B-IDC A609183 BiochainDC 2 F140 44-B-IDG A609198 BiochainDG 2 F/77 45-B-IDC A609181 BiochainDC 2 F/58 48-B-IDG A609222 BiochainDG 2 F/44 49-B-IDC A609223 BiochainIDC 2 F/54 50-B-IDG A609224 BiochainIDG 2 F/69 53-B-IDC A605151 BiochainIDC 2 F/44 54-B-IDC A605353 BiochainIDC 2 F/41 55-B-IDC A609179 BiochainIDG 2 F/42 61-B-IDC A610029 BiochainIDC 2 F/46 62-B-IDC A609194 BiochainIDG 2 F/51 47-B-IDG A609221 BiochainIDC 2 46-B-CarciA609177 BiochainCarcinoma2 F/48 T2NOMstage 27-A-IDC 49070200727ABS IDC 3 NA x T1cN0 42-A-IDG 60050200317ABS IDC 3 NA Mx 31-GG-IDG CG-154 IchilovIDC NA
32-A-Muc 171167 I ABS MucinousI I F/54 IT2NOM(stage I
Carci carcinoma 0 2A
Normal matched 35-A-N 7238N ABS o 6T F/60 M6 t Normal matched 36-A-N 7263N ABS to 7T F/43 Normal matched 39-A-N 7259N ABS to 15T F159 Normal matched 40-A-N 1432N ABS to 12T F/46 Normal matched 41-A-N 7249N ABS to 26T F/60 Normal 56-B-N A609235 BiochainPM F/59 Normal 57-B-N A609233 BiochainPM F/34 Normal 58-B-N A609232 BiochainPM F/65 Normal 59-B-N A607155 BiochainPM F/35 Normal 60-B-N A609234 BiochainPM FI36 63-Am-N 26486 AmbionNormal F/43 PS
Normal 64-Am-N 23036 AmbionPM F/57 65-Am-N 131410 IAmbionNormal I I F/63 ( zaa PM
Normal 66-Am-N 36678 AmbionPM F/45 073P0106020 Normal 67-Am-N 6A AmbionPM F/64 Table 2: Tissue samples in normal panel:
Lot no. ource issue Pathology Sex/Age 1-Am-Colon (C71 071P10B mbion Colon PM F/43 ) 2-B-Colon (C69) A411078 BiochainColon PM-Pool M&F
of 10 3-CI-Colon (C70)1110101 ClontechColon PM-Pool M&F
of 3 4-Am-Small Intestine091P0201mbion Small IntestinePM M/75 5-B-Small IntestineA501158 BiochainSmall IntestinePM M/63 6-B-Rectum A605138 BiochainRectum PM M/25 7-B-Rectum A610297 BiochainRectum PM M/24 8-B-Rectum A610298 BiochainRectum PM M/27 9-Am-Stomach 110P04A mbion Stomach PM M/16 10-B-Stomach A501159 BiochainStomach PM M/24 11-B-Esophagus A603814 BiochainEsophagus PM M/26 12-B-Esophagus A603813 BiochainEsophagus PM M141 13-Am-Pancreas 071P25C mbion Pancreas PM M/25 14-CG-Pancreas CG-255-2IchilovPancreas PM M/75 15-B-Lung A409363 BiochainLung PM F126 16-Am-Lung (L93)111P0103mbion Lung PM F161 17-B-Lung (L92) A503204 BiochainLung PM M/28 18-Am-Ovary (047)061P43A mbion Ovary PM F/16 19-B-Ovary (048)A504087 BiochainOvary PM F/51 20-B-Ovary (046)A504086 BiochainOvary PM F/41 21-Am-Cervix IOlPOlOlAmbion Cervix PM F/40 22-B-Cervix A408211 BiochainCervix PM i =~''~~fi 23-B-Cervix A504089 BiochainCervix PM-Pool Ni~i of 5 24-B-Uterus A411074 BiochainUterus PM-Pool M~~xF
of 10 25-B-Uterus A409248 BiochainUterus PM F/43 26-B-Uterus A504090 BiochainUterus PM-Pool M&F
of 5 27-B-Bladder A501157 BiochainBladder PM M/29 28-Am-Bladder 071P02C mbion Bladder PM M/20 29-B-Bladder A504088 BiochainBladder PM-Pool M&F
of 5 30-Am-Placenta 021P33A mbion Placenta PB F/33 31-B-Placenta A410165 BiochainPlacenta PB F/26 32-B-Placenta A411073 BiochainPlacenta PB-Pool M&F
of 5 33-B-Breast (B59)A607155 BiochainBreast PM F/36 34-Am-Breast 26486 mbion Breast PM F/43 (B63) 35-Am-Breast 23036 mbion Breast PM F/57 (B64) 36-CI-Prostate 1070317 ClontechProstate PB-Pool M&F
(P53) of 47 37-Am-Prostate 061P04A mbion Prostate PM M/47 (P42) 38-Am-Prostate 25955 mbion Prostate PM M/62 (P59) 39-Am-Testis 111P0104Ambion estis PM M/25 40-B-Testis A411147 Biochainestis PM MI74 41-CI-Testis 1110320 Clontechestis PB-Pool M&F
of 45 42-CG-Adrenal CG-184-101chilovdrenal PM F/81 43-B-Adrenal A610374 Biochaindrenal PM F/83 44-B-Heart A411077 BiochainHeart PB-Pool M&F
of 5 45-GG-Heart CG-255-9IchilovHeart PM M/75 46-CG-Heart CG-227-1IchilovHeart PM F/36 47-Am-Liver 081P0101mbion Liver PM M/64 48-CG-Liver CG-93-3 IchilovLiver PM F/19 49-CG-Liver CG-124-4IchilovLiver PM F/34 50-CI-BM 1110932 GlontechBone MarrowPM-Pool M&F
of 8 51-CGEN-Blood WBC#5 GLEN Blood M
52-CGEN-Blood WBC#4 CGEN Blood M
53-CGEN-Blood WBC#3 CGEN Blood M
54-CG-Spleen CG-267 chilovSpleen PM F/25 I
55-CG-Spleen 111P0106Bmbion Spleen PM M/25 56-CG-Spleen A409246 BiochainSpleen PM F/12 56-CG-Thymus CG-98-7 Ichilovhymus PM F/28 58-Am-Thymus IOIPOIOIAmbion hymns PM M/14 59-B-Thymus A409278 Biochainhymns PM M/28 60-B-Thyroid A610287 Biochainhyroid PM M/27 61-B-Thyroid A610286 Biochainhyroid PM M/24 62-CG-Thyroid CG-119-2Ichilovhyroid PM F/66 63-CI-Salivary 1070319 ClontechSalivary PM-Pool M&F
Gland Gland of 24 64-Am-Kidney 111PO1O1Bmbion Kidney PM-Pool M&F
of 14 65-CI-Kidney 1110970 ClontechKidney PM-Pool M&F
of 14 66-B-Kidney A411080 BiochainKidney PM-Pool M&F
of 5 67-CG-Cerebellum CG-183-5IchilovCerebellum PM MI74 68-CG-Gerebellum CG-212-5IchilovCerebellum PM M/54 69-B-Brain A411322 BiochainBrain PM M/28 70-CI-Brain 1120022 ClontechBrain PM-Pool M&F
of 2 71-B-Brain A411079 BiochainBrain PM-Pool M&F
of 2 72-CG-Brain CG-151-1IchilovBrain PM F/86, 73-Am-Skeletal 1O1P013Ambion Skeletal PM F/28 Muscle Muscle 74-CI-Skeletal 11061038IGIontechlSkeletal PM-Pool M&F
Muscle MuscIeI of 2 I
Materials aftd Experimental Procedacres RNA preparation - RNA was obtained from Clontech (Franklin Lakes, NJ LISA
07417, www.clontech.com), BioChain Inst. Inc. (Hayward, CA 94545 USA
www.biochain.com), ABS
(Wilmington, DE 19801, USA, http://www.absbioreagents.com) or Ambion (Austin, USA, http://www.ambion.com). Alternatively, RNA was generated from tissue samples using TRI-Reage nt (Molecular Research Center), according to Manufacturer's instructions. Tissue and RNA samples were obtained from patients or from postmortem. Total RNA samples were treated with DNaseI (Ambion) and purified using RNeasy columns (Qiagen).
RT PCR - Purified RNA (1 p.g) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 p.M dNTP in a total volume of 15.6 p.1. The mixture was incubated for 5 min at 65 °C and then quickly chilled on ice. Thereafter, 5 p.1 of SX
SuperscriptII first strand buffer (Invitrogen), 2.4.1 O.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 p l (200units) of SuperscriptII (Invitrogen) was added and the reaction (final volume of 25p1) was incubated for 50 min at 42 °C and then inactivated at 70 °C for l5min. The resulting cDNA was diluted 1:211 in TE buffer (10 mM Tris pH=8, 1 mM EDTA pH=8).
Real-TurTe RT-PCR analysis- cDNA (5p l), prepared as described above, was used as a template in Real-Time PCR reactions using the SYBR Green I assay (PE Applied Biosystem) with specific primers and IJNG 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 l5sec, 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 trarrcript quantity in the RT reactions. The relative quantity was calculated using the equation Q=efficiency~-~'. The efficiency of the PCR
reaction was calculated from a standard curve, created by using serial dilutions of several reverse transcription (RT) reactions. To minimize inherent differences in the RT reaction, the resulting relative quantities were normalized to the geometric mean of the relative quantities of several housekeeping (HSKP) genes. Schematic summary of quantitative real-time PCR
analysis is presented in Figure 3. As shov~m, 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
product signal is above the background level (passive dye RO~i) and still in the Geometric/Exponential phase (as shown, once the level of fluorescence crosses the measurement threshold, it has a geomeMcally 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 breast cancer panel were as follows:
G6PD .(GenBank Accession No. NM_000402) G6PD Forv,~ard primer: gaggccgtcaccaagaacat G6PD Reverse primer: ggacagccggtcagagctc G6PD-amplicon:
gaggccgtcaccaagaacattcacgagtcctgcatgagccagataggctggaaccgcatcatcgtggagaagcccttcg ggagggacct gcagagctctgaccggctgtcc SDHA (GenBank Accession No. NM_004168) SDHA Forward primer: TGGGAACAAGAGGGCATCTG
SDHA Reverse primer: CCACCACTGCATCAAATTCATG
SDHA-amplicon TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATCCAGT
AGTGGATCATGAATTTGATGCAGTGGTGG
PBGD (GenBank Accession No. BC019323), PBGD Forward primer: TGAGAGTGATTCGCGTGGG
PBGD Reverse primer: CCAGGGTACGAGGCTTTCAAT
PBGD-amplicon:
TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAGACGGAC
AGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG
HPRT 1 (GenBank Accession No. NM_000194), HPRT1 Forward primer: TGACACTGGCAAAACAATGCA
HPRT1 Reverse primer: GGTCCTTTTCACCAGCAAGCT
HPRT .l-amplicon:
TGAC.~.CTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAA
AGA'I'~~iGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
The sequences of the housekeeping genes measured in all the examples on normal tissue samples panel were as follows:
RPL19 (GenBanl: Accession No. NM_000981), RPL19 Forward primer: TGGCAAGAAGAAGGTCTGGTTAG
RPL 19 Reverse primer: TGATCAGCCCATCTTTGATGAG
RPL19 -amplicon:
TGGCAAGAAGAAGGTCTGGTTAGACCCCA.ATGAGACCAATGAAATCGCCAATGCCA
ACTCCCGTCAGCAGATCCGGAAGCTCATCAAAGATGGGCTGATCA
TATA box (GenBank Accession No. NM_003194), TATA box Forward primer : CGGTTTGCTGCGGTAATCAT
TATA box Reverse primer: TTTCTTGCTGCCAGTCTGGAC
TATA box -amplicon:
CGGTTTGCTGCGGTAATCATGAGGATAAGAGAGCCACGAACCACGGCACTGATTTT
CAGTTCTGGGAAAATGGTGTGCACAGGAGCCAAGAGTGAAGAACAGTCCAGACTG
GCAGCAAGAAA
UBC (GenBank Accession No. BC000449) UBC Forvvard primer: ATTTGGGTCGCGGTTCTTG
LTBC Reverse primer: TGCCTTGACATTCTCGATGGT
LTBC -amplicon:
ATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGACAATGCAGAT
CTTCGTGAAGACTCTGACTGGTAAGACCATCACCCTCGAGG
TTGAGCCCAGTGACACCATCGAGAATGTCAAGGCA
SDHA (GenBanl: Accession No. NM_004168) SDHA Forward primer: TGGGAACAAGAGGGCATCTG
SDHA Reverse primer: CCACCACTGCATCAAATTCATG
SDHA-amplicon TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATCCAGT
AGTGGATCATGAATTTGATGCAGTGGTGG
Oligorat.rcleotide-based micro-array experiment protocol-Microarray fabrication Microarrays (chips) were printed by pin deposition using the MicroGrid II MGII
robot from BioRobotics Limited (Cambridge, LTK). 50-mer oligonucleotides target sequences were designed by Compugen Ltd (Tel-Avviv, IL) as described by A. Shoshan et al, "Optical technologies and informatics", Proceedings of SPIE. Vol 4266, pp. 86-95 (2001). The designed oligonucleotides were synthesized and purified by desalting with the Sigma-Genosys system (The Woodlands, TX, US) and all of the oligonucleotides were joined to a C6 amino-modified linker at the 5' end, or being attached directly to CodeLink slides (Cat #25-6700-O1. Amersham Bioscience, Piscataway, NJ, US). T'he 50-mer oligonucleotides, forming the target sequences, were first suspended in Ultra-pure DDW (Cat # O1-866-lA Kibbutz Beit-Haemek, Israel) to a concentration of SON.M. Before printing the slides, the oligonucleotides were resuspended in 300mM sodium phosphate (pH 8.5) to final concentration of 150mM and printed at 35-40%
relative humidity at 21 °C.
Each slide contained a total of 9792 features in 32 subarrays. Of these features, 4224 features were sequences of interest according to the present invention and negative controls that were printed in duplicate. An additional 28S features (96 target sequences printed in triplicate) contained housekeeping genes from Human Evaluation Library2, Compugen Ltd, Israel.
Another 384 features are E.coli spikes 1-6, which are oligos to E Coli genes which are commercially available in the Array Control product (Array control- sense oligo spots, Ambion Inc. Austin, TX. Cat #1781, Lot #112K06).
Post-coupling processing of printed slides After the spotting of the oligonucleotides to the glass (CodeLinl:) slides, the slides were incubated for 24 hours in a sealed saturated NaCI humidification chamber (relativve humidity 70-75%).
Slides were treated for blocking of the residual reactive groups by incubating them in blocking solution at 50°C for 15 minutes (lOml/slide of buffer containing O.1M Tris, SOmM
ethanolamine, 0.1% SDS). The slides were then rinsed twice with Ultra-pure DDW
(double distilled water). The slides were then washed with wash solution (lOml/slide.
4X SSC, 0.1%
SDS)) at 50°C for 30 minutes on the shaker. The slides were then rinsed twice with Ultra-pure DDW, followed by drying by centrifugation for 3 minutes at 800 rpm.
Next, in order to assist in automatic operation of the hybridization protocol, the slides were treated with Ventana Discovery hybridization station barcode adhesives.
The printed slides were loaded on a Bio-Optica (Milan, Italy) hematology staining device and were incubated for 10 minutes in SOmI of 3-Aminopropyl Triethoxysilane (Sigma A3648 lot #122K589). Excess fluid was dried and slides were then incubated for three hours in 20 nvr>/Hg in a dark vacuum desiccator (Pelco 2251, Ted Pella, Inc. Redding CA).
The following protocol was then followed with the Genisphere 900-RP (random primer), with mini elute columns on the Ventana Discovery HybStationT"', to perform the microarray experiments. Briefly, the protocol was performed as described with regard to the instructions and information provided with the device itself. The protocol included cDNA
synthesis and labeling. cDNA concentration was measured with the TBS-380 (Turner Biosystems.
Sunnyvale, CA.) PicoFlour, which is used with the OliGreen ssDNA Quantitation reagent and kit.
Hybridization was performed with the Ventana Hybridization device, according to the provided protocols (Discovery Hybridization Station Tuscon AZ).
The slides were then scanned with GenePix 4000B dual laser scanner from Axon Instruments Inc, and analyzed by GenePix Pro 5.0 software.
Schematic summary of the oligonucleotide based microarray fabrication and the experimental flow is presented in Figures 4 and 5.
Briefly, as shown in Figure 4, DNA oligonucleotides at 25uM.were deposited (printed) onto Amersham 'CodeLinl:' glass slides generating a well defined 'spot'. These slides are covered with a long-chain, hydrophilic polymer chemistry that creates an active 3-D surface that covalently binds the DNA oligonucleotides 5'-end via the C6-amine modii~i _~.~tion. This binding ensures that the full length of the DNA oligonucleotides is available for hybr~iciization to the cDNA and also allows lower background, high sensitivity and reproducibility.
Figure 5 shows a schematic method for performing the microarray experiments.
It should be noted that stages on the left-hand or right-hand side may optionally be performed in any order, including in parallel, until stage 4 (hybridization). Briefly, on the left-hand side, the target oligonucleotides are being spotted on a glass microscope slide (although optionally other materials could be used) to form a spotted slide (stage 1). On the right hand side, control sample RNA and cancer sample RNA are Cy3 and Cy5 labeled, respectively (stage 2), to form labeled probes. It should be noted that the control and cancer samples come from corresponding tissues (for example, normal prostate tissue and cancerous prostate tissue).
Furthermore, the tissue from which the RNA was taken is indicated below in the specific examples of data for particular clusters, with regard to overexpression of an oligonucleotide from a "chip"
(microarray), as for example "prostate" for chips in which prostate cancerous tissue and normal tissue were tested as described above. In stage 3, the probes are mixed. In stage 4, hybridization is performed to form a processed slide. In stage 5, the slide is washed and scanned to form an image file, followed by data analysis in stage 6.
Cluster T10888 features 4 transcripts) and 8 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table 1 - Ti~arrscripts of rrzterest Transcript ' Sequence ID No.
I~Tame T10888_PEA_1T1 1 T10888_PEA_1T4 T10888_PEA_1TS
T10888_PEA_1T6 Table ? - Segnzertts of interest Se~tnent i Sequence ID Na. __ Name T10888PEA_1node_11 5 T10888PEA_1node_12 6 T10888_PEA_1node_17 7 T10888_PEA_1node 8 T10888_PEA_1node_6 9 T10888PEA node7 10 I
T10888_PEA_1node9 11 T10888_PEA_1node_IS 12 Table 3 - Proteins of irrterest Protein '-'- Szduence ID Na.
Name PEA_ PEA
PEA
T10888_PEA_1 P6 17 These sequences are variants of the known protein Carcinoembryonic antigen-related cell adhesion molecule 6 precursor (SwissProt accession identifier CEA6_HUMAN;
known also according to the synonyms Normal cross-reacting antigen; Nonspecific crossreacting antigen;
CD66c antigen), SEQ ID NO: 13, referred to herein as the previously known protein.
The sequence for protein Carcinaembryonic antigen-related cell adhesion molecule 6 precursor is given at the end of the application, as "Carcinoembryanic antigen-related cell adhesion molecule 6 precursor amino acid sequence". Known polymorphisms for this sequence are as shown in Table 4.
Table 4 - Arr2irlo acid rnnctations for Kraowrz Protein SNP positions) Comment an amino acid sequence 138 F->L
Protein Carcinoembryonic antigen-related cell adhesion molecule 6 precursor localization is believved to be Attached to the membrane by a GPI-anchor.
The previously known protein also has the following indications) and/or potential therapeutic use(s): Cancer. It has been investigated for clinicaUtherapeutic use in humans, for example as a target for an antibody or small molecule, and/or as a direct therapeutic; available information related to these investigations is as follows. Potential pharmaceutically related or therapeutically related activity or activities of the previously known protein are as follows:
Immunostimulant. A therapeutic role for a protein represented by the cluster has been predicted.
The cluster was assigned this field because there was information in the drug database or the public databases (e.g., described herein above) that this protein, or part thereof, is used or can be used for a potential therapeutic indication: Imaging agent; Anticancer;
Immunostimulant;
Immunoconjugate; Monoclonal antibody, murine; Antisense therapy; antibody.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: signal transduction; cell-cell signaling, which are.
annotations) related to Biological Process; and integral plasma membrane protein, which are annotations) related to Cellular Comporent.
The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from <http:i/wn?v.expasy.ch/sprot/>; or Locuslink, available from <http://www.ncbi.nlm.nih.gov/projects/LocusLink/>.
Cluster T10888 can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term "number" in the right hand column of the table and the numbers on the taxis of Figure 6 refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histograms in Figure 6 and Table 5. 'This cluster is overexpressed (at least at a minimum level) in the following pathological conditions: colorectal cancer, a mixture of malignant tumors from different tissues, pancreas carcinoma and gastric carcinoma.
Table 5 - Normal tissue distribution Name of Tissue Number Bladder 0 Colon 107 Epithelial 52 General 22 head and neck 40 Lung 237 Breast 0 pancreas 32 Prastate 12 Stomach 0 Table 6 - P values and ratios for expression in cancerous tissue Name of TissueP1 ~ p2 SP1 ~ _ ; SP2 4 . R
Bladder 5.4e-013.4e-01 5.6e-011.8 4.(e-011.9 Colon 1.2e-011.7e-01 2.Se-OS3.7 7.9e-042.8 epithelial 3.3e-022.1e-01 2.8e-202.8 4.8e-101.9 General 3.3e-052.2e-03 1.9e-444.9 4.6e-273.3 head and neck4.fie-O14.3e-01 1 0.8 7.Se-Ol1.0 Lung 7.6e-018.2e-01 8.9e-010.6 1 0.3 Breast 3.7e-024.1e-02 l.Se-O13.3 3.1e-012.4 pancreas 2.6e-012.4e-01 8.6e-232.8 1.5e-194.5 Prostate 9.1e-019.3e-01 4.1e-021.2 l.Oe-O11.0 Stomach 4.5e-0'25.6e-02 S.le-044.1 4.7e-046.3 LOO
As noted above, cluster T10888 features 4 transcript(s), which were listed in Table above. These transcripts) encode for proteins) which are variants) of protein Carcinoembryonic antigerrrelated cell adhesion molecule 6 precursor. A
description of each variant protein according to the present invention is now provided.
Variant protein T10888_PEA_1 P2 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10888 PEA_1 T1. An alignment is given to the known protein (Carcinoembryonic antigen related cell adhesion molecule 6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10888_PEA_1 P2 and CEA6 HUMAN:
l.An isolated chimeric polypeptide encoding for T10888_PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVI'PELPKPSISSNNSNPVEDKDAVAFTCEPEVVQNTTYL
WWVNGQSLPVSPRLQLSNGNMTLTLLSVkRIVDAGSYECEIQNPASANRSDPVTLNVLY
GPDVPTISPSkANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGS
I'MCQAHNSATGLNRTTVTMITVS corresponding to amino acids 1 - 319 of CEA6_ _HUhZAN, which also corresponds to amino acids 1 - 319 ofT10888 PEA_1 P2, 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 DWTRP corresponding to amino acids 320 - 324 of T1088S
PEA_1 P2, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of T1088S PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence DWTRP in T10888_PEA_1 P2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows v~rith regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T10888_PEA_1 P2 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 7, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs _in variant protein T10888_PEA_1 P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 7 - Anrirra acid mutcrtians SNP' position(s). Alteruai'ive an~ir~o~ Previously l.~nowin on amino acid acids) SNP'?
sequence 13 V -> No 232 N _> D No 324 P -> No 63 1-> No G _> ~ No Variant protein T10888_PEA_1 P2 is encoded by the following transcript(s):
T10888_PEA_1 T1, for which the sequences) is/are given at the end of the application. The coding portion of transcript T1088S PEA_1 T1 is shown in bold; this coding portion starts at position 151 and ends at position I 122. The transcript also has the following SNPs as listed in Table 8 (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 _irz variant protein T10888_PEA_1 P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 8 - Nucleic acid SNPs SNk' Position on nucleotideAlternative nucleic Previously Ionnocvn Seql~erlCe acid ' SNP? ~_ 119 C->T No 120 A -> T No 1062 A _> G Yes 1120 -- C _> No 1297- G _> T Yes 1501 A -> G Yes 1824 G -> A No 2036 A -> C No 2036 A -> G No 2095 A -> C No 2242 A -> C No 2245 A -> C No 189 C -> No 2'?Sp A -> T Yes 2339 C -> A Yes 276 G _> A Yes 338 - T -> No 424 G -> No 546 A -> G No 702 C -> T No 844 A -> G No 93p C -> T Yes Variant protein T10888_PEA_1 P4 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10888_PEA_1 T4. An alignment is given to the known protein (Carcinoembryonic antigen-related cell adhesion molecule 6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A briefdescription of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10888_PEA_1 P4 and CEA6_HUMAN:
l.An _isolated chimeric polypeptide encoding for T10888_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYItGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVY'PELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
WWVNGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNPASANRSDPVTLNVL
corresponding to amino acids 1 - 234 of CEA6_HUMAN, which also corresponds to amino acids 1 - 234 of T10888 PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence LLLSSQLWPPSASRLECWPGWL corresponding to amino acids 235 - 256 of T10888_PEA_I P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _isolated polypeptide encoding for a tail of T10888_PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence LLLSSQLWPPSASRLECWPGWL in T10888_PEA_1 P4.
Comparison report between T10888_PEA_1 P4 and Q13774 (SEQ ID N0:829):
l.An isolated chimeric polypeptide encoding for T10888_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWkEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGILEVLLLAHNLP
QNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FI'TLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
ATV'WNGQSLPVSPRLQLSNGNMTLTLLS'~Il'~:ItNDAGSYECEIQNPASANRSDPVTLNVL
corresponding to amino acids 1 - 234 of Q 137 i 4, which also corresponds to amino acids 1 - 234 of T10888_PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence LLLSSQLWPPSASRL,ECWFGWL
corresponding to amino acids 235 - 256 of T10888_PEA_1 P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _ _isolated polypeptide encoding for a tail of T10888 PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence LLLSSQLWPPSASRLECWPGWL in T10888_PEA_1 P4.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignaIP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T1088S PEA_1 P4 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10S88 PEA_1 P4 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 9 - Arnin~ acid mutations SNP positions) on Alternative,amino ' Previously known amino acid acids) SNP?
sequence 13 V -> No 232 N -> D No 63 I -> No ~2 G -> No.
Variant protein T1088R PEA_1 P4 is encoded by the following transcript(s):
T10888_PEA_1 T4, for which the sequences) is/are given at the end of the application. The coding portion of transcript T10888_PEA_1 T4 is shown in bold; this coding portion starts at position 151 and ends at position 918. The transcript also has the following SNPs as listed in Table 10 (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 knov~m SNPs in variant protein T10888_PEA_1 P4 sequence provides support for the deduced sequence of this variant protein according to the present invention).
TaL~le 10 - Nucleic acid SNPs SNF' position on nucleotideAlternative'rn~cleicI Preid.ously known sequence acid SNP? ____ 119 C->T No I 20 A -> T No Q78 C -> No 1155 G -> T Yes 1359 A -> G
Yes 1682 G -> A No 1894 A -> C No 1894 - A -> G No 1953 A -> C No 2100 A -> C No 2103 A -> C No ~ 108 A _> T Yes 189 G -> No C->A
Yes 276 G _> A Yes 338 T -> No 424 G -> No 546 A -> G No 702 C -> T No 844 A -> G No 958 G -> No Variant protein T10888_PEA_1 PS according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T1088S_PEA_1 T5. An alignment is given to the lenown protein (Carcinoembryryonic antigerE
related cell adhesion molecule 6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10888_PEA_1 PS and CEA6_HUMAN:
l.An _isolated chimeric polypeptide encoding for T10888_PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVL,LTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSV~TYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
WWVNGQSLPVSPRLQLSNGNMTLTLLS~~hRNDAGSYECEIQNPASANRSDPVTLNVLY
GPDVPTISPSKANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGS
YMCQAHNSATGLNRTTVTMITVSG corresponding to amino acids 1 - 320 of CEA6_HLJMAN, which also corresponds to amino acids 1 - 320 of T10888_PEA_1 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide havingthesequence KWIHEALASHFQVESGSQRRARI~KFSFPTCVQGAHANPKFSPEPSQFTSADSFPLVFLFF
VVFCFLISHV corresponding to amino acids 321 - 390 of T10888_PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _isolated polypeptide encoding for a tail of T10888_PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at 3east about 95%
homologous to the segue rice KWIFIEALASHFQVESGSQRRARICKFSFPTCVQGAH.a~I~dPKFSPEPSQFTSADSFPLVFLFF
VVFCFLISHV in T10888_PEA_1 P5.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
membrane. The protein localization is believed to be membrane because although both signal-peptide prediction programs agree that this protein has a signal.peptide, both trans-membrane region prediction programs predict that this protein has a trans-membrane region downstream of this signal peptide..
Variant protein T10888_PEA_1 PS also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 11, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is lmown _or not; the presence of known SNPs in variant protein T10888_PEA_1 PS
sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table I I - Amine acid fnzctatiofZs SNP:posilion(s) on 'Alternative amino i Previously knu~wn aimii~o acid acids) SNP'"?
sequence 13 V -> No 232 N -> D No 63 I -> No 92 G -> No Variant protein T10888_PEA_1 PS is encoded by the following transcript(s):
T10888_PEA_1 T5, for which the sequences) is/are given at the end of the application. The coding portion of transcript T10888_PEA_1 TS is shown in bold; this coding portion starts at position 151 and ends at position 1320. The transcript also has the following SNPs as listed in 2oa Table 12 (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 T1088S PEA_1 PS sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 1? -Nticleie acid SNPc SNP position on nu~teatid~Att~rnati.ve nucleic~ Previt~usly knav~m.
sequence acid SNP? -. -119 C->T No 120 A -> T No 1062 A -> G Yes 1943 C -> A Yes 2609 C -> T Yes X647 C -> G No 2701 C -> T Yes T -> C
es 1 s9 c -> No 276 G -> A Yes 338 T -> No 424 G -> No 546 A -> G No 702 C -> T No 844 A -> G No 930 C -> T Yes Variant protein T10888 PEA_1 P6 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10888_PEA_1 T6. An alignment is given to the known protein (Carcinoembryonic antigen-related cell adhesion molecule 6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application.
Comparison report between T10888_PEA_1 P6 and CEA6 l.An isolated chimeric polypeptide encoding for T10888_PEA_1 P6, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWILEVLLTASLLTFWNPPTTAKi,TIESTPFNVAEGIiEVLLLA
HNLPQNRIGYSWYILGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPN ASLLIQNVTQ
NDTGFYTLQVIIiSDLVNEEATGQFHVY
corresponding to amino acids 1 - 141 of CEA6 HUMAN, which also corresponds to amino acids 1 - 141 of T10888_PEA_1 P6, 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 REYFHMTSGCWGSVLLPTI'GIVRPGLCLWPSLHYIL,YQGLDI
corresponding to amino acids 142 - 183 of T10888_PEA_1 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of T10888 PEA_1 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95°,~o homologous to the sequence REYFHMTSGCWGSVLLPTYGIVRPGLCLWPSLHYILYQGLDI in T10888 PEA 1 P6.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T10888_PEA_1 P6 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 13, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10888_PEA_1 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 13 - Antdrto acid ntutatiorts SNP po~itian(s) on Alterdalive amine Previously tcnoi~~i amin.Q acid acxd(s) SNP's sequence 13 V -> No 63 I -> No 92 G -> No Variant protein T10888 PEA_1 P6 is encoded by the following transcript(s):
T1088s PEA_1 T6, for which the sequences) is/are given at the. end of the application. The coding portion of transcript T1088S PEA_1 T6 is shown in bold; this coding portion starts at position 151 and ends at position 6~9. 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 T1088S PEA_1 P6 sequence provides support for the deduced sequence of this variant protein according to tl~ present invention).
Table 14 - Nucleic acid SNPs SNP position on nucleotideAlternative nucleic Previously knowrx sequence acid SNF?
119 C -> T No 120 A -> T No 1 S9 C -> No 276 G -> A Yes 33R T-> No 424 G -> No 546 A -> G No As noted above, cluster T10888 features 8 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application. These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster T10888_PEA_1 node_11 according to the present invention is supported by 57 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T1 and T10888 PEA_1 T5. Table below describes the starting and ending position of this segment on each transcript.
Table 15 - Segment location on transcripts Transcript'iiame Seg~llent.starting S~gn~~nt ending position position T1088S PEA_1 TS 854 1108 15 Segment cluster T10888 PEA_1 node_12 according to the present invention is supported by 9 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T5. Table 16 below describes the starting and ending position of this segment on each transcript.
Table 16 - Segrnerrt location on transcripts Transcript name Segment starting positionSegment ending position T10888 PEA 1 TS 1109 '3004 Segment cluster T10888_PEA_1 node_17 according to the present invention is supported by 160 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T1 and T10888_PEA_1 T4. Table 17 below describes the starting and ending position of this se~rnent on each transcript.
Table 17 - Segment location on transcripts Transcript name Segment starCing Segment ex~~li~ag position position T10888_PEA_1 T4 967 2376 Segment cluster T10888_PEA_1 node 4 according to the present invention is supported by 61 libraries. The number of libraries was determined as previously described. This segment can be found in,the _ _following transcript(s): T10888_PEA_1 T1, T10888_PEA_1 T4, T10888_PEA_1 TS and T10888 PEA_1 T6. Table 18 below describes the starting and ending position of this segment on each transcript.
Table 18 - Segment locatiatt ort transcripts Transcript name .. ~eent starting position' Segment ending positron .
T10888 PEA_1 T4 1 214 Segment cluster T10888 PEA_1 node 6 according to the present invention is supported by 81 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 Tl, T10888_PEA_1 T4, T10888_PEA_1 TS and T10888_PEA_1 T6. Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on transcripts Transcript name Segment starting positionSegment ending position T10888 PEA_1 T1 215 574 T10888 PEA_1 T4 215 574 T10888_PEA_1 TS 215 574 T10S88 PEA_1 T6 215 574 Segment cluster T10888_PEA_1 node 7 according to the present invention is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T6. Table 20 below describes the starting and ending position of this segment on each transcript.
Table 20 - Segrnerrt location on transcripts TranscripCniam~ Se~h~nt starting ~ Segment ending ._ pcisi~on pASition T10888 PEA 1 T6 575 ~~ 1410 Segment cluster T1088F PEA_1 node 9 according to the present invention is supported by 72 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888 PEA_1 T1, T10888_PEA_1 T4 and T10888_PEA_1 T5. Table 21 below describes the starting and ending position of this segment on each transcript.
Table 2T - Segrrrertt location ort transcripts Transcript name Segment starting positionSegment ending position T10888 PEA 1 Tl 575 853 According to an optional embodiment of trie present invention, snort segments re~ateo to the abovve cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster T10888_PEA_1 node_15 according to the present invention is supported by 3A libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T4. Table 22 below describes the starting and ending position of this segment on each transcript.
Table ~2 - Segment location on transcripts Transcript name ~e~en'~ starting Segment ending position-position ~
T10888_PEA_1 T4 854 X66 Variant protein alignment to the previously known protein:
Sequence name: /tmp/tM4EgaoKvm/vuztUrlRc7:CEA6 HUMAN
Sequence documentation:
Alignment of: T10888-PEA-1_P2 x CEA6 HUMAN ..
Alignment segment 1/1:
Quality: 3163.00 Escore: 0 Matching length: 319 Total length: 319 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
51 VLLLAHNLFQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRE'~' i00 . . . . -~~~~~~~~~~~~I~~~~II
Sequence name: /tmg/Yj11gj7TCe/PgdufzLOIW:CEA6 HUMAN
Sequence documentation:
Alignment of: T10888-PEA 1-P4 x CEA6 HUMAN ..
Alignment segment 1/l:
Quality: 2310.00 Escore: 0 Matching length: 234 Total length: 234 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIIIIIIIilllllllllllllllllllllllllll IIIIIIIIIIIIilllllllllllllllllllllllllllllllllllll . . . .
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII.
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Illlllllilllllllllllllllllllllllll Sequence name: /tmp/Yj11gj7TCe/Pgduf,zLOlW:Q13774 Sequence documentation:
Alignment of: T10888 PEA 1 P4 x Q13774 ..
Alignment segment 1/1:
Quality: 2310.00 Encore : 0 Matching length: 234 Total length: 234 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIII~IIIIIIII~~IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~
III~IIIIIIIIIIII~III~IIIIIIIIIIIIIIII~IIIII~IIIII~
101 IYPNASLLIQNVTQNDTGF1'TLQVIKSDLVNEEATGQFHVYPELPKPSIS 150 3o IIIIIIIIIIIIIIIII~IIIIIIIIIIIIIIIIIIII~IIIIIIIIIIII
Sequence name: /tmp/x5xDBacdpj/rTXRGepv3y:CEA6 HUMAN
Sequence documentation:
Alignment of: T10888_PEA_1_P5 x CEA6 HUMAN ..
Alignment segment 1/1:
Quality: 3172.00 Encore: 0 Matching length: 320 Total length: 320 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
~°~1 VLLLAHNLPQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRET 100 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~IIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
201 TLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLYGPDVPTISPSKANYR ~50 IIIIillllillllllllllllllllllllllllllllllllllllllll 2o IIIIIilllllllllillllllllllllllllllllllllllllllllil IIIIIIIIIIIIIIIIIIII
~5 301 AHNSATGLNRTTVTMITVSG 320 Sequence name: /tmp/VAhvYFeatq/QNEM573uCo:CEA6 HUMAN
Sequence documentation:
Alignment of : T10888-PEA 1-P6 x CEA6 HCTI~fAN . .
yGO
Alignment segment 1/1:
Quality: 1393.00 Escore: 0 Matching length: 143 Total length: 143 Matching Percent Similarity: 99.30 Matching Percent Identity: 99.30 Total Percent Similarity: 99.30 Total Percent Identity: 99.30 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIillllllllllillllllllllllllllllllll IIIIIIIIIIIIIilllllllllllllllllllllllllllllillllll ?5 IIIIIIillllllllllllllllllllllllllllllllll Alignment of: T10888_PEA_1_P6 x CEA6 HUMAN ..
Alignment segment 1/l:
Quality: 101.00 Escore: 0 Matching length: 141 Total length: 183 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 77.05 Total Percent Identity: 77.05 Gaps: 1 Alignment: ' 101 IYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVY......... 141 141 ................................. 141 Expression of CEA6 I-IiIMAN
22~
Carcinoembryoniu antigen-related cell adhesion molecule 6 (T10888) transcripts which are detectable by artx~plicon as depicted in sequence name T10888 juncl l-17 in normal and cancerous Breast tissues Expression of CEA6-HUMAN Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts detectable by or according to juncl l-17, T10888junc1l-17 amplicon(s) and T108S8junc1l-17F and T10888junc1l-17R primers was measured by real time PCR. In parallel the expression of four housekeeping genes - Y13c~1~ (uent3anlc Accession wo.
BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM_000194;
amplicon - HPRT1-amplicon), and SDHA (GenBanl: Accession No. NM-004168;
amplicon -- .SDHA-amplicon), G6PD (GenBanl: Accession No. NM 000402; G6PD amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geomeMc mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 56-60, 63-67 Table 1, "Tissue samples in testing panel", above), to obtain a value of fold up-regulation for each sample relative to median of the normal PM
samples.
Figure 7 is a histogram showing over expression of the above-indicated CEA6-Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts in cancerous breast samples relative to the normal samples. Values represent the average of duplicate experiments.
Error bars indicate the minimal and maximal values obtained. The number and percentage of samples that exhibit at least 5 fold over-expression, out of the total number of samples tested, is indicated in the bottom.
As is evident from Figure 7, the expression of CEA6_HUl'~IAN Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts detectable by the above amplicon(s) in cancer samples was significantly higher than in the non-cancerous samples (Sample Nos. 56-60, 63-67 Table l, "Tissue samples in testing panel"). Notably an over-expression of at least 5 fold was found in 19 out of 28 adenocarcinoma samples.
Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of CEA6-HUMAN
Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts detectable by the above amplicon(s) in breast cancer samples versus the normal tissue samples was determined by T test as 2.00E-03.
Threshold of 5 fold overexpression was found to differentiate between cancer and normal samples with P value of S.44B-03 as checked by exact fisher test. The above values demonstrate statistical significance of the results.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: T10S8Sjuncll-17F
forward primer;
and T1088Sjuncll-17R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: T10S88junc1l-17.
T108SRjuncll-17F (SEQ ID N0:830) CCAGCAATCCACACAAGAGCT
T10888junc1l-17R (SEQ ID NO:S31) CAGGGTCTGGTCCAATCAGAG
T10888junc1l-17 (SEQ ID N0:832) CCAGCAATCCACACAAGAGCTCTTTATCCCCAACATCACTGTGAATAATAGC
GGATCCTATATGTGCCAAGCCCATAACTCAGCCACTGGCCTCAATAGGACCACAGT
CACGATGATCACAGTCTCTGATTGGACCAGACCCTG
Expression of CEA6 HUMAN
Carcinoembryonic antigen-related cell adhesion molecule 6T10SSR transcripts which are detectable by amplicon as depicted in sequence name T10S8Sjuncll-17 in different normal tissues.
Expression of CEA6 HUMAN Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts detectable by or according to T10SSS juncl l-17 amplicon(s) and T10SS8 juncl l-17F and T108S8 juncl l-17R was measured by real time PCR. In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No. NM-000981; RPL19 amplicon), TATA box (GenBank Accession No. NM-003194; TATA amplicon), UBC (GenBank Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM-004168; amplicon - SDHA-amplicon) was measured similarly. For each RT
sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantiy of each RT sample was then divided by the median of the quantities of the ovary samples (Sample Nos. 1 S-20, Table 2 "Tissue samples in normal panel" above), to obtain a value of relative expression of each sample relative to median of the ovary samples. Primers and amplicon are as above.
The results are presented in Figure 8, demonstrating the expression of CEA6-HUMAN
Carcinoembryonic antigen-related cell adhesion molecule 6 T10888 transcripts, which are detectable by amplicon as depicted in sequence name T10888junc1l-17, in different normal tissues.
Cluster T39971 features 4 transcripts) and 28 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table 1 - T)"CIITSCYIFtS Of 6111ei'ESl Transcript l~Tarne Sequence ID No.
T39971 TS ~1 Table ? - Segments of interest Se~nent Name ._ i Sequence ID Na.
T39971 node 0 22 T39971node1& 23 T39971node21 24 T39971node22 25 T39971node23 26 T39971node31 27 T39971node33 2S
T39971node7 29 T39971node1 30 T39971node10 31 T39971node11 32 T39971node12 33 T39971node15 34 T39971node16 35 T39971node17 36 T39971node26 37 T39971node27 38 T39971node28 39 T39971node29 40 T39971node3 41 T39971node30 42 T39971node34 43 T39971node35 44 T39971node36 45 T39971node4 46 T39971node5 47 T39971node8 48 T39971node9 49 Tahle 3 - P~~oteins of intef~est ProteinName , Sequence ID No.
T39971 P12 ~54 These sequences are variants of the known protein Vitronectin precursor (SwissProt accession identifier VTNC_HUMAN; known also according to the synonyms Serum spreading factor; S-protein; V75), SEQ ID NO: 50, referred to herein as the previously known protein.
Protein Vitronectin precursor is known or believed to have the following function(s):
Vitronectin is a cell adhesion and spreading factor found in serum and tissues. Vitronectin interacts with glycosaminoglycans and proteoglycans. Is recognized by certain members of the integrin family and serves as a cell-to-substrate adhesion molecule. Inhibitor of the me mbrane-damaging effect of the terminal cytolytic complement pathway. 'The sequence for protein Vitronectin precursor is given at the end of the application, as "Vitronectin precursor amino acid sequence" (SEQ ID NO:50). Hnown polymorphisms for this sequence are as shown in Table 4.
Table 4 - Antirto acid natttations for Known Protein SNP positions CommenE
j on amino acid sequence 122 A-> S. /FTId=VAR_012983.
268 R-> Q. /FTId=VAR_012984.
400 T -> M. /FTId=VAR_012985.
5p C _> N
225 S -> N
366 A -> T
Protein Vitronectin precursor localization is believed to be Extracellular.
The previously known protein also has the following indications) and/or potential therapeutic use(s): Cancer, melanoma. It has been investigated for clinical/therapeutic use in humans, for example as a target for an antibody or small molecule, and/or as a direct therapeutic; available information related to these investigations is as follows. Potential pharmaceutically related or therapeutically related activity or activities of the previously known protein are as follows: Alphavbeta3 integrin antagonist; Apoptosis agonist. A
therapeutic role for a protein represented by the cluster has been predicted. The cluster was assigned this field because there was information in the drug database or the public databases (e.g., described herein above) that this protein, or part thereof,, is used or can be used for a potential therapeutic indication: Anticancer.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: immune response; cell adhesion, which are annotations) related to Biological Process; protein binding; heparin binding, which are annotations) related to Molecular Function; and extracellular space, which are annotations) related to Cellular Component.
The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from <http:l/wwyv.expasy.ch/sprot/>; or Locuslink, available from <http://www.ncbi.nlm.nih.gov/projects/LocusLink/>.
Cluster T39971 can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term "number" in the right hand column of 2C1 the table and the numbers on the y axis of Figure 9 refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histograms in Figure 9 and Table 5. This cluster is overexpressed (at least at a minimum level) in the following pathological conditions: liver cancer, lung malignant tumors and pancreas carcinoma.
Table S - Nor°rttal tissue distribution Name of Tissue Number adrenal 60 ., -, $
bladder 0 Bone 0 Brain 9 Colon 0 epithelial 79 general ~9 Liver 2164 Lung 0 lymph nodes 0 breast 0 pancreas 0 prostate 0 Skin 0 uterus 0 Table 6 - P values acrd ratios for expr-essiort in cancerous tissue Name of TissueP1 p~ SPl R3 SP2 R4 ..
adrenal 6.9e-017.4e-012.0e-02 2.3 5.3e-021.8 bladder 5.4e-016.0e-015.6e-01 1.8 6.8e-011.5 Bone I 6.7e-011 1.0 7.0e-011.4 Brain B.Oe-O18.6e-013.0e-01 1.9 5.3e-011.2 Colon 4.2e-014.8e-017.0e-01 1.6 7.7e-011.4 epithelial 6.6e-015.7e-01l.Oe-O1 0.8 8.7e-010.6 general S.le-O13.8e-019.2e-08 1.6 8.3e-041.3 Liver 1 6.7e-012.3e-03 0.3 1 0.2 Lung 2.4e-0 9. l 1.7e-0 4.3 8. l 5.0 l e-02 1 e-03 lymph nodes 1 5.7e-011 1.0 5.8e-012.3 breast 1 6.7e-01I 1.0 8.2e-011.2 pancreas 9.5e-021.8e-011.5e-11 6.5 8.2e-094.6 prostate 7.3e-01 6.0e-016.7e-011.5 5.6e-011.7 Skin 1 4.4e-011 1.0 6.4e-011.6 uterus S.Oe-O 2.6e-0 1 1.1 B.Oe-O 1.4 AS noted above. 39971 ted cluster T features in 4 transcriotfsl. Table which were lis above. These transcripts) encode for proteins) which are variants) of protein Vitronectin precursor. A description of each variant protein according to the present invention is now provided.
Variant protein T39971 P6 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T39971 T5. An alignment is given to the known protein (Vitronectin precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T39971 P6 and VTNC_HUMAN:
l.An isolated chimeric polypeptide encoding for T39971 P6, comprising a ftrst amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAVJVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTV1'DDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKG corresponding to amino acids 1 - 276 of VTNC_HUMAN, which also corresponds to amino acids 1 - 276 of T39971 P6, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90°,~o and most preferably at least 95%
homologous to a polypeptide having the sequence TQGVVGD corresponding to amino acids 277 - 2S3 of T39971 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of T39971 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably -~ r, at least about 90% and most preferably at least about 95% homologous to the sequence TQGVVGD in T39971 P6.
The location of the variant protein was deterrtiined according to results from a number of different sof~vare programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T39971 P6 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 7, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T39971 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 7 - Amino acid nzactations 'SNP positions) on Alternative amino Prev7iously known amino acid acid(s). SNP?
sequence 122 A -> S Yes 145 G -> No 268 R -> Q yes 280 V -> A Yes 1 SO C -> No 180 C -> ~, No 192 Y -> No 209 A -> No ~ 11 T -> No 267 G -> No 267 G -> A No 268 ~ R -> No Variant protein T39971 P6 is encoded by the following transcript(s): T39971 T5, for which the sequences) is/are given at the end of the application. The coding portion of transcript T39971 TS is shown in bold; this coding portion starts at position 756 and ends at position 1604. The transcript also has the following SNPs as listed in Table 8 (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 T39971 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 8 - Nascleic acid SNPs SNP position'on nucleatxde,A.lternative nucleic'Preiriously kizown ' acid SNl'?
sequence 417 G -> C Yes 459 T -> C Yes 1387 C -> No 1406 -> A No 1406 -> G No 1555 G -> No 1555 G -> C No 1558 G -> No 1558 G -> A 1'es 1594 T -> C lres 1642 T -> C Yes 1770 C -> T Yes 529 G -> T Yes 1982 A -> G No 2007 G -> No 2029 T -> C No 2094 T -> C No 2117 C -> G No 2123 C -> T Yes 2152 C -> T YYes 2182 G -> T No ~ 1 SS A -> C No 2297 T -> C Yes 1119 G -> T Yes 2411 G -> No 2411 G -> T No 2487 T -> C Yes 1188 G -> No 1295 C -> No 1295 C -> G No 1324 -> T No 1331 C -> No 1381 C -> No Variant protein T39971 P9 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T39971 T10. An alignment is given to the known protein (Vitronectin precursor) at the end of the application. One or more alignments to one or mare previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows: .
Comparison report between T39971 P9 and VTNC_HCJMAN:
l.An isolated chimeric polypeptide encoding for T39971 P9, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCS~~QSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LILPEEEAPAPEVGASKPEGIDSRPETLHI'GRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQ1'-WR.FEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWE1'QFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRT corresponding to amino acids 1 - 325 of VTNC_HUMAN, which also corresponds to amino acids 1 - 325 of T39971_P9, and a second amino acid sequence being at least 90 % homologous to SGMAPRPSLAKKQRFRHIZNRILGY'RSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGA
NNYDDYRN>DWLVPATCEPIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGC
PAPGHL corresponding to amino acids 357 - 478 of VT'NC_HLJMAN, which also corresponds to amino acids 326 - 447 of T39971 P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _isolated chimeric polypeptide encoding for an edge portion of T39971 P9;
comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in Length, wherein at least two amino acids comprise TS, hawing a structure as follows: a sequence starting from any of amino acid numbers 325-x to 325; and ending at any of amino acid numbers 326 + ((n-2) - x), in which x varies from 0 to n-2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is belied to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T39971 P9 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T39971 P9 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 9 - Anzirzo acid mutatioras SNF position(s) on Artezr~h~ive am.zno Previously,known SNF2 amino acid acids) SeC~UEIICE , 122 A -> 5 Yes 145 G -> Na 268 R -> Q Yes 328 Ivl -> T No 350 5 -> p No 369 T-> Nl Yes 379 S -> T Na 380 N -> T No 180 C -> No 180 C -> W No 192 Y -> No 209 A -> No 211 T-> No 267 G -> No 267 G -> A Na 268 R -> No Variant protein T39971 P9 is encoded by tlae following transcript(s):
T39971_T10, for which the sequences) is/are given at the end of tlae application. The coding portion of transcript T39971 T10 is shown in bald; this coding portion starts at position 756 and ends at position 2096. The transcript also has the following SNPs as listed in Table 10 (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 T39971 P9 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 10 - Nzrcleic acid SNFs SNP position on nucleotideAlternative nucleic Previously Down SNP's sequence acid 417 G -> C Yes 459 T -> C Yes 1387 C -> No 1406 -> A No 1406 -> G No 1555 G -> No 1555 G -> C No 1558 G -> No 1558 G -> A Yes 1738 T -> C No 1803 T -> C No 1826 C -> G No 529 G -> T Yes 1832 C -> T Yes 1861 C -> T Yes 1891 G -> T No 1894 A -> C No 2006 T -> C Yes 2120 G -> No 2120 G -> T No 2196 T -> C Yes 1119 G -> T Yes 1188 G -> No C _> NO
1295 C -> G No 1324 -> T No 1331 C -> No 1381 C -> Na Variant protein T39971 PI 1 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T39971 T12. An alignment is given to the known protein (Vitronectin precursor) at the end of the application.
One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T39971 P 11 and VTNC_HUMAN:
l.An _isolated chimeric polypeptide encoding for T39971 F11, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHS1'SGRERVY'FFKGKQYWEYQFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTS corresponding to amino acids 1 - 326 of VTNC' -HUMAN, which also corresponds to amino acids 1 - 326 of T39971 PI 1, and a second amino acid sequence being at least 90 % homologous to DKI'YRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL corresponding to amino acids 442 - 478 of VT'NC_HUMAN, which also corresponds to amino acids 327 - 363 of T39971 PI 1, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated chimeric polypeptide encoding for an edge portion of T39971 PI
1, 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 SD, having a structure as follows: a sequence starting from any of amino acid numbers 326-x to 326; and ending at any of amino acid numbers 327 + ((n-2) - x), in which x varies from 0 to n-2.
Comparison report between T39971 P11 and Q9BSH7 (SEQ ID NO:S33):
l.An -isolated chimeric polypeptide encoding for T39971 P11, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLIL,ALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVI-iEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGAShPEG)DSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPIiLIRDVWGIEGPff~AAFTRINCQGItTYLFKGSQYWRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEE
CEGSSLSAVFEHFAIVfluIQRDSWEDIFELLFWGRTS corresponding to amino acids 1 - 326 of Q9BSH7, which also corresponds to amino acids 1 - 326 of T39971 P11, and a second amino acid sequence being at least 90 % homologous to DKI'YRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL corresponding to amino acids 442 - 473 of Q9BSH7; which also corresponds to amino acids 327 - 363 of T39971 P11, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated chimeric polypeptide encoding for an edge portion of T39971 P11, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 4C1 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise SD, having a structure as follows: a sequence starting from any of amino acid numbers 326-x to 326; and ending at any of amino acid numbers 327 + ((n-2) - x), in which x varies from 0 to n-2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither traps-membrane region prediction program predicts that this protein has a traps-membrane region..
Variant protein T39971 P11 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 11, (given according to their positions) on the amino acid ~38 sequence, with the alternative amino acids) listed; the last column indicates whutliPr the SNP is known or not; the presence of known SNPs in variant protein T39971 P 11 sequerac.n provides support for the deduced sequence of this variant protein according to the present i~Yv~antion).
Table II -Antino acid nu~tations SNP positions) on Alternative ammo acids)Pce~liously known amino acid SNP?
$eC~11~11Ce 122 A -> S Yes 145 G -> No 268 R -> Q 1'es 180 C -> No 180 C -> W No 192 Y -> No 209 A -> No 211 T-> No 267 G -> No 267 G -> A No 268 R -> No Variant protein T39971 P11 is encoded by the following transcript(s): T39971 T12, for which the sequences) is/are given at the end of the application. The coding portion of transcript T39971 T12 is shown in bold; this coding portion starts at position 756 and ends at position 1844. The transcript also has the following SNPs as listed in Table 12 (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 T39971 PI 1 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table I L - Nucleic acid SNPs SNP position on uucleutide Alternative nucleic acid Previously known SNP?
sequence 417 G -> C Yes 459 T -> C Yes 1387 C -> No 1406 -> A No 1406 -> G No 1555 G -> No 1555 G -> C No 1558 G -> No 1558 G -> A Yes 1754 T -> C Yes 1868 G -> No 1868 G -> T No 529 G -> T Yes 1944 T -> C Yes 1119 G -> T Yes 1188 G -> No 1295 C -> No 1295 C -> G No 1324 -> T No 1331 C -> No 1381 C -> No Variant protein T39971 P12 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T39971 T16. An alignment is given to the known protein (Vitronectin precursor) at the end of the application.
One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T39971 P12 and VTNC_HUMAN:
l.An _isolated chimeric polypeptide encoding for T39971 P12, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLIL,ALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFK corresponding to amino acids 1 - 223 of VTNC_HUMAN, which also corresponds to amino acids 1 -223 of T39971 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS°./o, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence VPGAVGQGRKHLGRV corresponding to amino acids 224 - 238 of T39971 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _isolated polypeptide encoding for a tail of T39971 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VPGAVGQGRKHLGRV in T39971 P12.
Comparison report between T39971 P12 and Q9BSH7:
l.An _isolated chimer-ic polypeptide encoding for T39971 P12, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSI'YQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGkPFDAFTDLKNGSLFAFR
GQ1'C1'ELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFIL corresponding to amino acids 1 - 223 of Q9BSH7, _which also corresponds to amino acids 1 - 223 ofT39971 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VPGAVGQGRKHLGRV corresponding to amino acids 238 of T39971 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of T39971 P12, comprising a polypepti~
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 VPGAVG(2GRhHLGRV in T39971 P12.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T39971 P12 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 13, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T39971 P 12 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 13 - Amino acid nzutcztions SNP positions) on Alternative amino Previously Down SNP?
amino acid acids) sequence 122 A -> S Yes 145 G -> No 180 C -> No 180 C -> W No 192 Y -> No 209 A -> No 211 T-> No Variant protein T39971 P12 is encoded by the following transcript(s): T39971 T16, for which the sequences) is/are given at the end of the application. The coding portion of transcript T39971 T16 is shown in bold; this coding portion starts at position 756 and ends at position 24~
1469. 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 T39971 P12 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 14 - Nucleic acid SNPs SNP posi~ian on nucleotideAlternative xtucleiei pxeviously known.
sequence acid S.I~I'?' 417 G -> C Yes 459 T -> C Yes 1387 C -> No 1406 -> A No 1406 -> G No 529 G -> T Yes 1119 G -> T Yes 1188 G -> No 1295 C -> No 1295 C -> G No 1324 -> T No 1331 C -> No 1381 C -> No As noted above, cluster T39971 features 28 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application.
These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster T39971 node 0 according to the present invention is supported by 76 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 15 below describes the starting and ending position of this segment on each transcript.
Table 1 S - Segment location on transcripts Transcript name ~~~eht starting positionSegment ending position ~~~
Segment cluster T39971 node_18 according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described.
Tlvs segment can be found in the following transcript(s): T39971 T 16. Table 16 below describes the starting and ending position of this segment on each transcript.
Table 16 - Segment location on transcripts Transcript name Segmeat starting Segment errdin~ xtasition position ~
Segment cluster T39971 node_21 according to the present invention is supported by 99 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5.
Table 17 below describes the starting and ending position of this segment on each transcript.
Table 17 - Segrnerrt location ora transcripts Transcript name Segment starting positionSegment ending position Segment cluster T39971 node 22 according to the present invention is supported by 7 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T5. Table 18 below describes the starting and ending position of this segment on each transcript.
Table 18 - Segment location on tr"ar2SCrIptS
Transcript nanne ~eg~~ht starkiing Segznirnt ending position pbsitaan Segment cluster T39971 node 23 according to the present invention is supported by 101 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12 and T'39971 T5.
Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on transcripts Transcripk naaoe Segmenk startingpositionSe~rn.ent ending positiari.
Segment cluster T39971 node 31 according to the present invention is supported by 94 libraries. The number of libraries was deterniined as previously described.
This segment can be found in the following transcript(s): T39971 T10 and T39971 T5. Table 20 below describes the starting and ending position of this segment on each transcript.
Table 20 - Segment location on transcripts Transcript name, Segment starting Segment ending position position Segment cluster T39971 node 33 according to the present invention is supported by 77 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5.
Table 21 below describes the starting and ending position of this segment on each transcript.
Table 21 - Segment location on transcripts Transc.~pt name Segment starting positaa~n~Se~tneait ending position Segment cluster T39971 node 7 according to the present invention is supported by 87 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 22 below describes the starting and ending position of this segment on each transcript.
Table ?2 - Segment location on transcripts T~s~pn name-- ~e~m~nt starting positionSegment ending position ~
T39971_T10 940 1162 T39971_T12 940 1162 T3997I_TS 940 1162 According to an optional embodiment of the present mventton, short segments reiatea to the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster T39971 node_1 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 23 below describes the starting and ending position of this segment on each transcript.
Table 33 - Segment location on transcripts Transcci~t name S~~le~nt stadrting Segment ending positiau position T39971 T10 811 ~ 819 T39971 TS 811 [819 Segment cluster T39971 node_10 according to the present invention is supported by 77 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s)T39971 TIO, T39971 T12, T39971 T16 and T39971 T5.
Table 24 below describes the starting and ending position of this segment on each transcript.
Table 24 - Segment location on transcripts Transcript name Segment starting position.Segment ending positicin Segment cluster T39971 node_I 1 according to the present invention is supported by 79 libraries. The number of libraries vvas determined as previously described.
This segment can be found in the following transcript(s)T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 25 below describes the starting and ending position of this segment on each transcript.
Table 25 - Segment location orr transcripts Transcript name Segment starting positionSegment ending positipn Segment cluster T39971 node_12 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 26 below describes the starting and ending position of this segment on each transcript.
Table '~ 6 - Segnze~at locatio~a on trarascriFts Transcript. name Segment startling i'Se~menr ending position' position Segment cluster T39971_node_15 according to the present invention is supported by 79 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 27 below describes the starting and ending position of this segment on each transcript.
Table 27 - Segment location on transcripts Trat~scnipt naic~e Segment,starting position~ Segment ending position , Segment cluster T39971 node_16 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 28 below describes the starting and ending position of this segment on each transcript.
Table 28 - Segment locr~:iort on tr-anscr-ipts Transcript naive Segment sfi.~rtiug Segment ending position ~ position T39971 T10 -~ 1317 1340 Segment cluster T39971 node_17 according to the present invention is supported by 86 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s)T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 29 below describes the starting and ending position of this segment on each transcript.
Table ?Q - Segment location on transcripts Transcript name Segix~ent starting Segment ending posihion position Segment cluster T39971 node 26 according to the present invention is supported by 85 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T5. Table 30 below describes the starting and ending position of this segment on each transcript.
Table 30 - Segment location on transcripts Transcript name Segment starting Segment. ending position position Segment cluster T39971 node 27 according to the present invention is supported by 90 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T5. Table 31 below describes the starting and ending position of this segment on each transcript.
Table 31 - Segment lOCatl011 Oit trarISCr"I~tS
Transcript name Segment stazting Segment ending position.
position Segment cluster T39971 node_28 according to the present invention can be found in the following transcript(s): T39971 T10 and T39971 T5. Table 32 below describes the starting and ending position of this segment on each transcript.
Table 32 - Segrnerzt location on transcripts Transcript name Segment starting Segment ending position ; position Segment cluster T39971 node 29 according to the present invention is supported by 99 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10 and T39971 T5. Table 33 below describes the starting and ending position of this segment on each transcript.
Table 33 - Segment lacatiort ort transcripts Trarrseript naive Segment starling ' Segment ending position position T39971 TS 2035 ~ 2129 Segment cluster T39971 node 3 according to the present invention is supported by 78 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 34 below describes the starting and ending position of this segment on each transcript.
Table 34 - Segrnent location on transcripts Transcript name ~egm.~t~t starting I segment ~n.~ling pasition~ --_.. . position T39971 TS 820 ~ 861 Segment cluster T39971 node_30 according to the present invention can be found in the following transcript(s): T39971 T10 and T39971 T~. Table 35 below describes the starting and ending position of this segment on each transcript.
Table 35 - Segrnerat location on transcripts Transcript name; Segment starting Segment ending position position II T39971 TS 2130 ~ 2137 Segment cluster T39971 node_34 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5. Table 36 below describes the starting and ending position of this segment on each transcript.
Table 36 - Segment location on transcripts Transcript name Se,~m.ent starting Segment ending position position Segment cluster T39971 node_35 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5. Table 37 below describes the starting and ending position of this segment on each transcript.
Table 37 - Segment locativrt an transcripts Trazascript name Segment starting positionSegment ~ncli~ig position -Segment cluster T39971 node 36 according to the present invention is supported by 51 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5.
Table 38 below describes the starting and ending position of this segment on each transcript.
Table 38 - Segtrzettt location on transcripts Tramscrlpl name -- Segment starting positionS~gu~ent ending position ~
Segment cluster T39971 node 4 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 39 below describes the starting and ending position of this segment on each transcript.
Table 39 - Segtnent location can transcripts Transcript name Segment starting Segment ending position position T39971 T10 862 881' ~i TS 862 881 Segment cluster T39971_node_5 according to the present invention is supported by 80 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 TIO, T39971 T12, T39971 T16 and T39971 T5.
Table 40 below describes the starting and ending position of this segment on each transcript.
Table 40 - Segment location orz transcripts Transcript name . Segment starting positionSegment ending position Segment cluster T39971 node_8 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971_T16 and T39971 T5.
Table 41 below describes the starting and ending position of this segment on each transcript.
Table 41 - Segment locatiota on transcripts Transcript name ' Segment starting position. Segruent eniding position IS
Segment cluster T39971 node 9 according to the present invention can be found in the following transcript(s)T39971 T10, T39971 T1~, T39971 T16 and T39971 T5. Table below describes the starting and ending-position of this segment on each transcript.
Table ~12 - Segment location on transcriFts Transu~ipt name ferment starting i segment ending position position ~
T39971 T10 1169 ~ 1188 T39971_TS 1169 1188 Variant protein alignment to the previously known protein:
Sequence name: /tmp/xkraCL20cZ/43L7YcPH7x.:VTNC HUMAN
Sequence documentation:
Alignment of: T39971-P6 x VTNC HUMAN ..
Alignment segment 1/1:
Quality: 2774.00 Escore: 4 Matching length: 278 Total length: ~78 Matching Percent Similarity: 99.64 Matching Percent Identity: 99.64 Total Percent Similarity: 99.64 Total Percent Identity: 99.64 ~54 Gaps: 0 Alignment:
$ 1 MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSC 50 151 AEEELCSGKPFDAFTDLhTIGSLFAFRGQYCYELDEKAVRPGYPTCLIRDVW 200 251 PDNVDAALALPAHSYSGRERVYFFKGTQ 2~8 '.'51 PDNVDAALALPAHSYSGRERVYFFKGKQ
Sequence name: /tmp/X4DeeuSlB4/yMubSRSFPs:VTNC HUNLAN
Sequence documentation:
Alignment of: T39971-P9 x VTNC HUMAN ..
Alignment segment 1/1:
Quality: 4430.00 Escore: 0 Matching length: 447 Total length: 478 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 93.51 Total Percent Identity: 93.51 Gaps: 1 Alignment:
. - . . .
101 DLQAQSKGNPEQTPVLKPEEEAPAPEV~~ASKPEGIDSRPETLHFGRPQPP 150 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllllllllll Illlllllllllllllllllllllllllllllllllllllllllllllll . - - - -301 VFEHFAMMQRDSWEDIFELLFWGRT...............-....----. 325 IIIIIIIIIIIIIIIIIIIIIIIII
326 ......SGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRAT 369 IIIIIIIIIIIIIIIIilllllllllllllllllllllllllll 370 WLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQSVFFFSGDh'YYRVNLR 419 ?5 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
420 TRRV'DTVDPPYPRSIAQYWLGCPAPGHL 447 IIIillllllllllllllllllllllll Sequence name: /tmp/jvplVtnxNy/wxNSeFVZZw:VTNC HUMAN
Sequence documentation:
Alignment of: T39971 P11 x VTNC HT1MAN ..
Alignment segment 1/1:
Quality: 3576.00 Escore : 0 Matching length: 363 Total length: 478 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 ?0 Total Percent Similarity: 75.94 Total Percent Identity: 75.94 Gaps: 1 Alignment:
51 CTDYTAECh.PQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTS 100 151 AEEELCSGKPFDAFTDLh'I~1GSLFAFRGQYCYELDEKAVRPGYPKLIRDVW 200 . . . - -301 VFEHFAMMQRDSWEDIFELLFWGRTS........................ 326 3~6 .................................................. 326 327 .........................................DKYYRVNLR 335 Sequence name: /tmp/jvplVtn.~cNy/wxNSeFVZZw:Q9BSH7 Sequence documentation:
Alignment of: T39971-P11 x Q9BSH7 ..
Alignment segment 1/1:
Quality: 3576.00 Encore: 0 Matching length: 363 Total length: 478 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 75.94 Total Percent Identity: 75.94 Gaps : 1 Alignment:
Iillllllllllllllllllllllllllllllllllllllllllllllll 151 AEEELCSGKPFDAFTDLkTTGSLFAFRGQYCYELDEKAVRPGYPKLIRDVW200 151 AEEELCSGKPFDAFTDLY~TGSLFAFRGQYCYELDEKAVRPGYPKLIRDVW200 201 GIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVI~DPDYPRNISDGFDGI250 301 VFEHFAMMQRDSWEDIFELLFWGRTS........................ 326 . . . . .
32G .................................................. 32G
327 .........................................DKYYRVNLR 335 ~61 Sequence name: /tmp/fgebv7ir4i/48bTBMziJO:VTNC HUMAN
Sequence documentation:
Alignment of: T39971_P12 x VTNC HUMAN ..
Alignment segment 1/1:
Quality: 2237.00 Escore: 0 Matching length: 223 Total length: 223 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
1 MAPLRPLLILALLAV~V'ALADQESCKGRCTEGFNVDKKCQCDELCSYYQSC 50 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
51 CTDYTAECKPQVTRGDVFTMPEDEYTVYDDGEEhT7NATVHEQVGGPSLTS 100 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
l0 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Iillllllllllllllllllllllllllllllllllllllllllllllll 151 AEEELCSGKPFDAFTDLkTIGSLFAFRGQYCYELDEKAVRPGYPKLIRDVW 200 2_01 GIEGPIDAAFTRTNCQGKTYLFK 223 IIIIIIIIIIIIIIIIIIIIIII
Sequence name: /tmp/fgebv7ir4i/48bTBMziJ0:Q9BSH7 Sequence documentation:
Alignment of: T39971-P12 x Q9BSH7 ..
Alignment segment 1/1:
Quality: 2237.00 Encore: 0 Matching length: 223 Total length: 223 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Illllllllllilllllillllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
?5 101 DLQAQSKGNPEQTPVLKPEEEAPAFEVGASKPEGIDSRPETLHPGRPQPP 150 Iillllllllllllllllllilllllllllllllllllllllllllilll 151 AEEELCSGKPFDAFTDLI~TGSLFAFRGQYCYELDEKAVRPGYPKLIRDVW 200 201 GIEGPIDAAFTRINCQGh'TYLFK 223 Expression of VTNC-HUMAN vitronectin (serum spreading factor, somatamedin B, complement S-pratein) T39971 transcripts, which are detectable by amplicon as depicted in sequence name T39971 junc23-33 in normal and cancerous breast tissues E,r-pression of VTNC-HUIvIAN vitronectin (serum spreading factor, somatomedin B, complement S-protein) transcripts detectable by or according to junc23-33, T39971 junc23-33 amplicon and T39971 junc23-33F and T39971 junc23-33R primers was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No.
BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM_000194;
amplicon - HPRT1-amplicon), SDHA (GenBank Accession No. NM-004168; amplican -SDHA-amplicon), and G6PD (GenBank Accession No. NM-000402; G6PD amplicon), was measured similarly. For each RT sample, the expression of the above amplican was normalised to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 56-60, 63-67, Table 1, above, "Tissue samples in testing panel"), to obtain a value of fold differetial expression for each sample relative to median of the normal PM
samples.
Figure 10 is a histogram showing dawn regulation of the above-indicated VTNC_HCJMAN vitranectin (serum spreading factor, samatornedin B, complement S-protein) transcripts in cancerous breast samples relative to the normal samples.
As is evident from Figure 10, the expression of VTNC-HUMAN vitronectin (serum spreading factor, somatomedin B, complement Sprotein) transcripts detectable by the above amplicon in cancer samples was significantly lower than in the non-cancerous samples (Sample Nos. 56-60, 63-67 Table 1, "Tissue samples in testing panel").
Primer pairs are also aptionally and preferably encompassed within the present invention; for example, for the above experiment, the follawing primer pair was used as a non-limiting illustrative example only of a suitable primer pair: T39971 junc23-33F forward primer;
and T39971 junc23-33R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: T39971 junc23-33.
T39971junc22-33F (SEQ )D N0:834): GGGGCAGAACCTCTGACAAG
T39971junc22-33R (SEQ )D N0:835): GGGCAGCCCAGCCAGTA
T39971junc22-33 amplicon (SEQ )D N0:836):
GGGGCAGAACCTCTGACAAGTACTACCGAGTCAATCTTCGCACACGGCGAGTGGAC
ACTGTGGACCCTCCCTACCCACGCTCCATCGCTCAGTACTGGCTGGGCTGCCC
Expression of VTNC_HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein), antisense to SARM1 (T23434), T39971 transcripts which are detectable by amplicon as depicted in sequence name T39971 junc23-33 in different normal tissues Expression of VTNC-HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein), transcripts detectable by or according to T39971ji~rtc23-33 amplicon(s) and T39971junc?3-33F and T39971ji~nc23-33R was measured by real time PCR. In parallel the expression of four housekeeping genes -RPL19 (GenBank Accession No.
NM_000981;
RPL19 amplicon), TATA box (GenBank Accession No. NM_003194; TATA amplicon), UBC
(GenBanl: Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA
(GenBank Accession No. NM-004168; amplicon - SDHA-amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT
sample was then divided by the median of the quantities of the breast samples (Sample Nos. 33-35, Table 2, "Tissue samples in normal panel" above), to obtain a value of relative expression of each sample relative to median of the breast samples. Primers and amplicon are as above.
The results are presented in Figure 11, demonstrating the expression of VTNC-HUMAN
vitronectin .(serum spreading factor, somatomedin B, complement S-protein), antisense to SARM1 (T23434), T39971 transcripts, which are detectable by amplicon as depicted in sequence name T39971junc23-33, in different normal tissues.
Expression of VTNC-HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein) T39971 transcripts which are detectable by amplicon as depicted in sequence name T39971 seg22 in normal and cancerous breast tissues Expression of VTNC-HUI~~IAN vitronectin (serum spreading factor, somatomedin B, complement S-protein) transcripts detectable by or accoramg to seg~~, 1 J77 /
1 SCg amplicon(s) and primers T39971 seg22F and T39971 seg22R was measured by real time PCR.
In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No.
BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM_000194;
amplicon - HPRT1-amplicon), SDHA (GenBank Accession No. NM 004165; amplicon -SDHA-amplicon), G6PD (GenBank Accession No. NM_000402; G6PD amplicon), was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post mortem (PM) samples (Sample Nos. 56-60, 63-67, Table 1: Tissue samples in testing panel, above), to obtain a value of fold differential expression for each sample relative to median of the normal PM samples.
In one experiment that was carried out no differential expression in the cancerous samples relative to the normal PM samples was observed. However, this may be due to a problem that is specific to this particular experiment.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a no,i-limiting illustrative example only of a suitable primer pair: T39971 seg22F
forward primer; an ad T39971 seg22R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: T39971 seg22.
Forward primer T39971 seg22F (SEQ ID NO :837):
GCAGTCTTGGATTCCTTTCACATT
Reverse primer T39971 seg22R (SEQ ID NO :838):
GAGGCTGTTGAAGTTAGGATCTCC
Amplicon T39971 seg22 (SEQ ID NO :839):
GCAGTCTTGGATTCCTTTCACATTTCACTGGGGACAGGCCTCAGCATGTGCCCACCC
CTGACCCCCACCTCATGCTGGGAGATCCTAACTTCAACAGCCTC
Cluster 221368 features 7 transcripts) and 34 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table I - Transcripts of interest Transcript Sequence TD No.
N'am~e 221368 PEA_1T10 55 221368 PEA_1TS 59 221365 PEA_1T6 60 Z21368_PEA_1 T9 ~ 61 Table ~ - Segments of iraterest _~ ~
Segment - I Seduence IU hTo:
Narne Z21368_PEA_1 0 62 node_ Z21368_PEA_1 15 63 node_ Z21368_PEA_1 19 64 node_ 221368PEA_1 2 65 node 221368PEA_1 21 66 node Z21368_PEA_1 33 67 node Z21368_PEA_1 36 68 node Z21368_PEA_1 37 69 node 221368PEA_1 39 74 node Z21368_PEA_1 4 71 node Z21368_PEA_1 41 72 node Z21368_PEA_1 43 73 node Z21368_PEA_1 _45 74 node Z21368_PEA_1 _53 75 node Z21368_PEA_1 56 76 node Z21368_PEA_1 58 77 node Z2136SPEA_1 66 78 node Z21368_PEA_1 _67 79 node Z21368_PEA_1 69 80 node Z2136SPEA_1 _11 81 node Z21368_PEA_1 _12 82 node 221368PEA _1 _16 83 node Z21368_PEA_1 _17 84 node Z21368_PEA_1 23 85 node 221368PEA _1 _24 86 node Z21368_PEA_1 node30 87 221368PEA1 node31 88 Z21368_PEA_1 node38 89 221368PEA_1 node47 90 221368PEA1 node49 91 Z21368_PEA_1 node51 92 221368PEA1 node61 93 Z2136SPEA1 node68 94 Z21368_PEA_1 node7 95 Table 3 - Prc~teifis of interest Protein ! Sequence ID No. _ . . .
Name ~
PEA
PEA
PEA
PEA
PEA
PEA
These sequences are variants of the known protein Extracellular sulfatase Sulf 1 precursor (SwissProt accession identifier SLJL1 IILTNIAN; known also according to the synonyms EC
3.1.6.-; HSulf 1), SEQ ID NCB: 96, referred to herein as the previously known protein.
Protein Extracellular sulfatase Sulf 1 precursor is known or believed to have the following function(s): Exhibits arylsulfatase activity and highly specific endoglucosamine-6-sulfatase activity. It can remove sulfate from the C-6 position of glucosamine within specific subregions of intact heparin. Diminishes HSPG (heparan sulfate proteoglycans) sulfation, inhibits signaling by heparin-dependent growth factors, diminishes proliferation, and facilitates apoptosis in response to exogenous stimulation. The sequence for protein Extracellular sulfatase Sulf 1 precursor is given at the end of the application, as "Extracellular sulfatase Sulf 1 precursor ?70 amino acid sequence" (SEQ ID N0:96). Known polymorphisms for this sequence are as shown in Table 4.
Table ~l - Arnifzo acid nt2ttatiorts for Knc~u~n Protein SM' pasition(s) Cvmme~nt on amine acid sequence 87 - 88 CC->AA: LOSS OF ARYLSULFATASE ACTMTY
AND LOSS OF ABILITY TO MODULATE APOPTOSIS.
49 L -> P
728 K -> R
Protein Extracellular sulfatase Sulf 1 precursor localization is believed to be Endoplasmic reticulum and Golgi stack; also localized on the cell surface (By similarity).
The. following GO Annotations) apply to the previously known protein. The following annotations) were found: apoptosis; metabolism; heparan sulfate proteoglycan metabolism, which are annotations) related to Biological Process; arylsulfatase;
hydrolase, which are annotations) related to Molecular Function; and extracellular space;
endoplasmic reticulum;
Golgi apparatus, which are annotations) 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/>.
Cluster 221368 can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term "number" in the right hand column of the table and the numbers on the taxis of Figure 12 refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histograms in Figure 12 and Table 5. This cluster is overexpressed (at least at a minimum level) in the following laathological conditions: epithelial malignant tumors, a mit;ture of malignant tumors from different tissues and pancreas carcinoma.
Table 5 - Nut~nal tissue distribution Name afTissue ' Nor bladder 123 Bone 557 Brain 34 Colon 94 epithelial 56 general 6S
head and neck 0 kidney 35 Lung 22 lymph nodes 0 breast 52 muscle 31 ovary 0 pancreas 0 prostate 44 skin 67 stomach 109 T cells 0 Thyroid 0 uterus 140 Table 6 - P values afid ratios for expression in caricer~zrs tissue Name ~f TissueP l 1'2 SP1 R3 SP2 R4 bladder 5.4e-01 6.6e-016.4e-011.0 S.Se-O10.7 bone ( 4.5e-01~ S.2e-O1~ 9.1e-01~ 0.4 ~ 1 ~ 0.3 brain 5.5e-017.3e-01l.Se-O1 1.5 S.Oe-O10.9 colon 1.4e-012.8e-01l.Oe-O1 2.0 3.0e-011.4 epithelial 1.1 1.5e-011.2e-07 2.1 l.Oe-O 1.1 e-03 l general 1.4e-055.3e-021.9e-06 1.6 6.7e-010.8 head and neck2.4e-027.1e-024.6e-01 2.5 7.5e-011.4 kidney 8.9e-0 9.0e-0 1 0.4 1 0.4 lung 3.5e-014.1e-017.2e-03 2.6 l.Oe-O11.6 lymph nodes 7.7e-023.1e-012.3e-02 8.5 1.9e-013.2 breast 4.0e-0 6.1 5.4e-02 2.3 3.0e-0 1.3 1 e-O 1 muscle 7.5e-023.5e-021 1.0 1.7e-011.7 ovary 3.8e-014.2e-012.2e-01 2.9 3.4e-012.2 pancreas 2.2e-026.9e-021.4e-08 6.5 1.4e-064.6 prostate 8.3e-018.9e-013.1e-01 1.4 5.2e-011.1 skin 6.1e-018.1e-016.0e-01 1.2 1 0.3 stomach 4.4e-02S.Oe-O1S.Oe-O1 0.8 9.7e-010.4 T cells S.Oe-O16.7e-013.3e-01 3.1 7.2e-011.4 Thyroid 3.6e-013.6e-011 1.1 1 1.1 uterus 3.5e-017.8e-014.6e-01 0.9 9.1e-010.5 As noted above, cluster 221368 features 7 transcript(s), which were listed in Table 1 above. These transcripts) encode for protein(sl which are variants) of protein Extracellular sulfatase Sulf 1 precursor. A description of each variant protein according to the present invention is now provided.
Variant protein Z21368_PEA_1 P2 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) Z21368_PEA_1 T5. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 221368 PEA_1 P2 and SUL1 HUMAN:
l.An -isolated chimeric polypeptide encoding for Z2136S_PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELGSL
QVIvvINKTRh11V1EHGGATFINAFVTTPMCCPSRSSMLTGKYVI~~THI~VYT1~NENCSSPSW
QAMFIEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYIPPGWREWLGLIKNSRFYNYTVCR
NGIKEKHGFDYAKDYFTDLITNESINYFKMSKRNIYPHRPVMMVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYNYYAPNMDKHWIMQYTGPMLPIHMEFTNIL,QRhRLQTLMSVDD
SVERLYNMLVETGELENTYIIYTADHGYHIGQFGLVKGKSMPYDFDLRVPFFIRGPSVEP
GSIVPQIVLNIDL,APTILDIAGLDTPPDVDGKSVLKLLDPEKPGNRFRTNKhAKIWRDTFL
VERGKFLRKKEESSKNIQQSNHI.PKYERVKELCQQARYQTACEQPGQKWQCIEDTSGK
LR1HKCKGPSDLLTVRQSTRNLYARGFHDKDKECSCRESGYRASRSQRIkSQRQFLRNQ
GTPKYKPRFV'I-ITRQTRSLSVEFEGEIYDINLEEEEELQVLQPRNIAKRHDEGHKGPRDLQ
ASSGGNRGRMLADSSNAVGPPTTVRVTHKCFILPNDSIHCERELIIQSARAWKDHKAYI
DKEIEALQDKIKNLREVRGHLKRRKPEECSCSKQSYYNkEKGVKKQEKLKSHLHPFKE
AAQEVDSKLQLFKENNRRRhKERKEKRRQRIiGEECSLPGLTCFTHDNNHWQTAPFWN
corresponding to amino acids 1 - 761 of SL1L1 HUMAN, which also corresponds to amino acids 1 - 761 of 221368 PEA_1_P2, 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°'o homologous to a polypeptide having the sequence PHKYSAHGRTRHFESATRTTNGAQKLSRI corresponding to amino acids 762 - 790 of 221368 PEA_1 P2, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated polypeptide encoding for a tail of 221365 PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence PHKYSAHGRTRHFESATRTTNGAQILLSRI _in 221368 PEA_1 P2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protr~~n localization is believed to be secreted because both signal-peptide prediction prograt~as predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein Z21368_PEA_1 P2 is encoded by the following transctipt(s):
Z2136S_PEA_1 T5, for which the sequences) is/are given at the end of the application. The coding portion of transcript 221368 PEA_1 TS is shown in bold; this coding portion starts at position 529 and ends at position 2898.
Variant protein 221368 PEA_1 PS according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) 221368 PEA_1 T9. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between Z2136fi PEA_1 PS and Q7Z2W2 (SEQ ID N0:840):
l.An -isolated chimeric polypeptide encoding for Z21368_PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIIL,VLTDDQDVEL
corresponding to amino acids 1 - 57 of Q7Z2W2, which also corresponds to amino acids 1 -.57 of Z21368_PEA_1 P5, second bridging amino acid sequence comprising A, and a third amino acid sequence being at least 90 % homologous to FFGKYLNEYNGSYIPPGWREWLGLIICNSRFYNYTVCRNGIKEKHGFDYAKDYF TDLITN
ESINYFKMSKR11~I~.'PHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNM
DKHWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDDSVERLYNMLVETGELENTYIIYT
ADHGYHIGQFGLVKGKSMPl'DFDIRVPFF1RGPSVEPGSIVPQIVLNIDLAPTILDIAGLDT
PPDVDGKSVLKLLDPEKPGNRFRTNKILAILIWRDTFLVERGKFL.RILKEESSKNIQQSNHL, PKYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRNLY
ARGFHDKDIkECSCRESGYRASRSQRKSQRQFLRNQGTPIiYKPRFVHTRQTRSLSVEFE
GEIYDINLEEEEELQVLQPRNIAKRI-ff~EGHhGPRDLQASSGGNRGRMLADSSNAVGPPT
TVRVTHKCFILPNDSIHCERELYQSARAWILDHKAYIDKEIEALQDKIKNLREVRGHLKR
RKPEECSCSKQS1'YNIkEKGVKKQEKLKSHLHPFKEAAQEVDSKLQLFKETfNRRRICILER
KEKRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNE
THNFLFCEFATGFLEYFDMNTDPYQLTNTVHT'VERGIL,NQLHVQLMELRSCQGYILQCN
PRPKNLDVGNKDGGSYDLHRGQLWDGWEG corresponding to amino acids 139 - 871 of Q7~2W2, which also corresponds to amino acids 59 - 791 of Z21368_PEA_1 P5, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
2.An _ _isolated polypeptide encoding for an edge portion of 221368 PEA_1 P5, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least three amino acids comprise LAF having a structure as follows (numbering according to X21368 PEA_1 PS): a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 59 +
((n-2) - x), in which x varies from 0 to n-2.
Comparison report between Z21368_PEA_1 PS and AAH12997 (SEQ ID NC~:841):
l.An _isolated chimeric polypeptide encoding for Z21368_PEA_1 P5, comprising a first amino acid sequence being at least 70°fo, optionally at least SO%, preferably at least 85%, more preferably at least 90% and most preferably at least 95°ro homologous to a polypeptide having the sequence MK1'SCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELAFF
GKYLNEYNGSYIPPGWREVvTL,GLIhNSRFYNYT'VCRNGIKEKHGFDYAKDYFTDLITNES
INYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNMDK
HWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDDSVERLYNMLVETGELENTYIIYTAD
HGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTILDIAGLDTPP
DVDGKSVLKLLDPEKPGNRFRTNKKA.IZIWRDTFLVERGKFLRKKEESSKNIQQSNHLP
KYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHILCKGPSDLLTVRQSTRNLYA
RGFHDKDKECSCRESGY~RASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEFEGE
IYDINLEEEEELQVLQPRNIAKRI-IDEGHKGPRDLQASSGGNRGRMLADSSNAVGPPTTV
RVTHKCFIL,PNDSIHCERELYQSARAWKDHKAYIDKEIEALQDKIkNLREVRGHLKRRK
PEECSCSKQSYYNKEKGVKKQEKLIiSHLHPFKEAAQEVDSKLQLFKENNR_RRICILERKE
KRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNETH
NFLFCEFATGFLEYFDMNTDPYQLTNTVI-ITVERGIL,NQLHVQLME corresponding to amino acids 1 - 751 of Z2136S PEA_1 P5, and a second amino acid sequence being at least 90 homologous to LRSCQGY~KQCNPRPKNLDVGNKDGGSYDLHRGQLWDGWEG
corresponding to amino acids 1 - 40 of AAH12997, which also corresponds to amino acids 752 -791 of 221368 PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated polypeptide encoding for a head of Z2136g PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence MKYSCCALVLAVL,GTELLGSLCSTVRSPRFRGRIQQERKNIRPNILLVLTDDQDVELAFF
GhYLNEYNGSYIPPGWREWLGLIKNSRFYNI'TVCRNGIkEKHGFDYAhDYFTDLITNES
INYFKMSK12MY~PHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNMDK
HWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDDSVERLYNMLVETGELENTYIIYTAD
HGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTIL,DIAGLDTPP
DVDGKSVLKLLDPEKPGNRFRTNKhAKIWRDTFLVERGKFLRKKEESSKNIQQSNHLP
KYERVKELCQQARYQTACEQPGQKWQCIEDTSGkLFIHKCKGPSDLLTVRQSTRNLYA
RGFHDKDKECSCRESGYRASRSQRKSQRQFLRNQGTPKYILPRFVHTRQTRSLSVEFEGE
IYDINLEEEEELQVLQPRNIAKRHDEGHKGPRDLQASSGGNRGRMLADSSNAVGPPTTV
RVTHKCFILPNDSIHCERELYQSARAWKDIiKAYIDKEIEALQDKIkNLREVRGHLKI~
PEECSCSKQSYYNKEKGVKKQEILLKSHLHPFKEAAQEVDSKLQLFKENNRRRhhERKE
IiRRQRKGEECSLPGLTCFTHDNNHWQTAPFVVNL,GSFCACTSSNNNTYWCLRTVNETH
NFLFCEFATGFLEYFDMNTDPYQLTNTVHTVERGILNQLHVQLME of Z2136g PEA_1 P5.
Comparison report between Z2136g PEA_1 PS and SUL1 HUMAN:
l.An - .isolated chimeric polypeptide encoding for Z2136s PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVEL
corresponding to amino acids 1 - 57 of SUL1 I-ICTMAN, which also corresponds to amino acids 1 - 57 of 22136& PEA_1 P5, and a second amino acid sequence being at least 90 homologous to AFFGIkYLNEYNGSYL"~fr~;iWREWLGLIKNSRFYNYTVCRNGIIiEKHGFDYAKDYFTDLIT
NESINYFKMSKRM1'P i~ I~PVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPN
MDKHWIMQYTGPMLF'1~~MEFTNIL,QRKRLQTLMSVDDSVERLYNN>ZVETGELENTYII
YTADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVL,NII7LAPTILDIAGL
DTPPDVDGKSVLKLLDPEKPGNRFRTNKKAIKiWRDTFLVERGKFLRKKEESSKNIQQSN
HLPKYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRN
LYARGFHI)KDKECSCRESGI'RASRSQRKSQRQFLRNQGTPKYhPRFVHTRQTRSLSVE
PTTVRVTHKCFILPNDSIHCERELYQSARAWKDHKAY)DKEIEALQDKIKNLREVRGHL
KRRKPEECSCSKQSYYNKEKGVKKQEKLKSHLHPFILEA,AQEVDSKLQLFKErfNRRRh KERKEKRRQRKGEECSLPGLTCFTHI~NNHVvIQTAPFWNLGSFCACTSSNNNTYWCLRT
VNETHNFLFCEFATGFLEYFDMNTDPYQLTNTVHTVERGILNQLHVQLMELRSCQGYK
QCNPRPKNLDVGNKDGGSYDLHRGQLWDGWEG corresponding to amino acids 138 - 871 of SULI -HUMAN, which also corresponds to amino acids 58 - 791 of 221368_PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated chimeric polypeptide encoding for an edge portion of Z21368_PEA_1 P5, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise LA, having a structure as follows: a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 58 + ((n-2) - x), in which x varies from 0 to n-2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein Z21368_PEA_1 PS is encoded by the following transcript(s):
Z21368_PEA_1 T9, for which the sequences) is/are given at the end of the application. The 27s coding portion of transcript Z21368_PEA_1 T9 is shown in bold; this coding portion starts at position 556 and ends at position 2928.
Variant protein Z21368_PEA_1 P15 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) Z21368_PEA_1 T23. An alignment is given to the lmown protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or mare alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 221368 PEA_1 P15 and SUL1 HUMAN:
l.An -isolated chimeric polypeptide encoding for Z21368_PEA_1 P15, comprising a first amino acid sequence being at least 90 °ro homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELGSL
QVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYVI-INHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYIPPGWREWLGLIKNSRFYN1'TVCR
NGIKEKHGFDYYAILDYFTDLITNES1NYYFKMShRMI'PHRPVMMVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYN1'APNNLDKHWIMQYTGPMLPIHMEFTNILQRhRLQTLMSVDD
SVERLYNMLVETGELENTYII1'TADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEP
GSIVPQIVLNIDLAPTILDIAGLDTPPDVDGKSVLILLLDPEKPGNRFRTNKKAKIWRDTFL
VERG -corresponding to amino acids 1 - 416 of SUL1 HUMAN, which also corresponds to amino acids 1 - 416 of Z21368_PEA_1 P15.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neitl~r traps-membrane region prediction program predicts that this protein has a traps-membrane region.
Variant protein Z2136S PEA_1 P15 is encoded by the following transcript(s):
Z21368_PEA_1 T23, for which the sequences) is/are given at the end of the application. The coding portion of transcript Z2136S PEA_1 T23 is shown in bold; this coding portion, starts at position 691 and ends at position 1938.
Variant protein X21365 PEA_1 P16 according to the present invention has an amino acid sequence as given at tl~ end of the application; it is encoded by transcripts) 221368 PEA_I T24. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 221368_PEA_1 P16 and SUL.1 FiLTMAN:
l.An _ -isolated chimeric polypeptide encoding for 221368 PEA_1 P16, comprising a first amino acid sequence being at least 90 % homologous to MhYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERhNIRPNIILVLTDDQDVELGSL
QVMNIkTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYVHNHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRTAFFGRYLNEYNGSYIPPGWREWLGLIILNSRFYNYTVCR
NGIKEICHGFDYAKDYFTDLITNESINYFIILMSKRM1'PHRPVMMVISHAAPHGPEDSAPQ
FSItLYPNASQHITPSYNYAPN1\~kHWIMQYTGPMLPIHMEFTNILQRItRLQTLMSVDD
SVERLYMMLVETGELENTYIIYTADHGYHIGQFGLVKGhSMPYDFDIRVPFFIRGPSVEP
GSIVPQIVLNIDLAPTILDIAGLDTPPDVDGItSVLICLLDPEIH'GNR corresponding to amino acids 1 - 397 of SUL1 HUMAN, which also corresponds to amino acids 1 - 397 of X21368 PEA_1 P16, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least RS%, more preferably at least 9p% and most preferably at least 95%
homologous to a polypeptide having the sequence CVIVPPLSQPQIH corresponding to amino acids 398 - 410 of Z21368_PEA_1 P16, wherein said first and second amino acid sequences are contiguous and in a segue ntial order.
2.An _isolated polypeptide encoding for a tail of 221368_PEA_1 P16, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence CVIVPPLSQPQIH in Z21368_PEA_1 P16.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignaIP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neitl~r trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein Z21368_PEA_1 P16 is encoded by the following transcript(s):
221368 PEA_1 T24, for which the sequences) is/are given at the end of the application. The coding portion of transcript Z21368_PEA_1 T24 is shown in bold; this coding portion starts at position 691 and ends at position 1920.
Variant protein Z21368_PEA_1 P22 according to the present invention has an amino acid sequence as given at tl~ end of the application; it is encoded by transcripts) 221368 PEA_1 T10. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between Z21368_PEA_1_P22 and SUL1 HUMAN:
l.An _isolated chimeric polypeptide encoding for Z21368_PEA_1 P22, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIR.PNIILVLTDDQDVELGSL
QVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYVHNHN~rY'TNNENCSSPSW
QAI~gIEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYYIPPGWREWLGLIKNSRFY'NYTVCR
NGIKEkHGFDYAK _corresponding to amino acids 1 - 188 of SUL1 HUMAN, which also corresponds to amino acids 1 - 188 of Z21368_PEA_1 F22, 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 AR~~'DGDQPRCAPRPRGLSPTVF corresponding to amino acids 189 - 210 of Z21368_PEA_1 P22, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of Z21368_PEA_1 P22, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, mare preferably at least about 90% and most preferably at least about 95%
homologous to the sequence ARYDGDQPRCAPRPRGLSPTVF in Z21368_PEA_1 P22.
The location of the variant protein was determined according to results from a number of different software programs and ar~lyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein 221368_PEA_1 P22 is encoded by the following transcript(s):
Z21368_PEA_1 T10, for which the sequences) is/are given at the end of the application. The coding portion of transcript Z21368_PEA_1 T10 is shown in bold; this coding portion starts at position 691 and ends at position 1320.
Variant protein Z21368_PEA_1 P23 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) 221368 PEA_1 T11. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as fo Mows:
Comparison report between Z21368_PEA_1 P23 and Q7Z2W2:
l.An _ _isolated chimeric polypeptide encoding for 221368 PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologous to IvIKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERILNIRPNIIL,VLTDDQDVELGSL
QAMI-iEPRTFAVYLNNTGYRT corresponding to amino acids 1 - 137 of Q7~2W2, which also corresponds to amino acids 1 - 137 of Z21368_PEA_1 P23, 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% hmnologous to a polypeptide having the sequence GLLHRLNH corresponding to amino acids 138 - 145 of Z21368_PEA_1 P23, wherein said first and second amino acid sequences are° contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of Z21368_PEA_1 P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°.~0, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence GLLHRLNH in Z21368_PEA_1 P23.
Comparison report between Z2136S PEA_1 P23 and SUL1 I-IIJIvIAN:
l.An isolated chimeric polypeptide encoding for Z21368_PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELGSL
QANIIIEPRTFAVYLNNTGYRT corresponding to amino acids 1 - 137 of SLTLI HUMAN, which also corresponds to amino acids 1 - 137 of Z21368_PEA_1 P23, and a second amino acid sequence being at least 70%, optionally at least 80°~0, preferably at least 85%, more preferably at least 90% and most preferably at least 95°~o homologous to a polypeptide having the sequence GLLHRLNH corresponding to amino acids 138 - 145 of Z31368_PEA_1 P23, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of Z31368_PEA_1 P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence GLLHRLNH in Z21368_PEA_1 P23.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignaIP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein 221368 PEA_1 P23 is encoded by the following transcript(s):
Z21368_PEA_1 T11, for which the sequences) is/are given at the end of the application. The coding portion of transcript 221368 PEA_1 T11 is shown in bold; this coding portion starts at position 691 and ends at position 1125.
As noted above, cluster 221368 features 34 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application.
These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster 221368 PEA_1 node 0 according to the present invention is supported by 8 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PE<A_1 T9. Table 7 below describes the starting and ending position of this segment on each transcript.
Table 7 - Segment location on transcripts Tz~anscript name Segment starting positionSegment endizy' position Segment cluster 221368 PEA_1 node_15 according to the present invention is supported by 26 libraries. The number of libraries was determined as previously described. This segment - -can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23, _ - -Z21368_PEA_1 T24, Z21368_PEA_1 T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 8 below describes the starting and ending position of this segment on each transcript.
Table 8 - Segrnerzt location on transcripts Transcript name Segment starting Segment ending position position Z21368_PEA_1 T10 631 80~
221368 PEA_l_T11 631 807 221368FEA_1 T23 631 807 Z21368_PEA_1 T24 631 807 221368PEA_1 TS 469 645 Z21368_PEA_1 T6 469 645 Z2136bPEA_1 T9 496 672 Segment cluster Z21368_PEA_1 node_19 according to the present invention is supported by 24 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z2136S PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_I_T23, Z21368_PEA_1 T24, _Z21368_PEA_1 TS and Z21368_PEA_1 T6.
Table 9 below describes the starting and ending position of this segment on each transcript.
Table 9 - Segrnertt location on transcripts Transcript name S~~rnent starting Segment ending positiau position 221368 PEA_1 T10 863 1102 Z21368_PEA_1 T23 863 1102 Z21368_PEA_1 T24 863 1102 221368 PEA_1 T5 701 940 Z21368_PEA_1 T6 ~ 701 ~ 940 Segment cluster Z21368_PEA_1 node 2 according to the present invention is supported byy 15 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23, _ -Z21368_PEA_1 T24, Z21368_PEA_1 TS and Z21368_PEA_1 T6.
Table 10 below describes the starting and ending position of this segment on each transcript.
Table 10 - Seg~rierat location on transcripts Transcriptnarile I Segment starting position I Segment ending position .'~'35 Z21368_PEA_1 T10 1 300 Z21368_PEA_1 T11 1 300 Z21368_PEA_1 T23 1 300 Z21368_PEA_1 T24 1 300 221365PEA_1 T5 1 300 221368PEA_1 T6 1 300 Segment cluster 221368 PEA_1 node_21 according to the present invention is supported by 37 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, Z21368_PEA_1 T23, 221368 PEA_1 724221368 PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 11 below describes the starting and ending position of this segment on each transcript.
Tale II - SegrrZent location orr transcripts Transcript ~eg~~nC starting pasitionSe~merit ending ~rosition r~~ne .
Z21368_PEA_1_T10 1103 1254 Z21368_PEA_1 1103 1254 221368PEA_I 1103 1254 Z21368_PEA_1 941 1092 Z21368_PEA_1 728 879 Segment cluster Z21368_PEA_1 node_33 according to the present invention is supported by 45 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 710, Z21368_PEA_1 711, Z21368_PEA_1 723, _ -Z21368_PEA_1 724, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 12 below describes the starting and ending position of this segment on each transcript.
Table I? - Segment location on transcripts 28b Transcript Segment starting positionSegment ending positian name Z21368_PEA_1 1502 1677 Z21368_PEA_1 1424 1599 Z21368_PEA_1 1576 1751 Z21368_PEA_1 1414 1589 TS
Z21368_PEA_1 1414 1589 Z21368_PEA_1 1201 1376 Segment cluster 221368 PEA_1 nude 36 according to the present invention is supported by 44 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s)Z2136S PEA_1 T10, 221368 PEA_1 T1 l, Z21368_PEA_1 T23, Z21368_PEA_1 T24, Z21368_PEA_1 T5, Z2136S PEA_1 T6 and Z21368_PEA_1 T9. Table 13 below describes the starting and ending position of this segment an each transcript.
Table 13 - Segment loeatiort ort tnanscr~ipts Transcript rJame Se~ent starting pasitionSegnnent ending pvsitiari ' Z21368_PEA_1 T10 167S 1806 221368 PEA_1 T11 1600 1728 Z21368_PEA_1 T23 1752 1880 Z21368_PEA_1 T24 1752 1880 Z21368_PEA_1 TS 1590 1718 Z21368_PEA_1 T6 1590 1718 Z21368_PEA_1 T9 1377 1505 Segment cluster 221368 PEA_1 node 37 according to the present invention is supported by 3 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T24. Table 14 below describes the starting and ending positian of this segment on each transcript.
Table 1 ~ - Segrnerzt location on transcripts Transcript name Segment starting j Se~~nt ending position posi~ian Segment cluster 221368 PEA_1 node 39 according to the present invention is supported by 5 libraries. The number of libraries was determined as previausly described. This segment can be found in the following transcript(s): Z21368_PEA_1 T23 and Z21368_PEA_1 T24.
Table 15 below describes the starting and ending position of this segment on each transcript.
Table I S - Seg»tent location ora transcripts Transcript zaarne Segment starting :Segment ending~~ositian ~aosition 221368 PEA_1 T23 1938 ~ 2790 Z21368_PEA_1 T24 2217 3069 Segment cluster 221368 PEA_1 node 4 according to the present invention is supported by 13 libraries. The number of libraries was determined as previously described. This segment can be found in the following _ -transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23 and Z21368_PEA_1 T24. Table 16 below describes the starting and ending position of this segment on each transcript.
Table 16 - Segnte»t location o» transcripts Transcript name Segment starting pasitionSegment ending position 221368 PEA_1 T10 301 462 Z21368_PEA_1 T11 301 462 Z21368_PEA_1 T23 301 462 221368 PEA_1 T24 301 462 zss Segment cluster Z21368_PEA_1 node 41 according to the present invention is supported by 49 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s)Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z2136S_PEA_1 T5; Z2136S_PEA_1 T6 and Z21368_PEA_1 T9. Table 17 below describes the starting and ending position of this segment on each transcript.
Table 1 ? - Segrraent locatiora can transcripts Tr~nscz~pt na~te , Segment starting position~ Segment endi~a;g position ~' Z21368_PEA_1_T10 1864 1993 221368 PEA_1 T11 1786 1915 Z21368_PEA_1 TS 1776 1905 Z21368_PEA_1 T6 1776 1905 Z21368_PEA_1 T9 1563 1692 Segment cluster Z2136S_PEA_1 node_43 according to the present invention is supported by 52 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T5; Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 18 below describes the starting and ending position of this segment on each transcript.
Table 18 - Segment location ora transcripts Transcript name Segment starting position:'~ Segment ending position Z21368_PEA_1 T10 1994 2210 Z21368_PEA_1 T1I 1916 2132 Z21368_PEA_1 T5 1906 2122 Z21368_PEA_1 T6 1901; 2122 221368 PEA_1 T9 ~ 1693 ~ 1909 Segment cluster Z2136S_PEA_1 node 45 according to the present invention is supported by 64 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z2136S_PEA_1 T10, Z2136S_PEA_1 T1 l, Z21368_PEA_1 T5, Z2136S_PEA_1 T6 and Z21368_PEA_1 T9. Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on transcripts Transt~ript name Segment starring positian~. ~egmeat ending pasitiaxr ~
221365 PEA_1 T10 2211 ~ 2466 Z21368_PEA_1 T11 2133 2358 221368 PEA_1 TS 2123 2375 Z21368_PEA_1 T6 2123 2375 Z2136S PEA_1 T9 1910 2165 Segment cluster 221368 PEA_1 node_53 according to the present invention is supported by 60 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, Z21368_PEA_1 T1 l, Z2136S _ _ -PEA_1 T5, 221368 PEA_1 T6 and Z21363_PEA_1 T9. Table 20 below describes the starting and ending position of this segment on each transcript.
Table ?0 - Segment location orr transcripts "firanscript xxame Segment starting positionI Segment end.ingpasition Z2136S_PEA_1 T10 2725 2900 Z2136S_PEA_1 T11 2647 2822 Z21368_PEA_1 TS X637 2812 Z21368_PEA_1 T6 2637 '812 Z21368_PEA_1 T9 2424 2599 ~90 Segment cluster Z31368_PEA_1 node 56 according to tho present invention is supported by 50 libraries. The number of libraries was determined as previoaasly described. This segment can be found in the following transcript(s): Z21368_PEA_1 Tlf), Z,21368_PEA_1 T11 and 221368 PEA_1 T9. Table 21 below describes the starting and ending position of this segment on each transcript.
Table 21 - Segment location on transcripts Transcript name Se~nxent starting Segment ending position position ~_.
221368 PEA_1_T10 2901 3043 221368 PEA_1 T11 2823 2965 Segment cluster 221368 PEA_1 node 58 according to the present invention is supported by 71 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s)221368 PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T5, _Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 22 below describes the starting and ending position of this segment on each transcript.
Table 22 - Segment location ova transcripts Ticanscri~t name Segment startiyg posifionSegment ending positiota Z2136S_PEA_1 T10 3044 3167 221368 PEA l Tll 2966 3089 Z21368_PEA_1 T5 2813 2936 Segment cluster 221368 PEA_1 node 66 according to the present invention is supported by 142 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, 221368 PEA_1_T5, Z21368_PEA_1 T6 and 221365 PEA_1 T9. Table 23 below describes the starting and ending position of this segment on each transcript.
Table 23 - Segnterrt location on transcripts Transcript name Segment starting position; segment endilzg pasitzcizt Segment cluster Z21368_PEA_1 node 67 according to the present invention is supported by 181 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_I T10, Z21368_PEA_1 T11, Z21368_PEA_1 T5, _Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 24 below describes the starting and ending position of this segment on each transcript.
Table 24 - SegnterZt location on transcripts Transcript nave se~rr~ent starting Segment ending position posiCian Z21368_PEA_1 T9 3489 4073 Segment cluster Z21368_PEA_1 node 69 according to the present invention is supported by 150 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, 221368-PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1_T9. Table 25 below describes the starting and ending position of this segment on each transcript.
Table 25 - Segment location ora transcripts Transept narae. Segment starting position' Segment ending posirion Z21368_PEA_1 T10 4428 4755 Z21368_PEA_1 T11 4350 4677 Z21368_PEA_1 TS 4197 5384 Z21368_PEA_1 T6 4197 4524 According to an optional embodiment or the present mvennon, snort segments re~a~CU w the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster 221368 PEA_1 node_11 according to the present invention is supported by 26 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1_T11, 221368 PEA_1 T23; Z21368_PEA_1 T24, Z2136fi PEA_1 T5, Z21368_PEA_1 T6 and 221368 PEA 1 T9. Table 26 below describes the starting and ending position of this segment on each transcript.
Table 26 - Segnaent location on transcripts Transcript ~ segment statrting Segn~eint ~z~ding naive pasition position Z21368_PEA_1 558 602 Z21368_PEA_1 558 602 221368PEA_1 396 440 TS
Z21368_PEA_1 396 440 Z21368_PEA_1 423 467 Segment cluster Z21368_PEA_1 node_12 according to the present invention is supported by 23 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA 1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T23, Z21368_PEA_1 T24, 221368 PEA_1 T5, Z2136b PEA_1 T6 and Z2136S PEA_1 T9. Table 27 below describes the starting and ending position of this segment on each transcript.
Table ?7 - Segment location on transcripts Transcript' Segment starting ppsitioinSe~rnent ending po~itic>n n~rne ~
TS
Segment cluster Z21368_PEA_1 node_16 according to the present invention can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23, _ _Z21368_PEA_1 T24, Z21368_PEA_1 T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 28 below describes the starting and ending position of this segment on each transcript.
Table ?8 - Segment location oj7 transcripts Transcript name Segment starting positiun~Segment ending position 221368 PEA_1TS 646 660 Z21368_PEA_1T6 646 660 Z2136S_PEA_1T9 673 687 Segment cluster Z21368_PEA_1 node_17 according to the present invention is supported by 19 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s)Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23, _ - -Z2136S_PEA_1 T24, Z2136S PEA_l T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 29 below describes the starting and ending position of this segment on each transcript.
Table 29 - Segment location on transcripts Trattscri~t carne; augment startirig ~ ~e,~,ment ending pasxtion positioca.
Z21368_PEA_1 T10 823 862 Z2136S PEA_1 T11 823 862 Z21368_PEA_1 T23 823 862 Z21368_PEA_1 T24 823 S62 Z21368_PEA_1 TS 661 700 Z2136fi PEA_1 T6 661 700 Segment cluster Z21368_PEA_1 node 23 according to the present invention is supported by 36 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T11, Z21368_PEA_1 T23, Z21368_PEA_1 T24Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 30 below describes the starting and ending position of this segment on each transcript.
Table 30 - SegnTent locatio~a on transcripts Transcript naive. Segment starting position ' Segment ending pasition Z21368_PEA_1 T9 880 ~ 953 Segment cluster Z21368_PEA_1 node 24 according to the present invention is supported by 36 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T23, Z21368_PEA_1 T24, Z21368_PEA_1_T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 31 below describes the starting and ending position of this segment on each transcript.
Table 31 - Segment locatiora an trarrscr-ipts Transcript tame Se~ent starting positionSegment ~~,ding position -Z21368_PEA_1 T9 ) 1049 Segment clusterZ21368_PEA_1 node_30 according to the present invention is supported by 39 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 T11, 221368 PEA_1 T23, Z2136b PEA_1 T24, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 32 below describes the starting and ending position of this segment on each transcript.
Table 32 - Segnrerrt location on transcripts Transcript name Segment starting segment ending position positioh Z21368_PEA_1 T11 1273 1331 Z21368_PEA_I T9 1050 1108 Segment cluster 221368 PEA_I node_31 according to the present invention is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 TI
I, 221368 PEA_I T23,, _ _Z21368_PEA_1 T24, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 33 below describes the starting and ending position of this segment on each transcript.
Table 33 - Segrnerrt location on trarrseripts Transcript Se,grr~eint starEing'pasitionSe~naent ending posatian name ~
-TIO ~~
221368PEA_1 1322 1413 Z2136SPEA_1 1323 1413 Z21368_PEA_1 1109 1200 Segment cluster Z21368_PEA_1 node_38 according to the present invention is supported by 45 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 Tl 1, Z21368_PEA_1 T23, Z21368_PEA_1 T24, Z21368_PEA_1_T5, Z21368_PEA_1_T6 and Z21368_PEA_1 T9. Table 34 below describes the starting and ending position of this segment on each transcript.
Table 34 - Segme~zt locatiota orZ transcripts Transcript nanne Segment starting positionSegment ending, position Z21368_PEA_1 T10 1807 1863 Z21368_PEA_1 T24 2160 2216 Z21368_PEA_1 T5 1719 1775 Z21368_PEA_1 T9 1506 1562 Segment cluster 221368 PEA_1 node 47 according to the present invention is supported by 61 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z2136S PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 35 below describes the starting and ending position of this segment on each transcript.
Table 35 - Segment locatio~i an trarascr-ipts Transcript name Segment starting positionSegment ending position Z21368_PEA_1 T10 2467 2563 Z21368_PEA_1 T11 2389 2485 Z21368_PEA 1 T5 2379 2475 221368 PEA_1 T6 2379 2475 Z21368_PEA_1 T9 ~ 2166 2262 Segment cluster Z21368_PEA_1 node 49 according to the present invention is supported by 57 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, 221368 PEA_1 T11, 221368 PEA_1 T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 36 below describes the starting and ending position of this segment on each transcript.
Table 36 - Segment location orz tr~anscniFts Transcript uam,e ~~gm~nl farting position' Segment ending ~osit~ozi Z21368_PEA_1 T10 2564 2658 221368 PEA_1 T11 2486 2580 Z21368_PEA_1 TS 2476 2570 221368 PEA_1 T6 2476 2570 221368 PEA_1 T9 2263 2357 Segment cluster 221368 PEA_1 node 51 according to the present invention is supported by 46 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 T11, 221368 PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 37 below describes the starting and ending position of this segment on each transcript.
Table 37 - Segrnerrt lc~catiara era tr-arrscriyts Transcript Segment starting : Segment ending posifiion name position Z2136S_PEA_1 2659 2724 Z21368_PEA_1 2581 2646 Z21368_PEA_1 2571 2636 TS
Z21368_PEA_1 2571 2636 Z21368_PEA_1 2358 2423 ~99 Segment cluster Z21368_PEA_1 node 61 according to the present invention is supported by 61 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 38 below describes the starting and ending position of this segment on each transcript.
Table 38 - Segment location ort transcripts Transc~nipt ~~- Seguaent starting Segment ending position za~rn~e position ~ ~~~ ~
221368PEA_1 3168 3201 Z21368_PEA_1 3090 3123 221368PEA_1 2937 2970 TS
221368PEA_1 2937 2970 Z21368_PEA_1 2867 2900 Segment cluster Z21368_PEA_1 node 68 according to the present invention is supported by 87 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and 221368 PEA_1 T9. Table 39 below describes the starting and ending position of this segment on each transcript.
Tale 39 - Segment location on transcripts Ticanscript Segment starting positionI Segment ending name position Z21368_PEA_1 4375 4427 Z21368_PEA_1 4297 4349 221368PEA_1 4144 4196 TS
Z21368_PEA_1 4144 4196 Z21368_PEA_1 4074 4126 Segment cluster 221368 PEA_1 node 7 according to the present invention is supported by 29 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T23, Z21368_PEA_1 T?4, Z21368_FEA_1 T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 40 below describes the starting and ending position of this segment on each transcript.
Transcript ~1 Segpent starting ~ Segmient ~ndarig name position posi~an ~~ ~r Z2136RPEA _1T10 463 557 ~~
221368PEA_1T11 463 557 Z21368_PEA_1T23 463 557 Z21368_PEA_1T24 463 557 Z21368_PEA_1TS 301 395 Z21368_PEA_1T6 301 395 Z21368_PEA_1T9 328 422 Overexpression of at least a portion of this cluster was determined according to oligonucleotides and one or more chips. The results were as follows:
Oligonucleotide 221368_0 0 61857 was on the TAA chip and was found to be overexpressed in breast cancer.
Variant protein alignment to the previously known protein:
Sequence name: /tmp/5ER3vIMKE2/9LOY71D1TQ:SULl HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P2 ~ SUL1 HUMAN ..
Table 4p - Segment location on tr~anseripts Alignment segment 1/1:
3oi Quality: 7664.00 Escore: 0 Matching length: 761 Total length: 761 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIilllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIilllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
. , , IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIilllllllllllllllllll IIIIIIIIIIilllilllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Illlllllllllllllllilllllllllllllllllllllllllllllll 501 DKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTR.SLSVEF 550 Iillllllllllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
701 PFKEA.AQEVDSKLQLFKENNRRRKKERKEKRRQRKGEECSLPGLTCFTHD 750 20 Sequence name: /tmp/tt3yfXIUKV/YxSTFWr66h:Q7Z2W2 Sequence documentation:
Alignment of: 221368 PEA 1 P5 x Q7Z2W2 ..
Alignment segment 1/1:
Quality: 7869.00 Encore: 0 Matching length: 791 Total length: 871 Matching Percent Similarity: 99.87 Matching Percent Tdentity: 99.87 Total Percent Similarity: 90.70 Total Percent Identity: 90.70 Gaps: 1 Alignment:
IIIIIII~IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
51 DDQDVELA......................,................... 58 1111!11 59 ......................................FFGKYLNEYNGS 70 IIIlllllllll ~ -7l YIPPGWREWLGLIKNSRFYNYTVCRNGIKEKHGFDYAKDYFTDLITNESI 120 llllllillllllllllllllllllllllllllllllllllllllllll.
illlllllllllllllllllllllllllllllll.Iillllllllllllll 20l NYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYN 250 3o IlllilllllllllllllllllllIIIIIIIIIIilllllllllllllll lllllllllllIllllIIIIIIIIIillllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIII
I
I
I
illlllllllllillllIII
PGSIVPQIVLNIDLAPTILDIAGLDTPPDVDGKSVLKLLDPEKPGNRFRT
IIIIIIIIIIIIIIIIIIIIII
I
I
I
lllllllllllIIIIIIIIIIII
NKKAKIWRDTFLVERGKFLRKKEESSKNIQQSNHLPKYERVKELCQQARY
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllllll 421. DKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEF470 lllllilllliilllliilllllli 501 Illllllllllllllllllllllll 550 DKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEF
Illlllllllllllllllllllllllllllllllllllllllllllllll - -IIIIII1111111(Illlllilllllllllillllllilliilillllll Illlllllllllllllllllllllllllllllllllllllllllllllil 111111lIIIIIIIIIIIIIIIIIIIIIillllllllIIIIIIIIIIIII
111111l111111lIIIIIIIiIiIIIIIIIIIIIillllllllllllll . . . . .
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
802 FDMNTDPYQLTNTVHTVERGILNQLHVQLMELRSCQGYKQCNPRPKNLDV $50 IIIIIIIIIIIIIIIIIIIII
Sequence name; /tmp/tt3yfXIUKV/YxSTFWr66h:AAH12997 Sequence documentation:
Alignment of: 221368 PEA 1-P5 x AAH12997 ..
Alignment segment 1/1:
Quality: 420.00 Escore: 0 Matching length: 40 Total length: 40 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
Sequence name: /tmp/tt3yfXIUKV/YxSTFWr66h:SUL1 HUMAN
Sequence documentation:
Alignment of: Z21368_PEA l_P5 x SUL1'HUMAN . ..
Alignment segment 1/l:
Quality: 7878.00 Escore: 0 Mai=thing length: 791 Total length: 871 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 90.82 Total Percent Identity: 90.82 Gaps: 1 Alignment:
. . . . .
iilililiiillllililliiiliillilliililllliilllliiilll 51 DDQDVEL........................................... 57 Ilillii 51~DDQDVELGSLQVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYV 100 58 .....................................AFFGKYLNEYNGS 70 2o iliiiiillliii liliiiillllilliliillillliililililillliillliiliiill illlliiiiililliiilllliliiiiiiillliilliiliiiiiiiili - - -IIII I i IIIII II II IIII IIII IIIII III III
Ililliiiilliililililliilliiliilillililliiillllilil Illll!!lllllill!IIiIlilllllllllllllllllllliillllll llll11111111!llllllllllll!!llllllllllillllilllllil IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Ilillilliliillilllliilililillliliilililllillllilll 1l1111(Illlllllill!llllllllilllillllllllllllllilll llllllllllll1111)l)lllllllllllilllillllllllllllll 701 PFKEAAQEVDSKLQLFKENNRRRKKER.KEKR.RQRKGEECSLPGLTCFTHD 750 Sequence name: /tmp/AVAZGWHuFO/RzHFOnHIsT:SULl HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P15 x SUL1 HUMAN .-Alignment segment 7_,% 1 Quality: 4174.00 Escore: 0 Matching length: 416 Total length: 416 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
2o IIIIIIIIIIIIIIIIIIIillllllllllllllllllllllllllllll IIIIIIIIIIilllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
-IIIIIIIIIIIIIIIIIIIIIIilllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllllllilll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIII
25 Sequence name: /tmp/JhwgR.dKc~nt/kqSmjxkWWk:SUL1 HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P16 x SULl HUMAN ..
Alignment segment 1/1:
Quality: 3985.00 Escore: 0 Matching length: 397 Total length: 397 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps : 0 Alignment:
~I~~I~~~~~~~~~~~~~~~~~~~~~~I~I~~~~~~~II~~~~~~~~~~I
5l DDQDVELGSLQVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYV 100 ~ - ' ' ~ ' 20 Sequence name: /tmp/GPlnIw3BOg/zXFdxqG4ow:SUL1 HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P22 x SUL1 HUMAN ..
Alignment segment 1/1:
Quality: 1897.00 Escore: 0 Matching length: 188 Total length: 188 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity. 100.00 Gaps: 0 Alignment:
51 DDQDVELGSLQVMNKTRKIMEHGGATFII~AFVTTPMCCPSRSSMLTGKYV 100 ~ ' ' 30 Sequence name: /tmp/oji5Fs74fB/8xeB9KrGjp:Q7Z2W2 Sequence documentation:
Alignment of: 221368 PEA 1 P23 x Q7Z2W2 ..
Alignment segment 1/1:
Quality: 1368.00 Encore: 0.000511 Matching length: 137 Total length: 137 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps : 0 Alignment:
2o Iillllllllllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIilllllllllllllllllllllllil Sequence name: /tmp/oji5Fs74fB/8xeB9KrGjp:SUL1 HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P23 x SULl HUMAN ..
Alignment segment 1/1:
Quality: 1368.00 Escore: 0.000511 Matching length: 137 Total length: 137 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
~Ol HNHNVYTNNENCSSPSWQAMHEPRTFAVYLNNTGYRT 137 l01 HNHNVYTNNENCSSPSWQAMHEPRTFAVYLNNTGYR.T l37 Expression of SLTL1 HCTMAN - Extracellular sulfatase Sulf 1221368 transcripts which are detectable by amplicon as depicted in sequence name Z21368seg39 in normal arid cancerous breast tissues Expression of SI1LI_HLTMAN - Extracellular sulfatase Sulf 1 transcripts detectable by or according to seg39, Z21368seg39 amplicon and Z21368seg39F and Z2I368seg39R
primers was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD
(GenBank Accession No. BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM_000194; amplicon - HPRTI-amplicon), SDHA (GenBank Accession No.
NM_004168; amplicon - SDHA-amplicon), and G6PD (GenBanle Accession No.
NM_000402;
G6PD amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes.
The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 56-60,63-67, Table 1 above, Tissue samples in testing panel), to obtain a value of fold up-regulation for each sample relative to median of the normal PM samples.
Figure 13 is a histogram showing over expression of the above-indicated SILL 1 HUMAN - Extracellular sulfatase Sulf 1 transcripts in cancerous breast samples relative to the normal samples. Values represent the average of duplicate experiments.
Error bars indicate the minimal and maximal values obtained. The number and percentage of samples that exhibit at least 5-fold over-expression, out of the total number of samples tested is indicated in the bottom.
As is evident from Figure 13, the expression of SUL 1 HUMAN - Extracellular sulfatase Sulf 1 transcripts detectable by the above amplicon(s) in cancer samples was significantly higher than in the non-cancerous samples (Sample Nos 56-60,63-67, Table 1 above, Tissue samples in testing panel). Notably an over-expressir:~n of at least 5 fold was found in 13 out of 28 adenocarcinoma samples.
Statistical analysis was applied to verify the spg~uficance of these results, as described below.
The P value for the difference in the expression levels of SUL 1 ~~JMAN -Extracellular sulfatase Sulf 1 transcripts detectable by the above amplicon(s) in breast cancer samples versus the normal tissue samples was determined by T test as 2.14E-03.
Threshold of 5 fold overexpression was found to differentiate between cancer and normal samples with P value of 6.911s03 as checked by exact fisher test. The above values demonstrate statistical significance of the results.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: Z21368seg39F
forward primer;
Z21368seg39R reverse primer.
The present invention also preferably encarnpasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following arnplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: Z21368seg39.
Z21368seg39F (SEQ ID N0:842)- GTTGCATTTCTCAGTGCTGGTTT
Z21368seg39R (SEQ ID N0:843)- AGGGTGCCGGGTGAGG
Z21368seg39 (SEQ m N0:844~
GTTGCATTTCTCAGTGCTGGTTTCTAATCAGACCAGTGGATTGAGTTTCTCTACCATC
CTCCCCACGTTCTTCTCTAAGCTGCCTCCAAGCCTCACCCGGCACCCT
Expression of SUL1 HUMAN - Extracellular sulfatase Sulf 1221368 transcripts which are detectable by amplicon as depicted in sequence name Z21368seg39 in different normal tissues Expression of SUL 1 HUMAN - Extracellular sulfatase Sulf I transcripts detectable by or according to Z21368seg39 amplicon and Z21368seg39F Z2I368seg39R was measured by real time PCR. In parallel the expression of four housekeeping genes -[ RPL19 (GenBank Accession No. NM_000981; RPL19 amplicon), TATA box (GenBank Accession No.
NM_003194; TATA amplicon), UBC (GenBank Accession No. BC000449; ampIicon -Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM_004168; amplicon - SDHA-amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the breast samples (sample nos. 33-35 in table 2 "Tissue samples in normal panel") to obtain a value of relative expression of each sample relative to median of the Normal samples. Primers and amplicon are as above.
The results are presented in Figure 14, demonstrating the expression of SLTL 1 ~iIIMAN
- Extracellular sulfatase Sulf 1221368 transcripts, which are detectable by amplicon as depicted in sequence name Z21368seg39, in different normal tissues.
Expression of SUL1 HCJMA.N - Extracellular sulfatase Sulf 1 221368 transcripts which are detectable by amplicon as depicted in sequence name Z21368juncl7-21 in normal and cancerous breast tissues Expression of SI1L 1HUMAN - Extracellular sulfatase Sulf 1 transcripts detectable by ' or according to Z21368juncl7-2I amplicon and Z21368junc17-21F and Z21368juncl7-primers was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No. BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM 000194; amplicon - HPRTI-amplicon), and SDHA (GenBank Accession No. NM_004168; amplicon - SDHA-amplicon), G6PD (GenBanle Accession No.
NM_000402;
G6PD amplicon) was measured sunilarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes.
The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos 56-60,63-67 Table 1 above, "Tissue samples in testing panel"), ~ obtain a value of fold up-regulation for each sample relative to median of the normal PM samples.
Figure IS is a histogram showing over expression of the above-indicated SITL 1 HUMAN - Extracellular sulfatase Sulf 1 transcripts in cancerous breast sample s relative to the normal samples. Values represent the average of duplicate experiments.
Error bars indicate the minimal and maximal values obtained. The number and percentage of samples that exhibit at least 5 fold over-expression, out of the total number of samples tested is indicated in the bottom.
As is evident from Figure 15, the expression of SLTL1 HCJMAN - Extracellular sulfatase Sulf 1 transcripts detectable by the above amplicon(s) in cancer samples was significantly higher than in the norrcancerous samples (Sample Nos 56-60,63-67, Table 1 above, Tissue samples in testing panel). Notably an over-expression of at least 5 fold was found in 11 out of 28 adenocarcinoma samples.
Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of SUL1 HUMAN -Extracellular sulfatase Sulf 1 transcripts detectable by the above amplicon(s) in breast cancer samples versus the normal tissue samples was determined by T test as 4.6E 03.
IS Threshold of 5 fold overexpression was found to differentiate between cancer and normal samples with P value of 1.78E-02 as checked by exact fisher test. The above values demonstrate statistical signif cance of the results. Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair:
Z21368juncl 7-21 F forward primer; Z21368junc17-~ 1 R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: ~21368junc17-~1 22I368junc17-2IF (SEQ ID N0:845)- GGACGGATACAGCAGGAACG
Z21368junc17-21R (SEQ ID N0:846}- TATTTTCCP~AAAAAGGCCAGCTC
Z21368junc17-21 (SEQ ID NO:847}-GGACGGATACAGCAGGAACGAAAAAACATCCGACCCAACATTATTCTTGTGCTTAC
CGATGATCAAGATGTGGAGCTGGCCTTTTTTGGAAAATA
Expression of SUL1 HUMAN - Extracellu.lar sulfatase Sulf 1 221368 transcripts which are detectable by amplicon .us depicted in sequence name Z21368juncl7-21 in different normal tissues Expression of SUL1 HLTIvIAN - Extracellular sulfatase Sulf 1 221368 transcripts detectable by or according to amplicon Z21368junc17-21 was measured by real time PCR. In parallel the expression of four housekeeping genes -RPL19 (GenBanl: Accession No.
NM-000981; RPL19 amplicon), TATA box (GenBank Accession No. NM_003194; TATA
amplicon); UBC (GenBank Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA -(GenBanl: Accession No. NM 004168; amplicon - SDHA-amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was norn~alized to the geometric mean of the quantities of the housekeeping genes, as above. The normalized quantity of each RT sample was then divided by the median of the quantities of the breast samples (Sample Nos. - 33-35 Table 2 above, 'Tissue samples on normal panel"), to obtain a value of relative expression of each sample relative to median of the breast samples.
Primers and amplicon are as above.
The results are presented in Figure 16, demonstrating the expression of SUL 1 HUMAN
- Extracellular sulfatase Sulf 1 221368 transcripts, which are detectable by amplicon as depicted in sequence name Z21368junc17-21, in different normal tissues.
''S Cluster T59832 features 6 transcripts) and 33 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3 Table 1 - Transcripts of interest Transcript Name ' ' Sequence ID No ,.
Table 2 - Segments of interest Segment.Nam~e i ~e,~uenc,~ I17 No T59832node1 109 T59832node22 110 T59832node23 111 T59832node24 112 T59832node29 113 T59832_node39 114 T59832_node7 115 T59832_node_10 116 T59832node_11 117 T59832_node_12 118 T59832_node_14 119 T59832_node_16 120 T59832_node_19 121 T59832node_2 122 T59832_node20 123 T59832_node25 124 T59832_node26 125 T59832_node27 126 T59832_node_28 127 T59832_node3 128 T59832_node30 129 T59832node31 130 T59832_node32 131 T59832_node34 132 T59832node_35 133 T59832node36 134 T59S32_node37 135 T59832node_38 136 T59832node_4 137 T5983'?_node5 13S
T59832_node6 139 T59832node8 140 T59832node9 141 Table s - Proteins of interest Protein Nataie ! Sequence LD No T59832_PS 143 T59832 P18 ~ 147 These sequences are variants of the known protein Gamma-interferon inducible lysosomal thiol reductase precursor (SwissProt accession identifier GILT HUIvIAN; known also according to the synonyms Gamma-interferon-inducible protein IP-30), SEQ ID NU: 142, referred to herein as the previously known protein.
Protein Garruna-interferon inducible lysosomal thiol reductase precursor is known or believed to have the following function(s): Cleaves disulfide bonds in proteins by reduction.
May facilitate the complet unfolding of proteins destined for lysosomal degradation. May be involved in MHC class II-restricted antigen processing. The sequence for protein Gamma-interferon inducible lysosomal thiol reductase precursor is given at the end of the application, as "Gamma-interferon inducible lysosomal thiol reductase precursor amino acid sequence". known polymorphisms for this sequence are as shown in Table 4.
Table d - Arnirro acid rnrctations far Known Protein ~,~ ~~sitic~n(s)Comment.
on amino acid sequence 109 L -> S
130 H -> L
Protein Gamma-interferon inducible lysosomal thiol reductase precursor localization is believed to be Lysosomal.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: extracellular; lysosome, wlvch are annotations) 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/>.
Cluster T59832 can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term "number" in the left hand column of the table and the numbers on the y axis of Figure 17 refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histograms in Figure 17 and Table 5. This cluster is overexpressed (at least at a minimum level) in the following pathological conditions: brain malignant tumors, breast malignant tumors, ovarian carcinoma and pancreas carcinoma.
Table S - Norrrzal tissue distribcrtiorr Name cif Tissue Dumber Adrenal 208 Bladder SOS
Bone X00 Brain 18 Colon 236 Epithelial 143 General 280 head and neck 192 Kidney 71 Liver 53 Lung 459 lymph nodes X48 Breast 0 bone marrow 94 Ovary 0 Pancreas ~0 Prostate 86 Skin 2 Stomach 109 T cells 557 Thyroid 0 Uterus 63 Table 6 - F values and ratios for expression in cancerous tissa~e Narne: o~'TissueP1 ~ P2 SP1 ~ SPA R4 adrenal 4.9e-015.9e-014.7e-031.1 2.9e-020.8 bladder 3.7e-015.6e-013.7e-021.3 2.5e-010.9 Bone 6.6e-0 6.7e-0 3.4e-0 0.6 9.1 0.4 1 1 1 e-O
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Gland Gland of 24 64-Am-Kidney 111PO1O1Bmbion Kidney PM-Pool M&F
of 14 65-CI-Kidney 1110970 ClontechKidney PM-Pool M&F
of 14 66-B-Kidney A411080 BiochainKidney PM-Pool M&F
of 5 67-CG-Cerebellum CG-183-5IchilovCerebellum PM MI74 68-CG-Gerebellum CG-212-5IchilovCerebellum PM M/54 69-B-Brain A411322 BiochainBrain PM M/28 70-CI-Brain 1120022 ClontechBrain PM-Pool M&F
of 2 71-B-Brain A411079 BiochainBrain PM-Pool M&F
of 2 72-CG-Brain CG-151-1IchilovBrain PM F/86, 73-Am-Skeletal 1O1P013Ambion Skeletal PM F/28 Muscle Muscle 74-CI-Skeletal 11061038IGIontechlSkeletal PM-Pool M&F
Muscle MuscIeI of 2 I
Materials aftd Experimental Procedacres RNA preparation - RNA was obtained from Clontech (Franklin Lakes, NJ LISA
07417, www.clontech.com), BioChain Inst. Inc. (Hayward, CA 94545 USA
www.biochain.com), ABS
(Wilmington, DE 19801, USA, http://www.absbioreagents.com) or Ambion (Austin, USA, http://www.ambion.com). Alternatively, RNA was generated from tissue samples using TRI-Reage nt (Molecular Research Center), according to Manufacturer's instructions. Tissue and RNA samples were obtained from patients or from postmortem. Total RNA samples were treated with DNaseI (Ambion) and purified using RNeasy columns (Qiagen).
RT PCR - Purified RNA (1 p.g) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 p.M dNTP in a total volume of 15.6 p.1. The mixture was incubated for 5 min at 65 °C and then quickly chilled on ice. Thereafter, 5 p.1 of SX
SuperscriptII first strand buffer (Invitrogen), 2.4.1 O.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 p l (200units) of SuperscriptII (Invitrogen) was added and the reaction (final volume of 25p1) was incubated for 50 min at 42 °C and then inactivated at 70 °C for l5min. The resulting cDNA was diluted 1:211 in TE buffer (10 mM Tris pH=8, 1 mM EDTA pH=8).
Real-TurTe RT-PCR analysis- cDNA (5p l), prepared as described above, was used as a template in Real-Time PCR reactions using the SYBR Green I assay (PE Applied Biosystem) with specific primers and IJNG 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 l5sec, 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 trarrcript quantity in the RT reactions. The relative quantity was calculated using the equation Q=efficiency~-~'. The efficiency of the PCR
reaction was calculated from a standard curve, created by using serial dilutions of several reverse transcription (RT) reactions. To minimize inherent differences in the RT reaction, the resulting relative quantities were normalized to the geometric mean of the relative quantities of several housekeeping (HSKP) genes. Schematic summary of quantitative real-time PCR
analysis is presented in Figure 3. As shov~m, 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
product signal is above the background level (passive dye RO~i) and still in the Geometric/Exponential phase (as shown, once the level of fluorescence crosses the measurement threshold, it has a geomeMcally 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 breast cancer panel were as follows:
G6PD .(GenBank Accession No. NM_000402) G6PD Forv,~ard primer: gaggccgtcaccaagaacat G6PD Reverse primer: ggacagccggtcagagctc G6PD-amplicon:
gaggccgtcaccaagaacattcacgagtcctgcatgagccagataggctggaaccgcatcatcgtggagaagcccttcg ggagggacct gcagagctctgaccggctgtcc SDHA (GenBank Accession No. NM_004168) SDHA Forward primer: TGGGAACAAGAGGGCATCTG
SDHA Reverse primer: CCACCACTGCATCAAATTCATG
SDHA-amplicon TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATCCAGT
AGTGGATCATGAATTTGATGCAGTGGTGG
PBGD (GenBank Accession No. BC019323), PBGD Forward primer: TGAGAGTGATTCGCGTGGG
PBGD Reverse primer: CCAGGGTACGAGGCTTTCAAT
PBGD-amplicon:
TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAGACGGAC
AGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG
HPRT 1 (GenBank Accession No. NM_000194), HPRT1 Forward primer: TGACACTGGCAAAACAATGCA
HPRT1 Reverse primer: GGTCCTTTTCACCAGCAAGCT
HPRT .l-amplicon:
TGAC.~.CTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAA
AGA'I'~~iGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
The sequences of the housekeeping genes measured in all the examples on normal tissue samples panel were as follows:
RPL19 (GenBanl: Accession No. NM_000981), RPL19 Forward primer: TGGCAAGAAGAAGGTCTGGTTAG
RPL 19 Reverse primer: TGATCAGCCCATCTTTGATGAG
RPL19 -amplicon:
TGGCAAGAAGAAGGTCTGGTTAGACCCCA.ATGAGACCAATGAAATCGCCAATGCCA
ACTCCCGTCAGCAGATCCGGAAGCTCATCAAAGATGGGCTGATCA
TATA box (GenBank Accession No. NM_003194), TATA box Forward primer : CGGTTTGCTGCGGTAATCAT
TATA box Reverse primer: TTTCTTGCTGCCAGTCTGGAC
TATA box -amplicon:
CGGTTTGCTGCGGTAATCATGAGGATAAGAGAGCCACGAACCACGGCACTGATTTT
CAGTTCTGGGAAAATGGTGTGCACAGGAGCCAAGAGTGAAGAACAGTCCAGACTG
GCAGCAAGAAA
UBC (GenBank Accession No. BC000449) UBC Forvvard primer: ATTTGGGTCGCGGTTCTTG
LTBC Reverse primer: TGCCTTGACATTCTCGATGGT
LTBC -amplicon:
ATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGACAATGCAGAT
CTTCGTGAAGACTCTGACTGGTAAGACCATCACCCTCGAGG
TTGAGCCCAGTGACACCATCGAGAATGTCAAGGCA
SDHA (GenBanl: Accession No. NM_004168) SDHA Forward primer: TGGGAACAAGAGGGCATCTG
SDHA Reverse primer: CCACCACTGCATCAAATTCATG
SDHA-amplicon TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATCCAGT
AGTGGATCATGAATTTGATGCAGTGGTGG
Oligorat.rcleotide-based micro-array experiment protocol-Microarray fabrication Microarrays (chips) were printed by pin deposition using the MicroGrid II MGII
robot from BioRobotics Limited (Cambridge, LTK). 50-mer oligonucleotides target sequences were designed by Compugen Ltd (Tel-Avviv, IL) as described by A. Shoshan et al, "Optical technologies and informatics", Proceedings of SPIE. Vol 4266, pp. 86-95 (2001). The designed oligonucleotides were synthesized and purified by desalting with the Sigma-Genosys system (The Woodlands, TX, US) and all of the oligonucleotides were joined to a C6 amino-modified linker at the 5' end, or being attached directly to CodeLink slides (Cat #25-6700-O1. Amersham Bioscience, Piscataway, NJ, US). T'he 50-mer oligonucleotides, forming the target sequences, were first suspended in Ultra-pure DDW (Cat # O1-866-lA Kibbutz Beit-Haemek, Israel) to a concentration of SON.M. Before printing the slides, the oligonucleotides were resuspended in 300mM sodium phosphate (pH 8.5) to final concentration of 150mM and printed at 35-40%
relative humidity at 21 °C.
Each slide contained a total of 9792 features in 32 subarrays. Of these features, 4224 features were sequences of interest according to the present invention and negative controls that were printed in duplicate. An additional 28S features (96 target sequences printed in triplicate) contained housekeeping genes from Human Evaluation Library2, Compugen Ltd, Israel.
Another 384 features are E.coli spikes 1-6, which are oligos to E Coli genes which are commercially available in the Array Control product (Array control- sense oligo spots, Ambion Inc. Austin, TX. Cat #1781, Lot #112K06).
Post-coupling processing of printed slides After the spotting of the oligonucleotides to the glass (CodeLinl:) slides, the slides were incubated for 24 hours in a sealed saturated NaCI humidification chamber (relativve humidity 70-75%).
Slides were treated for blocking of the residual reactive groups by incubating them in blocking solution at 50°C for 15 minutes (lOml/slide of buffer containing O.1M Tris, SOmM
ethanolamine, 0.1% SDS). The slides were then rinsed twice with Ultra-pure DDW
(double distilled water). The slides were then washed with wash solution (lOml/slide.
4X SSC, 0.1%
SDS)) at 50°C for 30 minutes on the shaker. The slides were then rinsed twice with Ultra-pure DDW, followed by drying by centrifugation for 3 minutes at 800 rpm.
Next, in order to assist in automatic operation of the hybridization protocol, the slides were treated with Ventana Discovery hybridization station barcode adhesives.
The printed slides were loaded on a Bio-Optica (Milan, Italy) hematology staining device and were incubated for 10 minutes in SOmI of 3-Aminopropyl Triethoxysilane (Sigma A3648 lot #122K589). Excess fluid was dried and slides were then incubated for three hours in 20 nvr>/Hg in a dark vacuum desiccator (Pelco 2251, Ted Pella, Inc. Redding CA).
The following protocol was then followed with the Genisphere 900-RP (random primer), with mini elute columns on the Ventana Discovery HybStationT"', to perform the microarray experiments. Briefly, the protocol was performed as described with regard to the instructions and information provided with the device itself. The protocol included cDNA
synthesis and labeling. cDNA concentration was measured with the TBS-380 (Turner Biosystems.
Sunnyvale, CA.) PicoFlour, which is used with the OliGreen ssDNA Quantitation reagent and kit.
Hybridization was performed with the Ventana Hybridization device, according to the provided protocols (Discovery Hybridization Station Tuscon AZ).
The slides were then scanned with GenePix 4000B dual laser scanner from Axon Instruments Inc, and analyzed by GenePix Pro 5.0 software.
Schematic summary of the oligonucleotide based microarray fabrication and the experimental flow is presented in Figures 4 and 5.
Briefly, as shown in Figure 4, DNA oligonucleotides at 25uM.were deposited (printed) onto Amersham 'CodeLinl:' glass slides generating a well defined 'spot'. These slides are covered with a long-chain, hydrophilic polymer chemistry that creates an active 3-D surface that covalently binds the DNA oligonucleotides 5'-end via the C6-amine modii~i _~.~tion. This binding ensures that the full length of the DNA oligonucleotides is available for hybr~iciization to the cDNA and also allows lower background, high sensitivity and reproducibility.
Figure 5 shows a schematic method for performing the microarray experiments.
It should be noted that stages on the left-hand or right-hand side may optionally be performed in any order, including in parallel, until stage 4 (hybridization). Briefly, on the left-hand side, the target oligonucleotides are being spotted on a glass microscope slide (although optionally other materials could be used) to form a spotted slide (stage 1). On the right hand side, control sample RNA and cancer sample RNA are Cy3 and Cy5 labeled, respectively (stage 2), to form labeled probes. It should be noted that the control and cancer samples come from corresponding tissues (for example, normal prostate tissue and cancerous prostate tissue).
Furthermore, the tissue from which the RNA was taken is indicated below in the specific examples of data for particular clusters, with regard to overexpression of an oligonucleotide from a "chip"
(microarray), as for example "prostate" for chips in which prostate cancerous tissue and normal tissue were tested as described above. In stage 3, the probes are mixed. In stage 4, hybridization is performed to form a processed slide. In stage 5, the slide is washed and scanned to form an image file, followed by data analysis in stage 6.
Cluster T10888 features 4 transcripts) and 8 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table 1 - Ti~arrscripts of rrzterest Transcript ' Sequence ID No.
I~Tame T10888_PEA_1T1 1 T10888_PEA_1T4 T10888_PEA_1TS
T10888_PEA_1T6 Table ? - Segnzertts of interest Se~tnent i Sequence ID Na. __ Name T10888PEA_1node_11 5 T10888PEA_1node_12 6 T10888_PEA_1node_17 7 T10888_PEA_1node 8 T10888_PEA_1node_6 9 T10888PEA node7 10 I
T10888_PEA_1node9 11 T10888_PEA_1node_IS 12 Table 3 - Proteins of irrterest Protein '-'- Szduence ID Na.
Name PEA_ PEA
PEA
T10888_PEA_1 P6 17 These sequences are variants of the known protein Carcinoembryonic antigen-related cell adhesion molecule 6 precursor (SwissProt accession identifier CEA6_HUMAN;
known also according to the synonyms Normal cross-reacting antigen; Nonspecific crossreacting antigen;
CD66c antigen), SEQ ID NO: 13, referred to herein as the previously known protein.
The sequence for protein Carcinaembryonic antigen-related cell adhesion molecule 6 precursor is given at the end of the application, as "Carcinoembryanic antigen-related cell adhesion molecule 6 precursor amino acid sequence". Known polymorphisms for this sequence are as shown in Table 4.
Table 4 - Arr2irlo acid rnnctations for Kraowrz Protein SNP positions) Comment an amino acid sequence 138 F->L
Protein Carcinoembryonic antigen-related cell adhesion molecule 6 precursor localization is believved to be Attached to the membrane by a GPI-anchor.
The previously known protein also has the following indications) and/or potential therapeutic use(s): Cancer. It has been investigated for clinicaUtherapeutic use in humans, for example as a target for an antibody or small molecule, and/or as a direct therapeutic; available information related to these investigations is as follows. Potential pharmaceutically related or therapeutically related activity or activities of the previously known protein are as follows:
Immunostimulant. A therapeutic role for a protein represented by the cluster has been predicted.
The cluster was assigned this field because there was information in the drug database or the public databases (e.g., described herein above) that this protein, or part thereof, is used or can be used for a potential therapeutic indication: Imaging agent; Anticancer;
Immunostimulant;
Immunoconjugate; Monoclonal antibody, murine; Antisense therapy; antibody.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: signal transduction; cell-cell signaling, which are.
annotations) related to Biological Process; and integral plasma membrane protein, which are annotations) related to Cellular Comporent.
The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from <http:i/wn?v.expasy.ch/sprot/>; or Locuslink, available from <http://www.ncbi.nlm.nih.gov/projects/LocusLink/>.
Cluster T10888 can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term "number" in the right hand column of the table and the numbers on the taxis of Figure 6 refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histograms in Figure 6 and Table 5. 'This cluster is overexpressed (at least at a minimum level) in the following pathological conditions: colorectal cancer, a mixture of malignant tumors from different tissues, pancreas carcinoma and gastric carcinoma.
Table 5 - Normal tissue distribution Name of Tissue Number Bladder 0 Colon 107 Epithelial 52 General 22 head and neck 40 Lung 237 Breast 0 pancreas 32 Prastate 12 Stomach 0 Table 6 - P values and ratios for expression in cancerous tissue Name of TissueP1 ~ p2 SP1 ~ _ ; SP2 4 . R
Bladder 5.4e-013.4e-01 5.6e-011.8 4.(e-011.9 Colon 1.2e-011.7e-01 2.Se-OS3.7 7.9e-042.8 epithelial 3.3e-022.1e-01 2.8e-202.8 4.8e-101.9 General 3.3e-052.2e-03 1.9e-444.9 4.6e-273.3 head and neck4.fie-O14.3e-01 1 0.8 7.Se-Ol1.0 Lung 7.6e-018.2e-01 8.9e-010.6 1 0.3 Breast 3.7e-024.1e-02 l.Se-O13.3 3.1e-012.4 pancreas 2.6e-012.4e-01 8.6e-232.8 1.5e-194.5 Prostate 9.1e-019.3e-01 4.1e-021.2 l.Oe-O11.0 Stomach 4.5e-0'25.6e-02 S.le-044.1 4.7e-046.3 LOO
As noted above, cluster T10888 features 4 transcript(s), which were listed in Table above. These transcripts) encode for proteins) which are variants) of protein Carcinoembryonic antigerrrelated cell adhesion molecule 6 precursor. A
description of each variant protein according to the present invention is now provided.
Variant protein T10888_PEA_1 P2 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10888 PEA_1 T1. An alignment is given to the known protein (Carcinoembryonic antigen related cell adhesion molecule 6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10888_PEA_1 P2 and CEA6 HUMAN:
l.An isolated chimeric polypeptide encoding for T10888_PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVI'PELPKPSISSNNSNPVEDKDAVAFTCEPEVVQNTTYL
WWVNGQSLPVSPRLQLSNGNMTLTLLSVkRIVDAGSYECEIQNPASANRSDPVTLNVLY
GPDVPTISPSkANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGS
I'MCQAHNSATGLNRTTVTMITVS corresponding to amino acids 1 - 319 of CEA6_ _HUhZAN, which also corresponds to amino acids 1 - 319 ofT10888 PEA_1 P2, 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 DWTRP corresponding to amino acids 320 - 324 of T1088S
PEA_1 P2, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of T1088S PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence DWTRP in T10888_PEA_1 P2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows v~rith regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T10888_PEA_1 P2 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 7, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs _in variant protein T10888_PEA_1 P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 7 - Anrirra acid mutcrtians SNP' position(s). Alteruai'ive an~ir~o~ Previously l.~nowin on amino acid acids) SNP'?
sequence 13 V -> No 232 N _> D No 324 P -> No 63 1-> No G _> ~ No Variant protein T10888_PEA_1 P2 is encoded by the following transcript(s):
T10888_PEA_1 T1, for which the sequences) is/are given at the end of the application. The coding portion of transcript T1088S PEA_1 T1 is shown in bold; this coding portion starts at position 151 and ends at position I 122. The transcript also has the following SNPs as listed in Table 8 (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 _irz variant protein T10888_PEA_1 P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 8 - Nucleic acid SNPs SNk' Position on nucleotideAlternative nucleic Previously Ionnocvn Seql~erlCe acid ' SNP? ~_ 119 C->T No 120 A -> T No 1062 A _> G Yes 1120 -- C _> No 1297- G _> T Yes 1501 A -> G Yes 1824 G -> A No 2036 A -> C No 2036 A -> G No 2095 A -> C No 2242 A -> C No 2245 A -> C No 189 C -> No 2'?Sp A -> T Yes 2339 C -> A Yes 276 G _> A Yes 338 - T -> No 424 G -> No 546 A -> G No 702 C -> T No 844 A -> G No 93p C -> T Yes Variant protein T10888_PEA_1 P4 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10888_PEA_1 T4. An alignment is given to the known protein (Carcinoembryonic antigen-related cell adhesion molecule 6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A briefdescription of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10888_PEA_1 P4 and CEA6_HUMAN:
l.An _isolated chimeric polypeptide encoding for T10888_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSWYItGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVY'PELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
WWVNGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNPASANRSDPVTLNVL
corresponding to amino acids 1 - 234 of CEA6_HUMAN, which also corresponds to amino acids 1 - 234 of T10888 PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence LLLSSQLWPPSASRLECWPGWL corresponding to amino acids 235 - 256 of T10888_PEA_I P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _isolated polypeptide encoding for a tail of T10888_PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence LLLSSQLWPPSASRLECWPGWL in T10888_PEA_1 P4.
Comparison report between T10888_PEA_1 P4 and Q13774 (SEQ ID N0:829):
l.An isolated chimeric polypeptide encoding for T10888_PEA_1 P4, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWkEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGILEVLLLAHNLP
QNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FI'TLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
ATV'WNGQSLPVSPRLQLSNGNMTLTLLS'~Il'~:ItNDAGSYECEIQNPASANRSDPVTLNVL
corresponding to amino acids 1 - 234 of Q 137 i 4, which also corresponds to amino acids 1 - 234 of T10888_PEA_1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence LLLSSQLWPPSASRL,ECWFGWL
corresponding to amino acids 235 - 256 of T10888_PEA_1 P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _ _isolated polypeptide encoding for a tail of T10888 PEA_1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence LLLSSQLWPPSASRLECWPGWL in T10888_PEA_1 P4.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignaIP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T1088S PEA_1 P4 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10S88 PEA_1 P4 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 9 - Arnin~ acid mutations SNP positions) on Alternative,amino ' Previously known amino acid acids) SNP?
sequence 13 V -> No 232 N -> D No 63 I -> No ~2 G -> No.
Variant protein T1088R PEA_1 P4 is encoded by the following transcript(s):
T10888_PEA_1 T4, for which the sequences) is/are given at the end of the application. The coding portion of transcript T10888_PEA_1 T4 is shown in bold; this coding portion starts at position 151 and ends at position 918. The transcript also has the following SNPs as listed in Table 10 (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 knov~m SNPs in variant protein T10888_PEA_1 P4 sequence provides support for the deduced sequence of this variant protein according to the present invention).
TaL~le 10 - Nucleic acid SNPs SNF' position on nucleotideAlternative'rn~cleicI Preid.ously known sequence acid SNP? ____ 119 C->T No I 20 A -> T No Q78 C -> No 1155 G -> T Yes 1359 A -> G
Yes 1682 G -> A No 1894 A -> C No 1894 - A -> G No 1953 A -> C No 2100 A -> C No 2103 A -> C No ~ 108 A _> T Yes 189 G -> No C->A
Yes 276 G _> A Yes 338 T -> No 424 G -> No 546 A -> G No 702 C -> T No 844 A -> G No 958 G -> No Variant protein T10888_PEA_1 PS according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T1088S_PEA_1 T5. An alignment is given to the lenown protein (Carcinoembryryonic antigerE
related cell adhesion molecule 6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10888_PEA_1 PS and CEA6_HUMAN:
l.An _isolated chimeric polypeptide encoding for T10888_PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWKEVL,LTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLP
QNRIGYSV~TYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTG
FYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYL
WWVNGQSLPVSPRLQLSNGNMTLTLLS~~hRNDAGSYECEIQNPASANRSDPVTLNVLY
GPDVPTISPSKANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGS
YMCQAHNSATGLNRTTVTMITVSG corresponding to amino acids 1 - 320 of CEA6_HLJMAN, which also corresponds to amino acids 1 - 320 of T10888_PEA_1 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide havingthesequence KWIHEALASHFQVESGSQRRARI~KFSFPTCVQGAHANPKFSPEPSQFTSADSFPLVFLFF
VVFCFLISHV corresponding to amino acids 321 - 390 of T10888_PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _isolated polypeptide encoding for a tail of T10888_PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about SS%, more preferably at least about 90% and most preferably at 3east about 95%
homologous to the segue rice KWIFIEALASHFQVESGSQRRARICKFSFPTCVQGAH.a~I~dPKFSPEPSQFTSADSFPLVFLFF
VVFCFLISHV in T10888_PEA_1 P5.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
membrane. The protein localization is believed to be membrane because although both signal-peptide prediction programs agree that this protein has a signal.peptide, both trans-membrane region prediction programs predict that this protein has a trans-membrane region downstream of this signal peptide..
Variant protein T10888_PEA_1 PS also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 11, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is lmown _or not; the presence of known SNPs in variant protein T10888_PEA_1 PS
sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table I I - Amine acid fnzctatiofZs SNP:posilion(s) on 'Alternative amino i Previously knu~wn aimii~o acid acids) SNP'"?
sequence 13 V -> No 232 N -> D No 63 I -> No 92 G -> No Variant protein T10888_PEA_1 PS is encoded by the following transcript(s):
T10888_PEA_1 T5, for which the sequences) is/are given at the end of the application. The coding portion of transcript T10888_PEA_1 TS is shown in bold; this coding portion starts at position 151 and ends at position 1320. The transcript also has the following SNPs as listed in 2oa Table 12 (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 T1088S PEA_1 PS sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 1? -Nticleie acid SNPc SNP position on nu~teatid~Att~rnati.ve nucleic~ Previt~usly knav~m.
sequence acid SNP? -. -119 C->T No 120 A -> T No 1062 A -> G Yes 1943 C -> A Yes 2609 C -> T Yes X647 C -> G No 2701 C -> T Yes T -> C
es 1 s9 c -> No 276 G -> A Yes 338 T -> No 424 G -> No 546 A -> G No 702 C -> T No 844 A -> G No 930 C -> T Yes Variant protein T10888 PEA_1 P6 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10888_PEA_1 T6. An alignment is given to the known protein (Carcinoembryonic antigen-related cell adhesion molecule 6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application.
Comparison report between T10888_PEA_1 P6 and CEA6 l.An isolated chimeric polypeptide encoding for T10888_PEA_1 P6, comprising a first amino acid sequence being at least 90 % homologous to MGPPSAPPCRLHVPWILEVLLTASLLTFWNPPTTAKi,TIESTPFNVAEGIiEVLLLA
HNLPQNRIGYSWYILGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPN ASLLIQNVTQ
NDTGFYTLQVIIiSDLVNEEATGQFHVY
corresponding to amino acids 1 - 141 of CEA6 HUMAN, which also corresponds to amino acids 1 - 141 of T10888_PEA_1 P6, 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 REYFHMTSGCWGSVLLPTI'GIVRPGLCLWPSLHYIL,YQGLDI
corresponding to amino acids 142 - 183 of T10888_PEA_1 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of T10888 PEA_1 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95°,~o homologous to the sequence REYFHMTSGCWGSVLLPTYGIVRPGLCLWPSLHYILYQGLDI in T10888 PEA 1 P6.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T10888_PEA_1 P6 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 13, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10888_PEA_1 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 13 - Antdrto acid ntutatiorts SNP po~itian(s) on Alterdalive amine Previously tcnoi~~i amin.Q acid acxd(s) SNP's sequence 13 V -> No 63 I -> No 92 G -> No Variant protein T10888 PEA_1 P6 is encoded by the following transcript(s):
T1088s PEA_1 T6, for which the sequences) is/are given at the. end of the application. The coding portion of transcript T1088S PEA_1 T6 is shown in bold; this coding portion starts at position 151 and ends at position 6~9. 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 T1088S PEA_1 P6 sequence provides support for the deduced sequence of this variant protein according to tl~ present invention).
Table 14 - Nucleic acid SNPs SNP position on nucleotideAlternative nucleic Previously knowrx sequence acid SNF?
119 C -> T No 120 A -> T No 1 S9 C -> No 276 G -> A Yes 33R T-> No 424 G -> No 546 A -> G No As noted above, cluster T10888 features 8 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application. These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster T10888_PEA_1 node_11 according to the present invention is supported by 57 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T1 and T10888 PEA_1 T5. Table below describes the starting and ending position of this segment on each transcript.
Table 15 - Segment location on transcripts Transcript'iiame Seg~llent.starting S~gn~~nt ending position position T1088S PEA_1 TS 854 1108 15 Segment cluster T10888 PEA_1 node_12 according to the present invention is supported by 9 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T5. Table 16 below describes the starting and ending position of this segment on each transcript.
Table 16 - Segrnerrt location on transcripts Transcript name Segment starting positionSegment ending position T10888 PEA 1 TS 1109 '3004 Segment cluster T10888_PEA_1 node_17 according to the present invention is supported by 160 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T1 and T10888_PEA_1 T4. Table 17 below describes the starting and ending position of this se~rnent on each transcript.
Table 17 - Segment location on transcripts Transcript name Segment starCing Segment ex~~li~ag position position T10888_PEA_1 T4 967 2376 Segment cluster T10888_PEA_1 node 4 according to the present invention is supported by 61 libraries. The number of libraries was determined as previously described. This segment can be found in,the _ _following transcript(s): T10888_PEA_1 T1, T10888_PEA_1 T4, T10888_PEA_1 TS and T10888 PEA_1 T6. Table 18 below describes the starting and ending position of this segment on each transcript.
Table 18 - Segment locatiatt ort transcripts Transcript name .. ~eent starting position' Segment ending positron .
T10888 PEA_1 T4 1 214 Segment cluster T10888 PEA_1 node 6 according to the present invention is supported by 81 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 Tl, T10888_PEA_1 T4, T10888_PEA_1 TS and T10888_PEA_1 T6. Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on transcripts Transcript name Segment starting positionSegment ending position T10888 PEA_1 T1 215 574 T10888 PEA_1 T4 215 574 T10888_PEA_1 TS 215 574 T10S88 PEA_1 T6 215 574 Segment cluster T10888_PEA_1 node 7 according to the present invention is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T6. Table 20 below describes the starting and ending position of this segment on each transcript.
Table 20 - Segrnerrt location on transcripts TranscripCniam~ Se~h~nt starting ~ Segment ending ._ pcisi~on pASition T10888 PEA 1 T6 575 ~~ 1410 Segment cluster T1088F PEA_1 node 9 according to the present invention is supported by 72 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888 PEA_1 T1, T10888_PEA_1 T4 and T10888_PEA_1 T5. Table 21 below describes the starting and ending position of this segment on each transcript.
Table 2T - Segrrrertt location ort transcripts Transcript name Segment starting positionSegment ending position T10888 PEA 1 Tl 575 853 According to an optional embodiment of trie present invention, snort segments re~ateo to the abovve cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster T10888_PEA_1 node_15 according to the present invention is supported by 3A libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10888_PEA_1 T4. Table 22 below describes the starting and ending position of this segment on each transcript.
Table ~2 - Segment location on transcripts Transcript name ~e~en'~ starting Segment ending position-position ~
T10888_PEA_1 T4 854 X66 Variant protein alignment to the previously known protein:
Sequence name: /tmp/tM4EgaoKvm/vuztUrlRc7:CEA6 HUMAN
Sequence documentation:
Alignment of: T10888-PEA-1_P2 x CEA6 HUMAN ..
Alignment segment 1/1:
Quality: 3163.00 Escore: 0 Matching length: 319 Total length: 319 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
51 VLLLAHNLFQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRE'~' i00 . . . . -~~~~~~~~~~~~I~~~~II
Sequence name: /tmg/Yj11gj7TCe/PgdufzLOIW:CEA6 HUMAN
Sequence documentation:
Alignment of: T10888-PEA 1-P4 x CEA6 HUMAN ..
Alignment segment 1/l:
Quality: 2310.00 Escore: 0 Matching length: 234 Total length: 234 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIIIIIIIilllllllllllllllllllllllllll IIIIIIIIIIIIilllllllllllllllllllllllllllllllllllll . . . .
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII.
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Illlllllilllllllllllllllllllllllll Sequence name: /tmp/Yj11gj7TCe/Pgduf,zLOlW:Q13774 Sequence documentation:
Alignment of: T10888 PEA 1 P4 x Q13774 ..
Alignment segment 1/1:
Quality: 2310.00 Encore : 0 Matching length: 234 Total length: 234 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIII~IIIIIIII~~IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~
III~IIIIIIIIIIII~III~IIIIIIIIIIIIIIII~IIIII~IIIII~
101 IYPNASLLIQNVTQNDTGF1'TLQVIKSDLVNEEATGQFHVYPELPKPSIS 150 3o IIIIIIIIIIIIIIIII~IIIIIIIIIIIIIIIIIIII~IIIIIIIIIIII
Sequence name: /tmp/x5xDBacdpj/rTXRGepv3y:CEA6 HUMAN
Sequence documentation:
Alignment of: T10888_PEA_1_P5 x CEA6 HUMAN ..
Alignment segment 1/1:
Quality: 3172.00 Encore: 0 Matching length: 320 Total length: 320 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
~°~1 VLLLAHNLPQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRET 100 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~IIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
201 TLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLYGPDVPTISPSKANYR ~50 IIIIillllillllllllllllllllllllllllllllllllllllllll 2o IIIIIilllllllllillllllllllllllllllllllllllllllllil IIIIIIIIIIIIIIIIIIII
~5 301 AHNSATGLNRTTVTMITVSG 320 Sequence name: /tmp/VAhvYFeatq/QNEM573uCo:CEA6 HUMAN
Sequence documentation:
Alignment of : T10888-PEA 1-P6 x CEA6 HCTI~fAN . .
yGO
Alignment segment 1/1:
Quality: 1393.00 Escore: 0 Matching length: 143 Total length: 143 Matching Percent Similarity: 99.30 Matching Percent Identity: 99.30 Total Percent Similarity: 99.30 Total Percent Identity: 99.30 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIillllllllllillllllllllllllllllllll IIIIIIIIIIIIIilllllllllllllllllllllllllllllillllll ?5 IIIIIIillllllllllllllllllllllllllllllllll Alignment of: T10888_PEA_1_P6 x CEA6 HUMAN ..
Alignment segment 1/l:
Quality: 101.00 Escore: 0 Matching length: 141 Total length: 183 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 77.05 Total Percent Identity: 77.05 Gaps: 1 Alignment: ' 101 IYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVY......... 141 141 ................................. 141 Expression of CEA6 I-IiIMAN
22~
Carcinoembryoniu antigen-related cell adhesion molecule 6 (T10888) transcripts which are detectable by artx~plicon as depicted in sequence name T10888 juncl l-17 in normal and cancerous Breast tissues Expression of CEA6-HUMAN Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts detectable by or according to juncl l-17, T10888junc1l-17 amplicon(s) and T108S8junc1l-17F and T10888junc1l-17R primers was measured by real time PCR. In parallel the expression of four housekeeping genes - Y13c~1~ (uent3anlc Accession wo.
BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM_000194;
amplicon - HPRT1-amplicon), and SDHA (GenBanl: Accession No. NM-004168;
amplicon -- .SDHA-amplicon), G6PD (GenBanl: Accession No. NM 000402; G6PD amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geomeMc mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 56-60, 63-67 Table 1, "Tissue samples in testing panel", above), to obtain a value of fold up-regulation for each sample relative to median of the normal PM
samples.
Figure 7 is a histogram showing over expression of the above-indicated CEA6-Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts in cancerous breast samples relative to the normal samples. Values represent the average of duplicate experiments.
Error bars indicate the minimal and maximal values obtained. The number and percentage of samples that exhibit at least 5 fold over-expression, out of the total number of samples tested, is indicated in the bottom.
As is evident from Figure 7, the expression of CEA6_HUl'~IAN Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts detectable by the above amplicon(s) in cancer samples was significantly higher than in the non-cancerous samples (Sample Nos. 56-60, 63-67 Table l, "Tissue samples in testing panel"). Notably an over-expression of at least 5 fold was found in 19 out of 28 adenocarcinoma samples.
Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of CEA6-HUMAN
Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts detectable by the above amplicon(s) in breast cancer samples versus the normal tissue samples was determined by T test as 2.00E-03.
Threshold of 5 fold overexpression was found to differentiate between cancer and normal samples with P value of S.44B-03 as checked by exact fisher test. The above values demonstrate statistical significance of the results.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: T10S8Sjuncll-17F
forward primer;
and T1088Sjuncll-17R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: T10S88junc1l-17.
T108SRjuncll-17F (SEQ ID N0:830) CCAGCAATCCACACAAGAGCT
T10888junc1l-17R (SEQ ID NO:S31) CAGGGTCTGGTCCAATCAGAG
T10888junc1l-17 (SEQ ID N0:832) CCAGCAATCCACACAAGAGCTCTTTATCCCCAACATCACTGTGAATAATAGC
GGATCCTATATGTGCCAAGCCCATAACTCAGCCACTGGCCTCAATAGGACCACAGT
CACGATGATCACAGTCTCTGATTGGACCAGACCCTG
Expression of CEA6 HUMAN
Carcinoembryonic antigen-related cell adhesion molecule 6T10SSR transcripts which are detectable by amplicon as depicted in sequence name T10S8Sjuncll-17 in different normal tissues.
Expression of CEA6 HUMAN Carcinoembryonic antigen-related cell adhesion molecule 6 transcripts detectable by or according to T10SSS juncl l-17 amplicon(s) and T10SS8 juncl l-17F and T108S8 juncl l-17R was measured by real time PCR. In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No. NM-000981; RPL19 amplicon), TATA box (GenBank Accession No. NM-003194; TATA amplicon), UBC (GenBank Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM-004168; amplicon - SDHA-amplicon) was measured similarly. For each RT
sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantiy of each RT sample was then divided by the median of the quantities of the ovary samples (Sample Nos. 1 S-20, Table 2 "Tissue samples in normal panel" above), to obtain a value of relative expression of each sample relative to median of the ovary samples. Primers and amplicon are as above.
The results are presented in Figure 8, demonstrating the expression of CEA6-HUMAN
Carcinoembryonic antigen-related cell adhesion molecule 6 T10888 transcripts, which are detectable by amplicon as depicted in sequence name T10888junc1l-17, in different normal tissues.
Cluster T39971 features 4 transcripts) and 28 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table 1 - T)"CIITSCYIFtS Of 6111ei'ESl Transcript l~Tarne Sequence ID No.
T39971 TS ~1 Table ? - Segments of interest Se~nent Name ._ i Sequence ID Na.
T39971 node 0 22 T39971node1& 23 T39971node21 24 T39971node22 25 T39971node23 26 T39971node31 27 T39971node33 2S
T39971node7 29 T39971node1 30 T39971node10 31 T39971node11 32 T39971node12 33 T39971node15 34 T39971node16 35 T39971node17 36 T39971node26 37 T39971node27 38 T39971node28 39 T39971node29 40 T39971node3 41 T39971node30 42 T39971node34 43 T39971node35 44 T39971node36 45 T39971node4 46 T39971node5 47 T39971node8 48 T39971node9 49 Tahle 3 - P~~oteins of intef~est ProteinName , Sequence ID No.
T39971 P12 ~54 These sequences are variants of the known protein Vitronectin precursor (SwissProt accession identifier VTNC_HUMAN; known also according to the synonyms Serum spreading factor; S-protein; V75), SEQ ID NO: 50, referred to herein as the previously known protein.
Protein Vitronectin precursor is known or believed to have the following function(s):
Vitronectin is a cell adhesion and spreading factor found in serum and tissues. Vitronectin interacts with glycosaminoglycans and proteoglycans. Is recognized by certain members of the integrin family and serves as a cell-to-substrate adhesion molecule. Inhibitor of the me mbrane-damaging effect of the terminal cytolytic complement pathway. 'The sequence for protein Vitronectin precursor is given at the end of the application, as "Vitronectin precursor amino acid sequence" (SEQ ID NO:50). Hnown polymorphisms for this sequence are as shown in Table 4.
Table 4 - Antirto acid natttations for Known Protein SNP positions CommenE
j on amino acid sequence 122 A-> S. /FTId=VAR_012983.
268 R-> Q. /FTId=VAR_012984.
400 T -> M. /FTId=VAR_012985.
5p C _> N
225 S -> N
366 A -> T
Protein Vitronectin precursor localization is believed to be Extracellular.
The previously known protein also has the following indications) and/or potential therapeutic use(s): Cancer, melanoma. It has been investigated for clinical/therapeutic use in humans, for example as a target for an antibody or small molecule, and/or as a direct therapeutic; available information related to these investigations is as follows. Potential pharmaceutically related or therapeutically related activity or activities of the previously known protein are as follows: Alphavbeta3 integrin antagonist; Apoptosis agonist. A
therapeutic role for a protein represented by the cluster has been predicted. The cluster was assigned this field because there was information in the drug database or the public databases (e.g., described herein above) that this protein, or part thereof,, is used or can be used for a potential therapeutic indication: Anticancer.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: immune response; cell adhesion, which are annotations) related to Biological Process; protein binding; heparin binding, which are annotations) related to Molecular Function; and extracellular space, which are annotations) related to Cellular Component.
The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from <http:l/wwyv.expasy.ch/sprot/>; or Locuslink, available from <http://www.ncbi.nlm.nih.gov/projects/LocusLink/>.
Cluster T39971 can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term "number" in the right hand column of 2C1 the table and the numbers on the y axis of Figure 9 refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histograms in Figure 9 and Table 5. This cluster is overexpressed (at least at a minimum level) in the following pathological conditions: liver cancer, lung malignant tumors and pancreas carcinoma.
Table S - Nor°rttal tissue distribution Name of Tissue Number adrenal 60 ., -, $
bladder 0 Bone 0 Brain 9 Colon 0 epithelial 79 general ~9 Liver 2164 Lung 0 lymph nodes 0 breast 0 pancreas 0 prostate 0 Skin 0 uterus 0 Table 6 - P values acrd ratios for expr-essiort in cancerous tissue Name of TissueP1 p~ SPl R3 SP2 R4 ..
adrenal 6.9e-017.4e-012.0e-02 2.3 5.3e-021.8 bladder 5.4e-016.0e-015.6e-01 1.8 6.8e-011.5 Bone I 6.7e-011 1.0 7.0e-011.4 Brain B.Oe-O18.6e-013.0e-01 1.9 5.3e-011.2 Colon 4.2e-014.8e-017.0e-01 1.6 7.7e-011.4 epithelial 6.6e-015.7e-01l.Oe-O1 0.8 8.7e-010.6 general S.le-O13.8e-019.2e-08 1.6 8.3e-041.3 Liver 1 6.7e-012.3e-03 0.3 1 0.2 Lung 2.4e-0 9. l 1.7e-0 4.3 8. l 5.0 l e-02 1 e-03 lymph nodes 1 5.7e-011 1.0 5.8e-012.3 breast 1 6.7e-01I 1.0 8.2e-011.2 pancreas 9.5e-021.8e-011.5e-11 6.5 8.2e-094.6 prostate 7.3e-01 6.0e-016.7e-011.5 5.6e-011.7 Skin 1 4.4e-011 1.0 6.4e-011.6 uterus S.Oe-O 2.6e-0 1 1.1 B.Oe-O 1.4 AS noted above. 39971 ted cluster T features in 4 transcriotfsl. Table which were lis above. These transcripts) encode for proteins) which are variants) of protein Vitronectin precursor. A description of each variant protein according to the present invention is now provided.
Variant protein T39971 P6 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T39971 T5. An alignment is given to the known protein (Vitronectin precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T39971 P6 and VTNC_HUMAN:
l.An isolated chimeric polypeptide encoding for T39971 P6, comprising a ftrst amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAVJVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTV1'DDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKG corresponding to amino acids 1 - 276 of VTNC_HUMAN, which also corresponds to amino acids 1 - 276 of T39971 P6, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90°,~o and most preferably at least 95%
homologous to a polypeptide having the sequence TQGVVGD corresponding to amino acids 277 - 2S3 of T39971 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of T39971 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably -~ r, at least about 90% and most preferably at least about 95% homologous to the sequence TQGVVGD in T39971 P6.
The location of the variant protein was deterrtiined according to results from a number of different sof~vare programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T39971 P6 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 7, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T39971 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 7 - Amino acid nzactations 'SNP positions) on Alternative amino Prev7iously known amino acid acid(s). SNP?
sequence 122 A -> S Yes 145 G -> No 268 R -> Q yes 280 V -> A Yes 1 SO C -> No 180 C -> ~, No 192 Y -> No 209 A -> No ~ 11 T -> No 267 G -> No 267 G -> A No 268 ~ R -> No Variant protein T39971 P6 is encoded by the following transcript(s): T39971 T5, for which the sequences) is/are given at the end of the application. The coding portion of transcript T39971 TS is shown in bold; this coding portion starts at position 756 and ends at position 1604. The transcript also has the following SNPs as listed in Table 8 (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 T39971 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 8 - Nascleic acid SNPs SNP position'on nucleatxde,A.lternative nucleic'Preiriously kizown ' acid SNl'?
sequence 417 G -> C Yes 459 T -> C Yes 1387 C -> No 1406 -> A No 1406 -> G No 1555 G -> No 1555 G -> C No 1558 G -> No 1558 G -> A 1'es 1594 T -> C lres 1642 T -> C Yes 1770 C -> T Yes 529 G -> T Yes 1982 A -> G No 2007 G -> No 2029 T -> C No 2094 T -> C No 2117 C -> G No 2123 C -> T Yes 2152 C -> T YYes 2182 G -> T No ~ 1 SS A -> C No 2297 T -> C Yes 1119 G -> T Yes 2411 G -> No 2411 G -> T No 2487 T -> C Yes 1188 G -> No 1295 C -> No 1295 C -> G No 1324 -> T No 1331 C -> No 1381 C -> No Variant protein T39971 P9 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T39971 T10. An alignment is given to the known protein (Vitronectin precursor) at the end of the application. One or more alignments to one or mare previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows: .
Comparison report between T39971 P9 and VTNC_HCJMAN:
l.An isolated chimeric polypeptide encoding for T39971 P9, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCS~~QSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LILPEEEAPAPEVGASKPEGIDSRPETLHI'GRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQ1'-WR.FEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWE1'QFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRT corresponding to amino acids 1 - 325 of VTNC_HUMAN, which also corresponds to amino acids 1 - 325 of T39971_P9, and a second amino acid sequence being at least 90 % homologous to SGMAPRPSLAKKQRFRHIZNRILGY'RSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGA
NNYDDYRN>DWLVPATCEPIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGC
PAPGHL corresponding to amino acids 357 - 478 of VT'NC_HLJMAN, which also corresponds to amino acids 326 - 447 of T39971 P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _isolated chimeric polypeptide encoding for an edge portion of T39971 P9;
comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in Length, wherein at least two amino acids comprise TS, hawing a structure as follows: a sequence starting from any of amino acid numbers 325-x to 325; and ending at any of amino acid numbers 326 + ((n-2) - x), in which x varies from 0 to n-2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is belied to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T39971 P9 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T39971 P9 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 9 - Anzirzo acid mutatioras SNF position(s) on Artezr~h~ive am.zno Previously,known SNF2 amino acid acids) SeC~UEIICE , 122 A -> 5 Yes 145 G -> Na 268 R -> Q Yes 328 Ivl -> T No 350 5 -> p No 369 T-> Nl Yes 379 S -> T Na 380 N -> T No 180 C -> No 180 C -> W No 192 Y -> No 209 A -> No 211 T-> No 267 G -> No 267 G -> A Na 268 R -> No Variant protein T39971 P9 is encoded by tlae following transcript(s):
T39971_T10, for which the sequences) is/are given at the end of tlae application. The coding portion of transcript T39971 T10 is shown in bald; this coding portion starts at position 756 and ends at position 2096. The transcript also has the following SNPs as listed in Table 10 (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 T39971 P9 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 10 - Nzrcleic acid SNFs SNP position on nucleotideAlternative nucleic Previously Down SNP's sequence acid 417 G -> C Yes 459 T -> C Yes 1387 C -> No 1406 -> A No 1406 -> G No 1555 G -> No 1555 G -> C No 1558 G -> No 1558 G -> A Yes 1738 T -> C No 1803 T -> C No 1826 C -> G No 529 G -> T Yes 1832 C -> T Yes 1861 C -> T Yes 1891 G -> T No 1894 A -> C No 2006 T -> C Yes 2120 G -> No 2120 G -> T No 2196 T -> C Yes 1119 G -> T Yes 1188 G -> No C _> NO
1295 C -> G No 1324 -> T No 1331 C -> No 1381 C -> Na Variant protein T39971 PI 1 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T39971 T12. An alignment is given to the known protein (Vitronectin precursor) at the end of the application.
One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T39971 P 11 and VTNC_HUMAN:
l.An _isolated chimeric polypeptide encoding for T39971 F11, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHS1'SGRERVY'FFKGKQYWEYQFQHQPSQEE
CEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTS corresponding to amino acids 1 - 326 of VTNC' -HUMAN, which also corresponds to amino acids 1 - 326 of T39971 PI 1, and a second amino acid sequence being at least 90 % homologous to DKI'YRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL corresponding to amino acids 442 - 478 of VT'NC_HUMAN, which also corresponds to amino acids 327 - 363 of T39971 PI 1, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated chimeric polypeptide encoding for an edge portion of T39971 PI
1, 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 SD, having a structure as follows: a sequence starting from any of amino acid numbers 326-x to 326; and ending at any of amino acid numbers 327 + ((n-2) - x), in which x varies from 0 to n-2.
Comparison report between T39971 P11 and Q9BSH7 (SEQ ID NO:S33):
l.An -isolated chimeric polypeptide encoding for T39971 P11, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLIL,ALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVI-iEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGAShPEG)DSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPIiLIRDVWGIEGPff~AAFTRINCQGItTYLFKGSQYWRFEDGV
LDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEE
CEGSSLSAVFEHFAIVfluIQRDSWEDIFELLFWGRTS corresponding to amino acids 1 - 326 of Q9BSH7, which also corresponds to amino acids 1 - 326 of T39971 P11, and a second amino acid sequence being at least 90 % homologous to DKI'YRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL corresponding to amino acids 442 - 473 of Q9BSH7; which also corresponds to amino acids 327 - 363 of T39971 P11, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated chimeric polypeptide encoding for an edge portion of T39971 P11, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 4C1 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise SD, having a structure as follows: a sequence starting from any of amino acid numbers 326-x to 326; and ending at any of amino acid numbers 327 + ((n-2) - x), in which x varies from 0 to n-2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither traps-membrane region prediction program predicts that this protein has a traps-membrane region..
Variant protein T39971 P11 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 11, (given according to their positions) on the amino acid ~38 sequence, with the alternative amino acids) listed; the last column indicates whutliPr the SNP is known or not; the presence of known SNPs in variant protein T39971 P 11 sequerac.n provides support for the deduced sequence of this variant protein according to the present i~Yv~antion).
Table II -Antino acid nu~tations SNP positions) on Alternative ammo acids)Pce~liously known amino acid SNP?
$eC~11~11Ce 122 A -> S Yes 145 G -> No 268 R -> Q 1'es 180 C -> No 180 C -> W No 192 Y -> No 209 A -> No 211 T-> No 267 G -> No 267 G -> A No 268 R -> No Variant protein T39971 P11 is encoded by the following transcript(s): T39971 T12, for which the sequences) is/are given at the end of the application. The coding portion of transcript T39971 T12 is shown in bold; this coding portion starts at position 756 and ends at position 1844. The transcript also has the following SNPs as listed in Table 12 (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 T39971 PI 1 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table I L - Nucleic acid SNPs SNP position on uucleutide Alternative nucleic acid Previously known SNP?
sequence 417 G -> C Yes 459 T -> C Yes 1387 C -> No 1406 -> A No 1406 -> G No 1555 G -> No 1555 G -> C No 1558 G -> No 1558 G -> A Yes 1754 T -> C Yes 1868 G -> No 1868 G -> T No 529 G -> T Yes 1944 T -> C Yes 1119 G -> T Yes 1188 G -> No 1295 C -> No 1295 C -> G No 1324 -> T No 1331 C -> No 1381 C -> No Variant protein T39971 P12 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T39971 T16. An alignment is given to the known protein (Vitronectin precursor) at the end of the application.
One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T39971 P12 and VTNC_HUMAN:
l.An _isolated chimeric polypeptide encoding for T39971 P12, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLIL,ALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFR
GQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFK corresponding to amino acids 1 - 223 of VTNC_HUMAN, which also corresponds to amino acids 1 -223 of T39971 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least SS°./o, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence VPGAVGQGRKHLGRV corresponding to amino acids 224 - 238 of T39971 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An _isolated polypeptide encoding for a tail of T39971 P12, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VPGAVGQGRKHLGRV in T39971 P12.
Comparison report between T39971 P12 and Q9BSH7:
l.An _isolated chimer-ic polypeptide encoding for T39971 P12, comprising a first amino acid sequence being at least 90 % homologous to MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSI'YQSCCTDYTAEC
KPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPV
LKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGkPFDAFTDLKNGSLFAFR
GQ1'C1'ELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFIL corresponding to amino acids 1 - 223 of Q9BSH7, _which also corresponds to amino acids 1 - 223 ofT39971 P12, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VPGAVGQGRKHLGRV corresponding to amino acids 238 of T39971 P12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of T39971 P12, comprising a polypepti~
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 VPGAVG(2GRhHLGRV in T39971 P12.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein T39971 P12 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 13, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T39971 P 12 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 13 - Amino acid nzutcztions SNP positions) on Alternative amino Previously Down SNP?
amino acid acids) sequence 122 A -> S Yes 145 G -> No 180 C -> No 180 C -> W No 192 Y -> No 209 A -> No 211 T-> No Variant protein T39971 P12 is encoded by the following transcript(s): T39971 T16, for which the sequences) is/are given at the end of the application. The coding portion of transcript T39971 T16 is shown in bold; this coding portion starts at position 756 and ends at position 24~
1469. 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 T39971 P12 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 14 - Nucleic acid SNPs SNP posi~ian on nucleotideAlternative xtucleiei pxeviously known.
sequence acid S.I~I'?' 417 G -> C Yes 459 T -> C Yes 1387 C -> No 1406 -> A No 1406 -> G No 529 G -> T Yes 1119 G -> T Yes 1188 G -> No 1295 C -> No 1295 C -> G No 1324 -> T No 1331 C -> No 1381 C -> No As noted above, cluster T39971 features 28 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application.
These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster T39971 node 0 according to the present invention is supported by 76 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 15 below describes the starting and ending position of this segment on each transcript.
Table 1 S - Segment location on transcripts Transcript name ~~~eht starting positionSegment ending position ~~~
Segment cluster T39971 node_18 according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described.
Tlvs segment can be found in the following transcript(s): T39971 T 16. Table 16 below describes the starting and ending position of this segment on each transcript.
Table 16 - Segment location on transcripts Transcript name Segmeat starting Segment errdin~ xtasition position ~
Segment cluster T39971 node_21 according to the present invention is supported by 99 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5.
Table 17 below describes the starting and ending position of this segment on each transcript.
Table 17 - Segrnerrt location ora transcripts Transcript name Segment starting positionSegment ending position Segment cluster T39971 node 22 according to the present invention is supported by 7 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T5. Table 18 below describes the starting and ending position of this segment on each transcript.
Table 18 - Segment location on tr"ar2SCrIptS
Transcript nanne ~eg~~ht starkiing Segznirnt ending position pbsitaan Segment cluster T39971 node 23 according to the present invention is supported by 101 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12 and T'39971 T5.
Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on transcripts Transcripk naaoe Segmenk startingpositionSe~rn.ent ending positiari.
Segment cluster T39971 node 31 according to the present invention is supported by 94 libraries. The number of libraries was deterniined as previously described.
This segment can be found in the following transcript(s): T39971 T10 and T39971 T5. Table 20 below describes the starting and ending position of this segment on each transcript.
Table 20 - Segment location on transcripts Transcript name, Segment starting Segment ending position position Segment cluster T39971 node 33 according to the present invention is supported by 77 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5.
Table 21 below describes the starting and ending position of this segment on each transcript.
Table 21 - Segment location on transcripts Transc.~pt name Segment starting positaa~n~Se~tneait ending position Segment cluster T39971 node 7 according to the present invention is supported by 87 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 22 below describes the starting and ending position of this segment on each transcript.
Table ?2 - Segment location on transcripts T~s~pn name-- ~e~m~nt starting positionSegment ending position ~
T39971_T10 940 1162 T39971_T12 940 1162 T3997I_TS 940 1162 According to an optional embodiment of the present mventton, short segments reiatea to the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster T39971 node_1 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 23 below describes the starting and ending position of this segment on each transcript.
Table 33 - Segment location on transcripts Transcci~t name S~~le~nt stadrting Segment ending positiau position T39971 T10 811 ~ 819 T39971 TS 811 [819 Segment cluster T39971 node_10 according to the present invention is supported by 77 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s)T39971 TIO, T39971 T12, T39971 T16 and T39971 T5.
Table 24 below describes the starting and ending position of this segment on each transcript.
Table 24 - Segment location on transcripts Transcript name Segment starting position.Segment ending positicin Segment cluster T39971 node_I 1 according to the present invention is supported by 79 libraries. The number of libraries vvas determined as previously described.
This segment can be found in the following transcript(s)T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 25 below describes the starting and ending position of this segment on each transcript.
Table 25 - Segment location orr transcripts Transcript name Segment starting positionSegment ending positipn Segment cluster T39971 node_12 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 26 below describes the starting and ending position of this segment on each transcript.
Table '~ 6 - Segnze~at locatio~a on trarascriFts Transcript. name Segment startling i'Se~menr ending position' position Segment cluster T39971_node_15 according to the present invention is supported by 79 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 27 below describes the starting and ending position of this segment on each transcript.
Table 27 - Segment location on transcripts Trat~scnipt naic~e Segment,starting position~ Segment ending position , Segment cluster T39971 node_16 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 28 below describes the starting and ending position of this segment on each transcript.
Table 28 - Segment locr~:iort on tr-anscr-ipts Transcript naive Segment sfi.~rtiug Segment ending position ~ position T39971 T10 -~ 1317 1340 Segment cluster T39971 node_17 according to the present invention is supported by 86 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s)T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 29 below describes the starting and ending position of this segment on each transcript.
Table ?Q - Segment location on transcripts Transcript name Segix~ent starting Segment ending posihion position Segment cluster T39971 node 26 according to the present invention is supported by 85 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T5. Table 30 below describes the starting and ending position of this segment on each transcript.
Table 30 - Segment location on transcripts Transcript name Segment starting Segment. ending position position Segment cluster T39971 node 27 according to the present invention is supported by 90 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T5. Table 31 below describes the starting and ending position of this segment on each transcript.
Table 31 - Segment lOCatl011 Oit trarISCr"I~tS
Transcript name Segment stazting Segment ending position.
position Segment cluster T39971 node_28 according to the present invention can be found in the following transcript(s): T39971 T10 and T39971 T5. Table 32 below describes the starting and ending position of this segment on each transcript.
Table 32 - Segrnerzt location on transcripts Transcript name Segment starting Segment ending position ; position Segment cluster T39971 node 29 according to the present invention is supported by 99 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10 and T39971 T5. Table 33 below describes the starting and ending position of this segment on each transcript.
Table 33 - Segment lacatiort ort transcripts Trarrseript naive Segment starling ' Segment ending position position T39971 TS 2035 ~ 2129 Segment cluster T39971 node 3 according to the present invention is supported by 78 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 34 below describes the starting and ending position of this segment on each transcript.
Table 34 - Segrnent location on transcripts Transcript name ~egm.~t~t starting I segment ~n.~ling pasition~ --_.. . position T39971 TS 820 ~ 861 Segment cluster T39971 node_30 according to the present invention can be found in the following transcript(s): T39971 T10 and T39971 T~. Table 35 below describes the starting and ending position of this segment on each transcript.
Table 35 - Segrnerat location on transcripts Transcript name; Segment starting Segment ending position position II T39971 TS 2130 ~ 2137 Segment cluster T39971 node_34 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5. Table 36 below describes the starting and ending position of this segment on each transcript.
Table 36 - Segment location on transcripts Transcript name Se,~m.ent starting Segment ending position position Segment cluster T39971 node_35 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5. Table 37 below describes the starting and ending position of this segment on each transcript.
Table 37 - Segment locativrt an transcripts Trazascript name Segment starting positionSegment ~ncli~ig position -Segment cluster T39971 node 36 according to the present invention is supported by 51 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 T10, T39971 T12 and T39971 T5.
Table 38 below describes the starting and ending position of this segment on each transcript.
Table 38 - Segtrzettt location on transcripts Tramscrlpl name -- Segment starting positionS~gu~ent ending position ~
Segment cluster T39971 node 4 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971 T16 and T39971 T5.
Table 39 below describes the starting and ending position of this segment on each transcript.
Table 39 - Segtnent location can transcripts Transcript name Segment starting Segment ending position position T39971 T10 862 881' ~i TS 862 881 Segment cluster T39971_node_5 according to the present invention is supported by 80 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T39971 TIO, T39971 T12, T39971 T16 and T39971 T5.
Table 40 below describes the starting and ending position of this segment on each transcript.
Table 40 - Segment location orz transcripts Transcript name . Segment starting positionSegment ending position Segment cluster T39971 node_8 according to the present invention can be found in the following transcript(s): T39971 T10, T39971 T12, T39971_T16 and T39971 T5.
Table 41 below describes the starting and ending position of this segment on each transcript.
Table 41 - Segment locatiota on transcripts Transcript name ' Segment starting position. Segruent eniding position IS
Segment cluster T39971 node 9 according to the present invention can be found in the following transcript(s)T39971 T10, T39971 T1~, T39971 T16 and T39971 T5. Table below describes the starting and ending-position of this segment on each transcript.
Table ~12 - Segment location on transcriFts Transu~ipt name ferment starting i segment ending position position ~
T39971 T10 1169 ~ 1188 T39971_TS 1169 1188 Variant protein alignment to the previously known protein:
Sequence name: /tmp/xkraCL20cZ/43L7YcPH7x.:VTNC HUMAN
Sequence documentation:
Alignment of: T39971-P6 x VTNC HUMAN ..
Alignment segment 1/1:
Quality: 2774.00 Escore: 4 Matching length: 278 Total length: ~78 Matching Percent Similarity: 99.64 Matching Percent Identity: 99.64 Total Percent Similarity: 99.64 Total Percent Identity: 99.64 ~54 Gaps: 0 Alignment:
$ 1 MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSC 50 151 AEEELCSGKPFDAFTDLhTIGSLFAFRGQYCYELDEKAVRPGYPTCLIRDVW 200 251 PDNVDAALALPAHSYSGRERVYFFKGTQ 2~8 '.'51 PDNVDAALALPAHSYSGRERVYFFKGKQ
Sequence name: /tmp/X4DeeuSlB4/yMubSRSFPs:VTNC HUNLAN
Sequence documentation:
Alignment of: T39971-P9 x VTNC HUMAN ..
Alignment segment 1/1:
Quality: 4430.00 Escore: 0 Matching length: 447 Total length: 478 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 93.51 Total Percent Identity: 93.51 Gaps: 1 Alignment:
. - . . .
101 DLQAQSKGNPEQTPVLKPEEEAPAPEV~~ASKPEGIDSRPETLHFGRPQPP 150 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllllllllll Illlllllllllllllllllllllllllllllllllllllllllllllll . - - - -301 VFEHFAMMQRDSWEDIFELLFWGRT...............-....----. 325 IIIIIIIIIIIIIIIIIIIIIIIII
326 ......SGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRAT 369 IIIIIIIIIIIIIIIIilllllllllllllllllllllllllll 370 WLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQSVFFFSGDh'YYRVNLR 419 ?5 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
420 TRRV'DTVDPPYPRSIAQYWLGCPAPGHL 447 IIIillllllllllllllllllllllll Sequence name: /tmp/jvplVtnxNy/wxNSeFVZZw:VTNC HUMAN
Sequence documentation:
Alignment of: T39971 P11 x VTNC HT1MAN ..
Alignment segment 1/1:
Quality: 3576.00 Escore : 0 Matching length: 363 Total length: 478 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 ?0 Total Percent Similarity: 75.94 Total Percent Identity: 75.94 Gaps: 1 Alignment:
51 CTDYTAECh.PQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTS 100 151 AEEELCSGKPFDAFTDLh'I~1GSLFAFRGQYCYELDEKAVRPGYPKLIRDVW 200 . . . - -301 VFEHFAMMQRDSWEDIFELLFWGRTS........................ 326 3~6 .................................................. 326 327 .........................................DKYYRVNLR 335 Sequence name: /tmp/jvplVtn.~cNy/wxNSeFVZZw:Q9BSH7 Sequence documentation:
Alignment of: T39971-P11 x Q9BSH7 ..
Alignment segment 1/1:
Quality: 3576.00 Encore: 0 Matching length: 363 Total length: 478 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 75.94 Total Percent Identity: 75.94 Gaps : 1 Alignment:
Iillllllllllllllllllllllllllllllllllllllllllllllll 151 AEEELCSGKPFDAFTDLkTTGSLFAFRGQYCYELDEKAVRPGYPKLIRDVW200 151 AEEELCSGKPFDAFTDLY~TGSLFAFRGQYCYELDEKAVRPGYPKLIRDVW200 201 GIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVI~DPDYPRNISDGFDGI250 301 VFEHFAMMQRDSWEDIFELLFWGRTS........................ 326 . . . . .
32G .................................................. 32G
327 .........................................DKYYRVNLR 335 ~61 Sequence name: /tmp/fgebv7ir4i/48bTBMziJO:VTNC HUMAN
Sequence documentation:
Alignment of: T39971_P12 x VTNC HUMAN ..
Alignment segment 1/1:
Quality: 2237.00 Escore: 0 Matching length: 223 Total length: 223 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
1 MAPLRPLLILALLAV~V'ALADQESCKGRCTEGFNVDKKCQCDELCSYYQSC 50 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
51 CTDYTAECKPQVTRGDVFTMPEDEYTVYDDGEEhT7NATVHEQVGGPSLTS 100 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
l0 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Iillllllllllllllllllllllllllllllllllllllllllllllll 151 AEEELCSGKPFDAFTDLkTIGSLFAFRGQYCYELDEKAVRPGYPKLIRDVW 200 2_01 GIEGPIDAAFTRTNCQGKTYLFK 223 IIIIIIIIIIIIIIIIIIIIIII
Sequence name: /tmp/fgebv7ir4i/48bTBMziJ0:Q9BSH7 Sequence documentation:
Alignment of: T39971-P12 x Q9BSH7 ..
Alignment segment 1/1:
Quality: 2237.00 Encore: 0 Matching length: 223 Total length: 223 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Illllllllllilllllillllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
?5 101 DLQAQSKGNPEQTPVLKPEEEAPAFEVGASKPEGIDSRPETLHPGRPQPP 150 Iillllllllllllllllllilllllllllllllllllllllllllilll 151 AEEELCSGKPFDAFTDLI~TGSLFAFRGQYCYELDEKAVRPGYPKLIRDVW 200 201 GIEGPIDAAFTRINCQGh'TYLFK 223 Expression of VTNC-HUMAN vitronectin (serum spreading factor, somatamedin B, complement S-pratein) T39971 transcripts, which are detectable by amplicon as depicted in sequence name T39971 junc23-33 in normal and cancerous breast tissues E,r-pression of VTNC-HUIvIAN vitronectin (serum spreading factor, somatomedin B, complement S-protein) transcripts detectable by or according to junc23-33, T39971 junc23-33 amplicon and T39971 junc23-33F and T39971 junc23-33R primers was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No.
BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM_000194;
amplicon - HPRT1-amplicon), SDHA (GenBank Accession No. NM-004168; amplican -SDHA-amplicon), and G6PD (GenBank Accession No. NM-000402; G6PD amplicon), was measured similarly. For each RT sample, the expression of the above amplican was normalised to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 56-60, 63-67, Table 1, above, "Tissue samples in testing panel"), to obtain a value of fold differetial expression for each sample relative to median of the normal PM
samples.
Figure 10 is a histogram showing dawn regulation of the above-indicated VTNC_HCJMAN vitranectin (serum spreading factor, samatornedin B, complement S-protein) transcripts in cancerous breast samples relative to the normal samples.
As is evident from Figure 10, the expression of VTNC-HUMAN vitronectin (serum spreading factor, somatomedin B, complement Sprotein) transcripts detectable by the above amplicon in cancer samples was significantly lower than in the non-cancerous samples (Sample Nos. 56-60, 63-67 Table 1, "Tissue samples in testing panel").
Primer pairs are also aptionally and preferably encompassed within the present invention; for example, for the above experiment, the follawing primer pair was used as a non-limiting illustrative example only of a suitable primer pair: T39971 junc23-33F forward primer;
and T39971 junc23-33R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: T39971 junc23-33.
T39971junc22-33F (SEQ )D N0:834): GGGGCAGAACCTCTGACAAG
T39971junc22-33R (SEQ )D N0:835): GGGCAGCCCAGCCAGTA
T39971junc22-33 amplicon (SEQ )D N0:836):
GGGGCAGAACCTCTGACAAGTACTACCGAGTCAATCTTCGCACACGGCGAGTGGAC
ACTGTGGACCCTCCCTACCCACGCTCCATCGCTCAGTACTGGCTGGGCTGCCC
Expression of VTNC_HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein), antisense to SARM1 (T23434), T39971 transcripts which are detectable by amplicon as depicted in sequence name T39971 junc23-33 in different normal tissues Expression of VTNC-HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein), transcripts detectable by or according to T39971ji~rtc23-33 amplicon(s) and T39971junc?3-33F and T39971ji~nc23-33R was measured by real time PCR. In parallel the expression of four housekeeping genes -RPL19 (GenBank Accession No.
NM_000981;
RPL19 amplicon), TATA box (GenBank Accession No. NM_003194; TATA amplicon), UBC
(GenBanl: Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA
(GenBank Accession No. NM-004168; amplicon - SDHA-amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT
sample was then divided by the median of the quantities of the breast samples (Sample Nos. 33-35, Table 2, "Tissue samples in normal panel" above), to obtain a value of relative expression of each sample relative to median of the breast samples. Primers and amplicon are as above.
The results are presented in Figure 11, demonstrating the expression of VTNC-HUMAN
vitronectin .(serum spreading factor, somatomedin B, complement S-protein), antisense to SARM1 (T23434), T39971 transcripts, which are detectable by amplicon as depicted in sequence name T39971junc23-33, in different normal tissues.
Expression of VTNC-HUMAN vitronectin (serum spreading factor, somatomedin B, complement S-protein) T39971 transcripts which are detectable by amplicon as depicted in sequence name T39971 seg22 in normal and cancerous breast tissues Expression of VTNC-HUI~~IAN vitronectin (serum spreading factor, somatomedin B, complement S-protein) transcripts detectable by or accoramg to seg~~, 1 J77 /
1 SCg amplicon(s) and primers T39971 seg22F and T39971 seg22R was measured by real time PCR.
In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No.
BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM_000194;
amplicon - HPRT1-amplicon), SDHA (GenBank Accession No. NM 004165; amplicon -SDHA-amplicon), G6PD (GenBank Accession No. NM_000402; G6PD amplicon), was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post mortem (PM) samples (Sample Nos. 56-60, 63-67, Table 1: Tissue samples in testing panel, above), to obtain a value of fold differential expression for each sample relative to median of the normal PM samples.
In one experiment that was carried out no differential expression in the cancerous samples relative to the normal PM samples was observed. However, this may be due to a problem that is specific to this particular experiment.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a no,i-limiting illustrative example only of a suitable primer pair: T39971 seg22F
forward primer; an ad T39971 seg22R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: T39971 seg22.
Forward primer T39971 seg22F (SEQ ID NO :837):
GCAGTCTTGGATTCCTTTCACATT
Reverse primer T39971 seg22R (SEQ ID NO :838):
GAGGCTGTTGAAGTTAGGATCTCC
Amplicon T39971 seg22 (SEQ ID NO :839):
GCAGTCTTGGATTCCTTTCACATTTCACTGGGGACAGGCCTCAGCATGTGCCCACCC
CTGACCCCCACCTCATGCTGGGAGATCCTAACTTCAACAGCCTC
Cluster 221368 features 7 transcripts) and 34 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table I - Transcripts of interest Transcript Sequence TD No.
N'am~e 221368 PEA_1T10 55 221368 PEA_1TS 59 221365 PEA_1T6 60 Z21368_PEA_1 T9 ~ 61 Table ~ - Segments of iraterest _~ ~
Segment - I Seduence IU hTo:
Narne Z21368_PEA_1 0 62 node_ Z21368_PEA_1 15 63 node_ Z21368_PEA_1 19 64 node_ 221368PEA_1 2 65 node 221368PEA_1 21 66 node Z21368_PEA_1 33 67 node Z21368_PEA_1 36 68 node Z21368_PEA_1 37 69 node 221368PEA_1 39 74 node Z21368_PEA_1 4 71 node Z21368_PEA_1 41 72 node Z21368_PEA_1 43 73 node Z21368_PEA_1 _45 74 node Z21368_PEA_1 _53 75 node Z21368_PEA_1 56 76 node Z21368_PEA_1 58 77 node Z2136SPEA_1 66 78 node Z21368_PEA_1 _67 79 node Z21368_PEA_1 69 80 node Z2136SPEA_1 _11 81 node Z21368_PEA_1 _12 82 node 221368PEA _1 _16 83 node Z21368_PEA_1 _17 84 node Z21368_PEA_1 23 85 node 221368PEA _1 _24 86 node Z21368_PEA_1 node30 87 221368PEA1 node31 88 Z21368_PEA_1 node38 89 221368PEA_1 node47 90 221368PEA1 node49 91 Z21368_PEA_1 node51 92 221368PEA1 node61 93 Z2136SPEA1 node68 94 Z21368_PEA_1 node7 95 Table 3 - Prc~teifis of interest Protein ! Sequence ID No. _ . . .
Name ~
PEA
PEA
PEA
PEA
PEA
PEA
These sequences are variants of the known protein Extracellular sulfatase Sulf 1 precursor (SwissProt accession identifier SLJL1 IILTNIAN; known also according to the synonyms EC
3.1.6.-; HSulf 1), SEQ ID NCB: 96, referred to herein as the previously known protein.
Protein Extracellular sulfatase Sulf 1 precursor is known or believed to have the following function(s): Exhibits arylsulfatase activity and highly specific endoglucosamine-6-sulfatase activity. It can remove sulfate from the C-6 position of glucosamine within specific subregions of intact heparin. Diminishes HSPG (heparan sulfate proteoglycans) sulfation, inhibits signaling by heparin-dependent growth factors, diminishes proliferation, and facilitates apoptosis in response to exogenous stimulation. The sequence for protein Extracellular sulfatase Sulf 1 precursor is given at the end of the application, as "Extracellular sulfatase Sulf 1 precursor ?70 amino acid sequence" (SEQ ID N0:96). Known polymorphisms for this sequence are as shown in Table 4.
Table ~l - Arnifzo acid nt2ttatiorts for Knc~u~n Protein SM' pasition(s) Cvmme~nt on amine acid sequence 87 - 88 CC->AA: LOSS OF ARYLSULFATASE ACTMTY
AND LOSS OF ABILITY TO MODULATE APOPTOSIS.
49 L -> P
728 K -> R
Protein Extracellular sulfatase Sulf 1 precursor localization is believed to be Endoplasmic reticulum and Golgi stack; also localized on the cell surface (By similarity).
The. following GO Annotations) apply to the previously known protein. The following annotations) were found: apoptosis; metabolism; heparan sulfate proteoglycan metabolism, which are annotations) related to Biological Process; arylsulfatase;
hydrolase, which are annotations) related to Molecular Function; and extracellular space;
endoplasmic reticulum;
Golgi apparatus, which are annotations) 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/>.
Cluster 221368 can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term "number" in the right hand column of the table and the numbers on the taxis of Figure 12 refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histograms in Figure 12 and Table 5. This cluster is overexpressed (at least at a minimum level) in the following laathological conditions: epithelial malignant tumors, a mit;ture of malignant tumors from different tissues and pancreas carcinoma.
Table 5 - Nut~nal tissue distribution Name afTissue ' Nor bladder 123 Bone 557 Brain 34 Colon 94 epithelial 56 general 6S
head and neck 0 kidney 35 Lung 22 lymph nodes 0 breast 52 muscle 31 ovary 0 pancreas 0 prostate 44 skin 67 stomach 109 T cells 0 Thyroid 0 uterus 140 Table 6 - P values afid ratios for expression in caricer~zrs tissue Name ~f TissueP l 1'2 SP1 R3 SP2 R4 bladder 5.4e-01 6.6e-016.4e-011.0 S.Se-O10.7 bone ( 4.5e-01~ S.2e-O1~ 9.1e-01~ 0.4 ~ 1 ~ 0.3 brain 5.5e-017.3e-01l.Se-O1 1.5 S.Oe-O10.9 colon 1.4e-012.8e-01l.Oe-O1 2.0 3.0e-011.4 epithelial 1.1 1.5e-011.2e-07 2.1 l.Oe-O 1.1 e-03 l general 1.4e-055.3e-021.9e-06 1.6 6.7e-010.8 head and neck2.4e-027.1e-024.6e-01 2.5 7.5e-011.4 kidney 8.9e-0 9.0e-0 1 0.4 1 0.4 lung 3.5e-014.1e-017.2e-03 2.6 l.Oe-O11.6 lymph nodes 7.7e-023.1e-012.3e-02 8.5 1.9e-013.2 breast 4.0e-0 6.1 5.4e-02 2.3 3.0e-0 1.3 1 e-O 1 muscle 7.5e-023.5e-021 1.0 1.7e-011.7 ovary 3.8e-014.2e-012.2e-01 2.9 3.4e-012.2 pancreas 2.2e-026.9e-021.4e-08 6.5 1.4e-064.6 prostate 8.3e-018.9e-013.1e-01 1.4 5.2e-011.1 skin 6.1e-018.1e-016.0e-01 1.2 1 0.3 stomach 4.4e-02S.Oe-O1S.Oe-O1 0.8 9.7e-010.4 T cells S.Oe-O16.7e-013.3e-01 3.1 7.2e-011.4 Thyroid 3.6e-013.6e-011 1.1 1 1.1 uterus 3.5e-017.8e-014.6e-01 0.9 9.1e-010.5 As noted above, cluster 221368 features 7 transcript(s), which were listed in Table 1 above. These transcripts) encode for protein(sl which are variants) of protein Extracellular sulfatase Sulf 1 precursor. A description of each variant protein according to the present invention is now provided.
Variant protein Z21368_PEA_1 P2 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) Z21368_PEA_1 T5. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 221368 PEA_1 P2 and SUL1 HUMAN:
l.An -isolated chimeric polypeptide encoding for Z2136S_PEA_1 P2, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELGSL
QVIvvINKTRh11V1EHGGATFINAFVTTPMCCPSRSSMLTGKYVI~~THI~VYT1~NENCSSPSW
QAMFIEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYIPPGWREWLGLIKNSRFYNYTVCR
NGIKEKHGFDYAKDYFTDLITNESINYFKMSKRNIYPHRPVMMVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYNYYAPNMDKHWIMQYTGPMLPIHMEFTNIL,QRhRLQTLMSVDD
SVERLYNMLVETGELENTYIIYTADHGYHIGQFGLVKGKSMPYDFDLRVPFFIRGPSVEP
GSIVPQIVLNIDL,APTILDIAGLDTPPDVDGKSVLKLLDPEKPGNRFRTNKhAKIWRDTFL
VERGKFLRKKEESSKNIQQSNHI.PKYERVKELCQQARYQTACEQPGQKWQCIEDTSGK
LR1HKCKGPSDLLTVRQSTRNLYARGFHDKDKECSCRESGYRASRSQRIkSQRQFLRNQ
GTPKYKPRFV'I-ITRQTRSLSVEFEGEIYDINLEEEEELQVLQPRNIAKRHDEGHKGPRDLQ
ASSGGNRGRMLADSSNAVGPPTTVRVTHKCFILPNDSIHCERELIIQSARAWKDHKAYI
DKEIEALQDKIKNLREVRGHLKRRKPEECSCSKQSYYNkEKGVKKQEKLKSHLHPFKE
AAQEVDSKLQLFKENNRRRhKERKEKRRQRIiGEECSLPGLTCFTHDNNHWQTAPFWN
corresponding to amino acids 1 - 761 of SL1L1 HUMAN, which also corresponds to amino acids 1 - 761 of 221368 PEA_1_P2, 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°'o homologous to a polypeptide having the sequence PHKYSAHGRTRHFESATRTTNGAQKLSRI corresponding to amino acids 762 - 790 of 221368 PEA_1 P2, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated polypeptide encoding for a tail of 221365 PEA_1 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence PHKYSAHGRTRHFESATRTTNGAQILLSRI _in 221368 PEA_1 P2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protr~~n localization is believed to be secreted because both signal-peptide prediction prograt~as predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein Z21368_PEA_1 P2 is encoded by the following transctipt(s):
Z2136S_PEA_1 T5, for which the sequences) is/are given at the end of the application. The coding portion of transcript 221368 PEA_1 TS is shown in bold; this coding portion starts at position 529 and ends at position 2898.
Variant protein 221368 PEA_1 PS according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) 221368 PEA_1 T9. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between Z2136fi PEA_1 PS and Q7Z2W2 (SEQ ID N0:840):
l.An -isolated chimeric polypeptide encoding for Z21368_PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIIL,VLTDDQDVEL
corresponding to amino acids 1 - 57 of Q7Z2W2, which also corresponds to amino acids 1 -.57 of Z21368_PEA_1 P5, second bridging amino acid sequence comprising A, and a third amino acid sequence being at least 90 % homologous to FFGKYLNEYNGSYIPPGWREWLGLIICNSRFYNYTVCRNGIKEKHGFDYAKDYF TDLITN
ESINYFKMSKR11~I~.'PHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNM
DKHWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDDSVERLYNMLVETGELENTYIIYT
ADHGYHIGQFGLVKGKSMPl'DFDIRVPFF1RGPSVEPGSIVPQIVLNIDLAPTILDIAGLDT
PPDVDGKSVLKLLDPEKPGNRFRTNKILAILIWRDTFLVERGKFL.RILKEESSKNIQQSNHL, PKYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRNLY
ARGFHDKDIkECSCRESGYRASRSQRKSQRQFLRNQGTPIiYKPRFVHTRQTRSLSVEFE
GEIYDINLEEEEELQVLQPRNIAKRI-ff~EGHhGPRDLQASSGGNRGRMLADSSNAVGPPT
TVRVTHKCFILPNDSIHCERELYQSARAWILDHKAYIDKEIEALQDKIKNLREVRGHLKR
RKPEECSCSKQS1'YNIkEKGVKKQEKLKSHLHPFKEAAQEVDSKLQLFKETfNRRRICILER
KEKRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNE
THNFLFCEFATGFLEYFDMNTDPYQLTNTVHT'VERGIL,NQLHVQLMELRSCQGYILQCN
PRPKNLDVGNKDGGSYDLHRGQLWDGWEG corresponding to amino acids 139 - 871 of Q7~2W2, which also corresponds to amino acids 59 - 791 of Z21368_PEA_1 P5, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
2.An _ _isolated polypeptide encoding for an edge portion of 221368 PEA_1 P5, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least three amino acids comprise LAF having a structure as follows (numbering according to X21368 PEA_1 PS): a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 59 +
((n-2) - x), in which x varies from 0 to n-2.
Comparison report between Z21368_PEA_1 PS and AAH12997 (SEQ ID NC~:841):
l.An _isolated chimeric polypeptide encoding for Z21368_PEA_1 P5, comprising a first amino acid sequence being at least 70°fo, optionally at least SO%, preferably at least 85%, more preferably at least 90% and most preferably at least 95°ro homologous to a polypeptide having the sequence MK1'SCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELAFF
GKYLNEYNGSYIPPGWREVvTL,GLIhNSRFYNYT'VCRNGIKEKHGFDYAKDYFTDLITNES
INYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNMDK
HWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDDSVERLYNMLVETGELENTYIIYTAD
HGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTILDIAGLDTPP
DVDGKSVLKLLDPEKPGNRFRTNKKA.IZIWRDTFLVERGKFLRKKEESSKNIQQSNHLP
KYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHILCKGPSDLLTVRQSTRNLYA
RGFHDKDKECSCRESGY~RASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEFEGE
IYDINLEEEEELQVLQPRNIAKRI-IDEGHKGPRDLQASSGGNRGRMLADSSNAVGPPTTV
RVTHKCFIL,PNDSIHCERELYQSARAWKDHKAYIDKEIEALQDKIkNLREVRGHLKRRK
PEECSCSKQSYYNKEKGVKKQEKLIiSHLHPFKEAAQEVDSKLQLFKENNR_RRICILERKE
KRRQRKGEECSLPGLTCFTHDNNHWQTAPFWNLGSFCACTSSNNNTYWCLRTVNETH
NFLFCEFATGFLEYFDMNTDPYQLTNTVI-ITVERGIL,NQLHVQLME corresponding to amino acids 1 - 751 of Z2136S PEA_1 P5, and a second amino acid sequence being at least 90 homologous to LRSCQGY~KQCNPRPKNLDVGNKDGGSYDLHRGQLWDGWEG
corresponding to amino acids 1 - 40 of AAH12997, which also corresponds to amino acids 752 -791 of 221368 PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated polypeptide encoding for a head of Z2136g PEA_1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence MKYSCCALVLAVL,GTELLGSLCSTVRSPRFRGRIQQERKNIRPNILLVLTDDQDVELAFF
GhYLNEYNGSYIPPGWREWLGLIKNSRFYNI'TVCRNGIkEKHGFDYAhDYFTDLITNES
INYFKMSK12MY~PHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPNMDK
HWIMQYTGPMLPIHMEFTNILQRKRLQTLMSVDDSVERLYNMLVETGELENTYIIYTAD
HGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVLNIDLAPTIL,DIAGLDTPP
DVDGKSVLKLLDPEKPGNRFRTNKhAKIWRDTFLVERGKFLRKKEESSKNIQQSNHLP
KYERVKELCQQARYQTACEQPGQKWQCIEDTSGkLFIHKCKGPSDLLTVRQSTRNLYA
RGFHDKDKECSCRESGYRASRSQRKSQRQFLRNQGTPKYILPRFVHTRQTRSLSVEFEGE
IYDINLEEEEELQVLQPRNIAKRHDEGHKGPRDLQASSGGNRGRMLADSSNAVGPPTTV
RVTHKCFILPNDSIHCERELYQSARAWKDIiKAYIDKEIEALQDKIkNLREVRGHLKI~
PEECSCSKQSYYNKEKGVKKQEILLKSHLHPFKEAAQEVDSKLQLFKENNRRRhhERKE
IiRRQRKGEECSLPGLTCFTHDNNHWQTAPFVVNL,GSFCACTSSNNNTYWCLRTVNETH
NFLFCEFATGFLEYFDMNTDPYQLTNTVHTVERGILNQLHVQLME of Z2136g PEA_1 P5.
Comparison report between Z2136g PEA_1 PS and SUL1 HUMAN:
l.An - .isolated chimeric polypeptide encoding for Z2136s PEA_1 P5, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVEL
corresponding to amino acids 1 - 57 of SUL1 I-ICTMAN, which also corresponds to amino acids 1 - 57 of 22136& PEA_1 P5, and a second amino acid sequence being at least 90 homologous to AFFGIkYLNEYNGSYL"~fr~;iWREWLGLIKNSRFYNYTVCRNGIIiEKHGFDYAKDYFTDLIT
NESINYFKMSKRM1'P i~ I~PVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYNYAPN
MDKHWIMQYTGPMLF'1~~MEFTNIL,QRKRLQTLMSVDDSVERLYNN>ZVETGELENTYII
YTADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEPGSIVPQIVL,NII7LAPTILDIAGL
DTPPDVDGKSVLKLLDPEKPGNRFRTNKKAIKiWRDTFLVERGKFLRKKEESSKNIQQSN
HLPKYERVKELCQQARYQTACEQPGQKWQCIEDTSGKLRIHKCKGPSDLLTVRQSTRN
LYARGFHI)KDKECSCRESGI'RASRSQRKSQRQFLRNQGTPKYhPRFVHTRQTRSLSVE
PTTVRVTHKCFILPNDSIHCERELYQSARAWKDHKAY)DKEIEALQDKIKNLREVRGHL
KRRKPEECSCSKQSYYNKEKGVKKQEKLKSHLHPFILEA,AQEVDSKLQLFKErfNRRRh KERKEKRRQRKGEECSLPGLTCFTHI~NNHVvIQTAPFWNLGSFCACTSSNNNTYWCLRT
VNETHNFLFCEFATGFLEYFDMNTDPYQLTNTVHTVERGILNQLHVQLMELRSCQGYK
QCNPRPKNLDVGNKDGGSYDLHRGQLWDGWEG corresponding to amino acids 138 - 871 of SULI -HUMAN, which also corresponds to amino acids 58 - 791 of 221368_PEA_1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated chimeric polypeptide encoding for an edge portion of Z21368_PEA_1 P5, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise LA, having a structure as follows: a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 58 + ((n-2) - x), in which x varies from 0 to n-2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein Z21368_PEA_1 PS is encoded by the following transcript(s):
Z21368_PEA_1 T9, for which the sequences) is/are given at the end of the application. The 27s coding portion of transcript Z21368_PEA_1 T9 is shown in bold; this coding portion starts at position 556 and ends at position 2928.
Variant protein Z21368_PEA_1 P15 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) Z21368_PEA_1 T23. An alignment is given to the lmown protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or mare alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 221368 PEA_1 P15 and SUL1 HUMAN:
l.An -isolated chimeric polypeptide encoding for Z21368_PEA_1 P15, comprising a first amino acid sequence being at least 90 °ro homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELGSL
QVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYVI-INHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYIPPGWREWLGLIKNSRFYN1'TVCR
NGIKEKHGFDYYAILDYFTDLITNES1NYYFKMShRMI'PHRPVMMVISHAAPHGPEDSAPQ
FSKLYPNASQHITPSYN1'APNNLDKHWIMQYTGPMLPIHMEFTNILQRhRLQTLMSVDD
SVERLYNMLVETGELENTYII1'TADHGYHIGQFGLVKGKSMPYDFDIRVPFFIRGPSVEP
GSIVPQIVLNIDLAPTILDIAGLDTPPDVDGKSVLILLLDPEKPGNRFRTNKKAKIWRDTFL
VERG -corresponding to amino acids 1 - 416 of SUL1 HUMAN, which also corresponds to amino acids 1 - 416 of Z21368_PEA_1 P15.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neitl~r traps-membrane region prediction program predicts that this protein has a traps-membrane region.
Variant protein Z2136S PEA_1 P15 is encoded by the following transcript(s):
Z21368_PEA_1 T23, for which the sequences) is/are given at the end of the application. The coding portion of transcript Z2136S PEA_1 T23 is shown in bold; this coding portion, starts at position 691 and ends at position 1938.
Variant protein X21365 PEA_1 P16 according to the present invention has an amino acid sequence as given at tl~ end of the application; it is encoded by transcripts) 221368 PEA_I T24. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 221368_PEA_1 P16 and SUL.1 FiLTMAN:
l.An _ -isolated chimeric polypeptide encoding for 221368 PEA_1 P16, comprising a first amino acid sequence being at least 90 % homologous to MhYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERhNIRPNIILVLTDDQDVELGSL
QVMNIkTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYVHNHNVYTNNENCSSPSW
QAMHEPRTFAVYLNNTGYRTAFFGRYLNEYNGSYIPPGWREWLGLIILNSRFYNYTVCR
NGIKEICHGFDYAKDYFTDLITNESINYFIILMSKRM1'PHRPVMMVISHAAPHGPEDSAPQ
FSItLYPNASQHITPSYNYAPN1\~kHWIMQYTGPMLPIHMEFTNILQRItRLQTLMSVDD
SVERLYMMLVETGELENTYIIYTADHGYHIGQFGLVKGhSMPYDFDIRVPFFIRGPSVEP
GSIVPQIVLNIDLAPTILDIAGLDTPPDVDGItSVLICLLDPEIH'GNR corresponding to amino acids 1 - 397 of SUL1 HUMAN, which also corresponds to amino acids 1 - 397 of X21368 PEA_1 P16, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least RS%, more preferably at least 9p% and most preferably at least 95%
homologous to a polypeptide having the sequence CVIVPPLSQPQIH corresponding to amino acids 398 - 410 of Z21368_PEA_1 P16, wherein said first and second amino acid sequences are contiguous and in a segue ntial order.
2.An _isolated polypeptide encoding for a tail of 221368_PEA_1 P16, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence CVIVPPLSQPQIH in Z21368_PEA_1 P16.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignaIP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neitl~r trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein Z21368_PEA_1 P16 is encoded by the following transcript(s):
221368 PEA_1 T24, for which the sequences) is/are given at the end of the application. The coding portion of transcript Z21368_PEA_1 T24 is shown in bold; this coding portion starts at position 691 and ends at position 1920.
Variant protein Z21368_PEA_1 P22 according to the present invention has an amino acid sequence as given at tl~ end of the application; it is encoded by transcripts) 221368 PEA_1 T10. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between Z21368_PEA_1_P22 and SUL1 HUMAN:
l.An _isolated chimeric polypeptide encoding for Z21368_PEA_1 P22, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIR.PNIILVLTDDQDVELGSL
QVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYVHNHN~rY'TNNENCSSPSW
QAI~gIEPRTFAVYLNNTGYRTAFFGKYLNEYNGSYYIPPGWREWLGLIKNSRFY'NYTVCR
NGIKEkHGFDYAK _corresponding to amino acids 1 - 188 of SUL1 HUMAN, which also corresponds to amino acids 1 - 188 of Z21368_PEA_1 F22, 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 AR~~'DGDQPRCAPRPRGLSPTVF corresponding to amino acids 189 - 210 of Z21368_PEA_1 P22, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of Z21368_PEA_1 P22, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, mare preferably at least about 90% and most preferably at least about 95%
homologous to the sequence ARYDGDQPRCAPRPRGLSPTVF in Z21368_PEA_1 P22.
The location of the variant protein was determined according to results from a number of different software programs and ar~lyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein 221368_PEA_1 P22 is encoded by the following transcript(s):
Z21368_PEA_1 T10, for which the sequences) is/are given at the end of the application. The coding portion of transcript Z21368_PEA_1 T10 is shown in bold; this coding portion starts at position 691 and ends at position 1320.
Variant protein Z21368_PEA_1 P23 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) 221368 PEA_1 T11. An alignment is given to the known protein (Extracellular sulfatase Sulf 1 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as fo Mows:
Comparison report between Z21368_PEA_1 P23 and Q7Z2W2:
l.An _ _isolated chimeric polypeptide encoding for 221368 PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologous to IvIKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERILNIRPNIIL,VLTDDQDVELGSL
QAMI-iEPRTFAVYLNNTGYRT corresponding to amino acids 1 - 137 of Q7~2W2, which also corresponds to amino acids 1 - 137 of Z21368_PEA_1 P23, 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% hmnologous to a polypeptide having the sequence GLLHRLNH corresponding to amino acids 138 - 145 of Z21368_PEA_1 P23, wherein said first and second amino acid sequences are° contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of Z21368_PEA_1 P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85°.~0, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence GLLHRLNH in Z21368_PEA_1 P23.
Comparison report between Z2136S PEA_1 P23 and SUL1 I-IIJIvIAN:
l.An isolated chimeric polypeptide encoding for Z21368_PEA_1 P23, comprising a first amino acid sequence being at least 90 % homologous to MKYSCCALVLAVLGTELLGSLCSTVRSPRFRGRIQQERKNIRPNIILVLTDDQDVELGSL
QANIIIEPRTFAVYLNNTGYRT corresponding to amino acids 1 - 137 of SLTLI HUMAN, which also corresponds to amino acids 1 - 137 of Z21368_PEA_1 P23, and a second amino acid sequence being at least 70%, optionally at least 80°~0, preferably at least 85%, more preferably at least 90% and most preferably at least 95°~o homologous to a polypeptide having the sequence GLLHRLNH corresponding to amino acids 138 - 145 of Z31368_PEA_1 P23, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of Z31368_PEA_1 P23, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence GLLHRLNH in Z21368_PEA_1 P23.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignaIP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein 221368 PEA_1 P23 is encoded by the following transcript(s):
Z21368_PEA_1 T11, for which the sequences) is/are given at the end of the application. The coding portion of transcript 221368 PEA_1 T11 is shown in bold; this coding portion starts at position 691 and ends at position 1125.
As noted above, cluster 221368 features 34 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application.
These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster 221368 PEA_1 node 0 according to the present invention is supported by 8 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PE<A_1 T9. Table 7 below describes the starting and ending position of this segment on each transcript.
Table 7 - Segment location on transcripts Tz~anscript name Segment starting positionSegment endizy' position Segment cluster 221368 PEA_1 node_15 according to the present invention is supported by 26 libraries. The number of libraries was determined as previously described. This segment - -can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23, _ - -Z21368_PEA_1 T24, Z21368_PEA_1 T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 8 below describes the starting and ending position of this segment on each transcript.
Table 8 - Segrnerzt location on transcripts Transcript name Segment starting Segment ending position position Z21368_PEA_1 T10 631 80~
221368 PEA_l_T11 631 807 221368FEA_1 T23 631 807 Z21368_PEA_1 T24 631 807 221368PEA_1 TS 469 645 Z21368_PEA_1 T6 469 645 Z2136bPEA_1 T9 496 672 Segment cluster Z21368_PEA_1 node_19 according to the present invention is supported by 24 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z2136S PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_I_T23, Z21368_PEA_1 T24, _Z21368_PEA_1 TS and Z21368_PEA_1 T6.
Table 9 below describes the starting and ending position of this segment on each transcript.
Table 9 - Segrnertt location on transcripts Transcript name S~~rnent starting Segment ending positiau position 221368 PEA_1 T10 863 1102 Z21368_PEA_1 T23 863 1102 Z21368_PEA_1 T24 863 1102 221368 PEA_1 T5 701 940 Z21368_PEA_1 T6 ~ 701 ~ 940 Segment cluster Z21368_PEA_1 node 2 according to the present invention is supported byy 15 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23, _ -Z21368_PEA_1 T24, Z21368_PEA_1 TS and Z21368_PEA_1 T6.
Table 10 below describes the starting and ending position of this segment on each transcript.
Table 10 - Seg~rierat location on transcripts Transcriptnarile I Segment starting position I Segment ending position .'~'35 Z21368_PEA_1 T10 1 300 Z21368_PEA_1 T11 1 300 Z21368_PEA_1 T23 1 300 Z21368_PEA_1 T24 1 300 221365PEA_1 T5 1 300 221368PEA_1 T6 1 300 Segment cluster 221368 PEA_1 node_21 according to the present invention is supported by 37 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, Z21368_PEA_1 T23, 221368 PEA_1 724221368 PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 11 below describes the starting and ending position of this segment on each transcript.
Tale II - SegrrZent location orr transcripts Transcript ~eg~~nC starting pasitionSe~merit ending ~rosition r~~ne .
Z21368_PEA_1_T10 1103 1254 Z21368_PEA_1 1103 1254 221368PEA_I 1103 1254 Z21368_PEA_1 941 1092 Z21368_PEA_1 728 879 Segment cluster Z21368_PEA_1 node_33 according to the present invention is supported by 45 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 710, Z21368_PEA_1 711, Z21368_PEA_1 723, _ -Z21368_PEA_1 724, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 12 below describes the starting and ending position of this segment on each transcript.
Table I? - Segment location on transcripts 28b Transcript Segment starting positionSegment ending positian name Z21368_PEA_1 1502 1677 Z21368_PEA_1 1424 1599 Z21368_PEA_1 1576 1751 Z21368_PEA_1 1414 1589 TS
Z21368_PEA_1 1414 1589 Z21368_PEA_1 1201 1376 Segment cluster 221368 PEA_1 nude 36 according to the present invention is supported by 44 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s)Z2136S PEA_1 T10, 221368 PEA_1 T1 l, Z21368_PEA_1 T23, Z21368_PEA_1 T24, Z21368_PEA_1 T5, Z2136S PEA_1 T6 and Z21368_PEA_1 T9. Table 13 below describes the starting and ending position of this segment an each transcript.
Table 13 - Segment loeatiort ort tnanscr~ipts Transcript rJame Se~ent starting pasitionSegnnent ending pvsitiari ' Z21368_PEA_1 T10 167S 1806 221368 PEA_1 T11 1600 1728 Z21368_PEA_1 T23 1752 1880 Z21368_PEA_1 T24 1752 1880 Z21368_PEA_1 TS 1590 1718 Z21368_PEA_1 T6 1590 1718 Z21368_PEA_1 T9 1377 1505 Segment cluster 221368 PEA_1 node 37 according to the present invention is supported by 3 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T24. Table 14 below describes the starting and ending positian of this segment on each transcript.
Table 1 ~ - Segrnerzt location on transcripts Transcript name Segment starting j Se~~nt ending position posi~ian Segment cluster 221368 PEA_1 node 39 according to the present invention is supported by 5 libraries. The number of libraries was determined as previausly described. This segment can be found in the following transcript(s): Z21368_PEA_1 T23 and Z21368_PEA_1 T24.
Table 15 below describes the starting and ending position of this segment on each transcript.
Table I S - Seg»tent location ora transcripts Transcript zaarne Segment starting :Segment ending~~ositian ~aosition 221368 PEA_1 T23 1938 ~ 2790 Z21368_PEA_1 T24 2217 3069 Segment cluster 221368 PEA_1 node 4 according to the present invention is supported by 13 libraries. The number of libraries was determined as previously described. This segment can be found in the following _ -transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23 and Z21368_PEA_1 T24. Table 16 below describes the starting and ending position of this segment on each transcript.
Table 16 - Segnte»t location o» transcripts Transcript name Segment starting pasitionSegment ending position 221368 PEA_1 T10 301 462 Z21368_PEA_1 T11 301 462 Z21368_PEA_1 T23 301 462 221368 PEA_1 T24 301 462 zss Segment cluster Z21368_PEA_1 node 41 according to the present invention is supported by 49 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s)Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z2136S_PEA_1 T5; Z2136S_PEA_1 T6 and Z21368_PEA_1 T9. Table 17 below describes the starting and ending position of this segment on each transcript.
Table 1 ? - Segrraent locatiora can transcripts Tr~nscz~pt na~te , Segment starting position~ Segment endi~a;g position ~' Z21368_PEA_1_T10 1864 1993 221368 PEA_1 T11 1786 1915 Z21368_PEA_1 TS 1776 1905 Z21368_PEA_1 T6 1776 1905 Z21368_PEA_1 T9 1563 1692 Segment cluster Z2136S_PEA_1 node_43 according to the present invention is supported by 52 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T5; Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 18 below describes the starting and ending position of this segment on each transcript.
Table 18 - Segment location ora transcripts Transcript name Segment starting position:'~ Segment ending position Z21368_PEA_1 T10 1994 2210 Z21368_PEA_1 T1I 1916 2132 Z21368_PEA_1 T5 1906 2122 Z21368_PEA_1 T6 1901; 2122 221368 PEA_1 T9 ~ 1693 ~ 1909 Segment cluster Z2136S_PEA_1 node 45 according to the present invention is supported by 64 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z2136S_PEA_1 T10, Z2136S_PEA_1 T1 l, Z21368_PEA_1 T5, Z2136S_PEA_1 T6 and Z21368_PEA_1 T9. Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on transcripts Transt~ript name Segment starring positian~. ~egmeat ending pasitiaxr ~
221365 PEA_1 T10 2211 ~ 2466 Z21368_PEA_1 T11 2133 2358 221368 PEA_1 TS 2123 2375 Z21368_PEA_1 T6 2123 2375 Z2136S PEA_1 T9 1910 2165 Segment cluster 221368 PEA_1 node_53 according to the present invention is supported by 60 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, Z21368_PEA_1 T1 l, Z2136S _ _ -PEA_1 T5, 221368 PEA_1 T6 and Z21363_PEA_1 T9. Table 20 below describes the starting and ending position of this segment on each transcript.
Table ?0 - Segment location orr transcripts "firanscript xxame Segment starting positionI Segment end.ingpasition Z2136S_PEA_1 T10 2725 2900 Z2136S_PEA_1 T11 2647 2822 Z21368_PEA_1 TS X637 2812 Z21368_PEA_1 T6 2637 '812 Z21368_PEA_1 T9 2424 2599 ~90 Segment cluster Z31368_PEA_1 node 56 according to tho present invention is supported by 50 libraries. The number of libraries was determined as previoaasly described. This segment can be found in the following transcript(s): Z21368_PEA_1 Tlf), Z,21368_PEA_1 T11 and 221368 PEA_1 T9. Table 21 below describes the starting and ending position of this segment on each transcript.
Table 21 - Segment location on transcripts Transcript name Se~nxent starting Segment ending position position ~_.
221368 PEA_1_T10 2901 3043 221368 PEA_1 T11 2823 2965 Segment cluster 221368 PEA_1 node 58 according to the present invention is supported by 71 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s)221368 PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T5, _Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 22 below describes the starting and ending position of this segment on each transcript.
Table 22 - Segment location ova transcripts Ticanscri~t name Segment startiyg posifionSegment ending positiota Z2136S_PEA_1 T10 3044 3167 221368 PEA l Tll 2966 3089 Z21368_PEA_1 T5 2813 2936 Segment cluster 221368 PEA_1 node 66 according to the present invention is supported by 142 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, 221368 PEA_1_T5, Z21368_PEA_1 T6 and 221365 PEA_1 T9. Table 23 below describes the starting and ending position of this segment on each transcript.
Table 23 - Segnterrt location on transcripts Transcript name Segment starting position; segment endilzg pasitzcizt Segment cluster Z21368_PEA_1 node 67 according to the present invention is supported by 181 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_I T10, Z21368_PEA_1 T11, Z21368_PEA_1 T5, _Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 24 below describes the starting and ending position of this segment on each transcript.
Table 24 - SegnterZt location on transcripts Transcript nave se~rr~ent starting Segment ending position posiCian Z21368_PEA_1 T9 3489 4073 Segment cluster Z21368_PEA_1 node 69 according to the present invention is supported by 150 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, 221368-PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1_T9. Table 25 below describes the starting and ending position of this segment on each transcript.
Table 25 - Segment location ora transcripts Transept narae. Segment starting position' Segment ending posirion Z21368_PEA_1 T10 4428 4755 Z21368_PEA_1 T11 4350 4677 Z21368_PEA_1 TS 4197 5384 Z21368_PEA_1 T6 4197 4524 According to an optional embodiment or the present mvennon, snort segments re~a~CU w the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster 221368 PEA_1 node_11 according to the present invention is supported by 26 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1_T11, 221368 PEA_1 T23; Z21368_PEA_1 T24, Z2136fi PEA_1 T5, Z21368_PEA_1 T6 and 221368 PEA 1 T9. Table 26 below describes the starting and ending position of this segment on each transcript.
Table 26 - Segnaent location on transcripts Transcript ~ segment statrting Segn~eint ~z~ding naive pasition position Z21368_PEA_1 558 602 Z21368_PEA_1 558 602 221368PEA_1 396 440 TS
Z21368_PEA_1 396 440 Z21368_PEA_1 423 467 Segment cluster Z21368_PEA_1 node_12 according to the present invention is supported by 23 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA 1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T23, Z21368_PEA_1 T24, 221368 PEA_1 T5, Z2136b PEA_1 T6 and Z2136S PEA_1 T9. Table 27 below describes the starting and ending position of this segment on each transcript.
Table ?7 - Segment location on transcripts Transcript' Segment starting ppsitioinSe~rnent ending po~itic>n n~rne ~
TS
Segment cluster Z21368_PEA_1 node_16 according to the present invention can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23, _ _Z21368_PEA_1 T24, Z21368_PEA_1 T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 28 below describes the starting and ending position of this segment on each transcript.
Table ?8 - Segment location oj7 transcripts Transcript name Segment starting positiun~Segment ending position 221368 PEA_1TS 646 660 Z21368_PEA_1T6 646 660 Z2136S_PEA_1T9 673 687 Segment cluster Z21368_PEA_1 node_17 according to the present invention is supported by 19 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s)Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z21368_PEA_1 T23, _ - -Z2136S_PEA_1 T24, Z2136S PEA_l T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 29 below describes the starting and ending position of this segment on each transcript.
Table 29 - Segment location on transcripts Trattscri~t carne; augment startirig ~ ~e,~,ment ending pasxtion positioca.
Z21368_PEA_1 T10 823 862 Z2136S PEA_1 T11 823 862 Z21368_PEA_1 T23 823 862 Z21368_PEA_1 T24 823 S62 Z21368_PEA_1 TS 661 700 Z2136fi PEA_1 T6 661 700 Segment cluster Z21368_PEA_1 node 23 according to the present invention is supported by 36 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T11, Z21368_PEA_1 T23, Z21368_PEA_1 T24Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 30 below describes the starting and ending position of this segment on each transcript.
Table 30 - SegnTent locatio~a on transcripts Transcript naive. Segment starting position ' Segment ending pasition Z21368_PEA_1 T9 880 ~ 953 Segment cluster Z21368_PEA_1 node 24 according to the present invention is supported by 36 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T23, Z21368_PEA_1 T24, Z21368_PEA_1_T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 31 below describes the starting and ending position of this segment on each transcript.
Table 31 - Segment locatiora an trarrscr-ipts Transcript tame Se~ent starting positionSegment ~~,ding position -Z21368_PEA_1 T9 ) 1049 Segment clusterZ21368_PEA_1 node_30 according to the present invention is supported by 39 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 T11, 221368 PEA_1 T23, Z2136b PEA_1 T24, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 32 below describes the starting and ending position of this segment on each transcript.
Table 32 - Segnrerrt location on transcripts Transcript name Segment starting segment ending position positioh Z21368_PEA_1 T11 1273 1331 Z21368_PEA_I T9 1050 1108 Segment cluster 221368 PEA_I node_31 according to the present invention is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 TI
I, 221368 PEA_I T23,, _ _Z21368_PEA_1 T24, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 33 below describes the starting and ending position of this segment on each transcript.
Table 33 - Segrnerrt location on trarrseripts Transcript Se,grr~eint starEing'pasitionSe~naent ending posatian name ~
-TIO ~~
221368PEA_1 1322 1413 Z2136SPEA_1 1323 1413 Z21368_PEA_1 1109 1200 Segment cluster Z21368_PEA_1 node_38 according to the present invention is supported by 45 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 Tl 1, Z21368_PEA_1 T23, Z21368_PEA_1 T24, Z21368_PEA_1_T5, Z21368_PEA_1_T6 and Z21368_PEA_1 T9. Table 34 below describes the starting and ending position of this segment on each transcript.
Table 34 - Segme~zt locatiota orZ transcripts Transcript nanne Segment starting positionSegment ending, position Z21368_PEA_1 T10 1807 1863 Z21368_PEA_1 T24 2160 2216 Z21368_PEA_1 T5 1719 1775 Z21368_PEA_1 T9 1506 1562 Segment cluster 221368 PEA_1 node 47 according to the present invention is supported by 61 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, Z21368_PEA_1 T11, Z2136S PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 35 below describes the starting and ending position of this segment on each transcript.
Table 35 - Segment locatio~i an trarascr-ipts Transcript name Segment starting positionSegment ending position Z21368_PEA_1 T10 2467 2563 Z21368_PEA_1 T11 2389 2485 Z21368_PEA 1 T5 2379 2475 221368 PEA_1 T6 2379 2475 Z21368_PEA_1 T9 ~ 2166 2262 Segment cluster Z21368_PEA_1 node 49 according to the present invention is supported by 57 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, 221368 PEA_1 T11, 221368 PEA_1 T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 36 below describes the starting and ending position of this segment on each transcript.
Table 36 - Segment location orz tr~anscniFts Transcript uam,e ~~gm~nl farting position' Segment ending ~osit~ozi Z21368_PEA_1 T10 2564 2658 221368 PEA_1 T11 2486 2580 Z21368_PEA_1 TS 2476 2570 221368 PEA_1 T6 2476 2570 221368 PEA_1 T9 2263 2357 Segment cluster 221368 PEA_1 node 51 according to the present invention is supported by 46 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 T11, 221368 PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 37 below describes the starting and ending position of this segment on each transcript.
Table 37 - Segrnerrt lc~catiara era tr-arrscriyts Transcript Segment starting : Segment ending posifiion name position Z2136S_PEA_1 2659 2724 Z21368_PEA_1 2581 2646 Z21368_PEA_1 2571 2636 TS
Z21368_PEA_1 2571 2636 Z21368_PEA_1 2358 2423 ~99 Segment cluster Z21368_PEA_1 node 61 according to the present invention is supported by 61 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and Z21368_PEA_1 T9. Table 38 below describes the starting and ending position of this segment on each transcript.
Table 38 - Segment location ort transcripts Transc~nipt ~~- Seguaent starting Segment ending position za~rn~e position ~ ~~~ ~
221368PEA_1 3168 3201 Z21368_PEA_1 3090 3123 221368PEA_1 2937 2970 TS
221368PEA_1 2937 2970 Z21368_PEA_1 2867 2900 Segment cluster Z21368_PEA_1 node 68 according to the present invention is supported by 87 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 221368 PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T5, Z21368_PEA_1 T6 and 221368 PEA_1 T9. Table 39 below describes the starting and ending position of this segment on each transcript.
Tale 39 - Segment location on transcripts Ticanscript Segment starting positionI Segment ending name position Z21368_PEA_1 4375 4427 Z21368_PEA_1 4297 4349 221368PEA_1 4144 4196 TS
Z21368_PEA_1 4144 4196 Z21368_PEA_1 4074 4126 Segment cluster 221368 PEA_1 node 7 according to the present invention is supported by 29 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z21368_PEA_1 T10, 221368 PEA_1 T11, Z21368_PEA_1 T23, Z21368_PEA_1 T?4, Z21368_FEA_1 T5, 221368 PEA_1 T6 and Z21368_PEA_1 T9. Table 40 below describes the starting and ending position of this segment on each transcript.
Transcript ~1 Segpent starting ~ Segmient ~ndarig name position posi~an ~~ ~r Z2136RPEA _1T10 463 557 ~~
221368PEA_1T11 463 557 Z21368_PEA_1T23 463 557 Z21368_PEA_1T24 463 557 Z21368_PEA_1TS 301 395 Z21368_PEA_1T6 301 395 Z21368_PEA_1T9 328 422 Overexpression of at least a portion of this cluster was determined according to oligonucleotides and one or more chips. The results were as follows:
Oligonucleotide 221368_0 0 61857 was on the TAA chip and was found to be overexpressed in breast cancer.
Variant protein alignment to the previously known protein:
Sequence name: /tmp/5ER3vIMKE2/9LOY71D1TQ:SULl HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P2 ~ SUL1 HUMAN ..
Table 4p - Segment location on tr~anseripts Alignment segment 1/1:
3oi Quality: 7664.00 Escore: 0 Matching length: 761 Total length: 761 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIilllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIilllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
. , , IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIilllllllllllllllllll IIIIIIIIIIilllilllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Illlllllllllllllllilllllllllllllllllllllllllllllll 501 DKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTR.SLSVEF 550 Iillllllllllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
701 PFKEA.AQEVDSKLQLFKENNRRRKKERKEKRRQRKGEECSLPGLTCFTHD 750 20 Sequence name: /tmp/tt3yfXIUKV/YxSTFWr66h:Q7Z2W2 Sequence documentation:
Alignment of: 221368 PEA 1 P5 x Q7Z2W2 ..
Alignment segment 1/1:
Quality: 7869.00 Encore: 0 Matching length: 791 Total length: 871 Matching Percent Similarity: 99.87 Matching Percent Tdentity: 99.87 Total Percent Similarity: 90.70 Total Percent Identity: 90.70 Gaps: 1 Alignment:
IIIIIII~IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
51 DDQDVELA......................,................... 58 1111!11 59 ......................................FFGKYLNEYNGS 70 IIIlllllllll ~ -7l YIPPGWREWLGLIKNSRFYNYTVCRNGIKEKHGFDYAKDYFTDLITNESI 120 llllllillllllllllllllllllllllllllllllllllllllllll.
illlllllllllllllllllllllllllllllll.Iillllllllllllll 20l NYFKMSKRMYPHRPVMMVISHAAPHGPEDSAPQFSKLYPNASQHITPSYN 250 3o IlllilllllllllllllllllllIIIIIIIIIIilllllllllllllll lllllllllllIllllIIIIIIIIIillllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIII
I
I
I
illlllllllllillllIII
PGSIVPQIVLNIDLAPTILDIAGLDTPPDVDGKSVLKLLDPEKPGNRFRT
IIIIIIIIIIIIIIIIIIIIII
I
I
I
lllllllllllIIIIIIIIIIII
NKKAKIWRDTFLVERGKFLRKKEESSKNIQQSNHLPKYERVKELCQQARY
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllllll 421. DKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEF470 lllllilllliilllliilllllli 501 Illllllllllllllllllllllll 550 DKECSCRESGYRASRSQRKSQRQFLRNQGTPKYKPRFVHTRQTRSLSVEF
Illlllllllllllllllllllllllllllllllllllllllllllllll - -IIIIII1111111(Illlllilllllllllillllllilliilillllll Illlllllllllllllllllllllllllllllllllllllllllllllil 111111lIIIIIIIIIIIIIIIIIIIIIillllllllIIIIIIIIIIIII
111111l111111lIIIIIIIiIiIIIIIIIIIIIillllllllllllll . . . . .
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
802 FDMNTDPYQLTNTVHTVERGILNQLHVQLMELRSCQGYKQCNPRPKNLDV $50 IIIIIIIIIIIIIIIIIIIII
Sequence name; /tmp/tt3yfXIUKV/YxSTFWr66h:AAH12997 Sequence documentation:
Alignment of: 221368 PEA 1-P5 x AAH12997 ..
Alignment segment 1/1:
Quality: 420.00 Escore: 0 Matching length: 40 Total length: 40 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
Sequence name: /tmp/tt3yfXIUKV/YxSTFWr66h:SUL1 HUMAN
Sequence documentation:
Alignment of: Z21368_PEA l_P5 x SUL1'HUMAN . ..
Alignment segment 1/l:
Quality: 7878.00 Escore: 0 Mai=thing length: 791 Total length: 871 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 90.82 Total Percent Identity: 90.82 Gaps: 1 Alignment:
. . . . .
iilililiiillllililliiiliillilliililllliilllliiilll 51 DDQDVEL........................................... 57 Ilillii 51~DDQDVELGSLQVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYV 100 58 .....................................AFFGKYLNEYNGS 70 2o iliiiiillliii liliiiillllilliliillillliililililillliillliiliiill illlliiiiililliiilllliliiiiiiillliilliiliiiiiiiili - - -IIII I i IIIII II II IIII IIII IIIII III III
Ililliiiilliililililliilliiliilillililliiillllilil Illll!!lllllill!IIiIlilllllllllllllllllllliillllll llll11111111!llllllllllll!!llllllllllillllilllllil IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Ilillilliliillilllliilililillliliilililllillllilll 1l1111(Illlllllill!llllllllilllillllllllllllllilll llllllllllll1111)l)lllllllllllilllillllllllllllll 701 PFKEAAQEVDSKLQLFKENNRRRKKER.KEKR.RQRKGEECSLPGLTCFTHD 750 Sequence name: /tmp/AVAZGWHuFO/RzHFOnHIsT:SULl HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P15 x SUL1 HUMAN .-Alignment segment 7_,% 1 Quality: 4174.00 Escore: 0 Matching length: 416 Total length: 416 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
2o IIIIIIIIIIIIIIIIIIIillllllllllllllllllllllllllllll IIIIIIIIIIilllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
-IIIIIIIIIIIIIIIIIIIIIIilllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllllllilll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIII
25 Sequence name: /tmp/JhwgR.dKc~nt/kqSmjxkWWk:SUL1 HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P16 x SULl HUMAN ..
Alignment segment 1/1:
Quality: 3985.00 Escore: 0 Matching length: 397 Total length: 397 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps : 0 Alignment:
~I~~I~~~~~~~~~~~~~~~~~~~~~~I~I~~~~~~~II~~~~~~~~~~I
5l DDQDVELGSLQVMNKTRKIMEHGGATFINAFVTTPMCCPSRSSMLTGKYV 100 ~ - ' ' ~ ' 20 Sequence name: /tmp/GPlnIw3BOg/zXFdxqG4ow:SUL1 HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P22 x SUL1 HUMAN ..
Alignment segment 1/1:
Quality: 1897.00 Escore: 0 Matching length: 188 Total length: 188 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity. 100.00 Gaps: 0 Alignment:
51 DDQDVELGSLQVMNKTRKIMEHGGATFII~AFVTTPMCCPSRSSMLTGKYV 100 ~ ' ' 30 Sequence name: /tmp/oji5Fs74fB/8xeB9KrGjp:Q7Z2W2 Sequence documentation:
Alignment of: 221368 PEA 1 P23 x Q7Z2W2 ..
Alignment segment 1/1:
Quality: 1368.00 Encore: 0.000511 Matching length: 137 Total length: 137 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps : 0 Alignment:
2o Iillllllllllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIilllllllllllllllllllllllil Sequence name: /tmp/oji5Fs74fB/8xeB9KrGjp:SUL1 HUMAN
Sequence documentation:
Alignment of: 221368 PEA 1 P23 x SULl HUMAN ..
Alignment segment 1/1:
Quality: 1368.00 Escore: 0.000511 Matching length: 137 Total length: 137 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
~Ol HNHNVYTNNENCSSPSWQAMHEPRTFAVYLNNTGYRT 137 l01 HNHNVYTNNENCSSPSWQAMHEPRTFAVYLNNTGYR.T l37 Expression of SLTL1 HCTMAN - Extracellular sulfatase Sulf 1221368 transcripts which are detectable by amplicon as depicted in sequence name Z21368seg39 in normal arid cancerous breast tissues Expression of SI1LI_HLTMAN - Extracellular sulfatase Sulf 1 transcripts detectable by or according to seg39, Z21368seg39 amplicon and Z21368seg39F and Z2I368seg39R
primers was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD
(GenBank Accession No. BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM_000194; amplicon - HPRTI-amplicon), SDHA (GenBank Accession No.
NM_004168; amplicon - SDHA-amplicon), and G6PD (GenBanle Accession No.
NM_000402;
G6PD amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes.
The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 56-60,63-67, Table 1 above, Tissue samples in testing panel), to obtain a value of fold up-regulation for each sample relative to median of the normal PM samples.
Figure 13 is a histogram showing over expression of the above-indicated SILL 1 HUMAN - Extracellular sulfatase Sulf 1 transcripts in cancerous breast samples relative to the normal samples. Values represent the average of duplicate experiments.
Error bars indicate the minimal and maximal values obtained. The number and percentage of samples that exhibit at least 5-fold over-expression, out of the total number of samples tested is indicated in the bottom.
As is evident from Figure 13, the expression of SUL 1 HUMAN - Extracellular sulfatase Sulf 1 transcripts detectable by the above amplicon(s) in cancer samples was significantly higher than in the non-cancerous samples (Sample Nos 56-60,63-67, Table 1 above, Tissue samples in testing panel). Notably an over-expressir:~n of at least 5 fold was found in 13 out of 28 adenocarcinoma samples.
Statistical analysis was applied to verify the spg~uficance of these results, as described below.
The P value for the difference in the expression levels of SUL 1 ~~JMAN -Extracellular sulfatase Sulf 1 transcripts detectable by the above amplicon(s) in breast cancer samples versus the normal tissue samples was determined by T test as 2.14E-03.
Threshold of 5 fold overexpression was found to differentiate between cancer and normal samples with P value of 6.911s03 as checked by exact fisher test. The above values demonstrate statistical significance of the results.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: Z21368seg39F
forward primer;
Z21368seg39R reverse primer.
The present invention also preferably encarnpasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following arnplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: Z21368seg39.
Z21368seg39F (SEQ ID N0:842)- GTTGCATTTCTCAGTGCTGGTTT
Z21368seg39R (SEQ ID N0:843)- AGGGTGCCGGGTGAGG
Z21368seg39 (SEQ m N0:844~
GTTGCATTTCTCAGTGCTGGTTTCTAATCAGACCAGTGGATTGAGTTTCTCTACCATC
CTCCCCACGTTCTTCTCTAAGCTGCCTCCAAGCCTCACCCGGCACCCT
Expression of SUL1 HUMAN - Extracellular sulfatase Sulf 1221368 transcripts which are detectable by amplicon as depicted in sequence name Z21368seg39 in different normal tissues Expression of SUL 1 HUMAN - Extracellular sulfatase Sulf I transcripts detectable by or according to Z21368seg39 amplicon and Z21368seg39F Z2I368seg39R was measured by real time PCR. In parallel the expression of four housekeeping genes -[ RPL19 (GenBank Accession No. NM_000981; RPL19 amplicon), TATA box (GenBank Accession No.
NM_003194; TATA amplicon), UBC (GenBank Accession No. BC000449; ampIicon -Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM_004168; amplicon - SDHA-amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the breast samples (sample nos. 33-35 in table 2 "Tissue samples in normal panel") to obtain a value of relative expression of each sample relative to median of the Normal samples. Primers and amplicon are as above.
The results are presented in Figure 14, demonstrating the expression of SLTL 1 ~iIIMAN
- Extracellular sulfatase Sulf 1221368 transcripts, which are detectable by amplicon as depicted in sequence name Z21368seg39, in different normal tissues.
Expression of SUL1 HCJMA.N - Extracellular sulfatase Sulf 1 221368 transcripts which are detectable by amplicon as depicted in sequence name Z21368juncl7-21 in normal and cancerous breast tissues Expression of SI1L 1HUMAN - Extracellular sulfatase Sulf 1 transcripts detectable by ' or according to Z21368juncl7-2I amplicon and Z21368junc17-21F and Z21368juncl7-primers was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No. BC019323; amplicon - PBGD-amplicon), HPRT1 (GenBank Accession No. NM 000194; amplicon - HPRTI-amplicon), and SDHA (GenBank Accession No. NM_004168; amplicon - SDHA-amplicon), G6PD (GenBanle Accession No.
NM_000402;
G6PD amplicon) was measured sunilarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes.
The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos 56-60,63-67 Table 1 above, "Tissue samples in testing panel"), ~ obtain a value of fold up-regulation for each sample relative to median of the normal PM samples.
Figure IS is a histogram showing over expression of the above-indicated SITL 1 HUMAN - Extracellular sulfatase Sulf 1 transcripts in cancerous breast sample s relative to the normal samples. Values represent the average of duplicate experiments.
Error bars indicate the minimal and maximal values obtained. The number and percentage of samples that exhibit at least 5 fold over-expression, out of the total number of samples tested is indicated in the bottom.
As is evident from Figure 15, the expression of SLTL1 HCJMAN - Extracellular sulfatase Sulf 1 transcripts detectable by the above amplicon(s) in cancer samples was significantly higher than in the norrcancerous samples (Sample Nos 56-60,63-67, Table 1 above, Tissue samples in testing panel). Notably an over-expression of at least 5 fold was found in 11 out of 28 adenocarcinoma samples.
Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of SUL1 HUMAN -Extracellular sulfatase Sulf 1 transcripts detectable by the above amplicon(s) in breast cancer samples versus the normal tissue samples was determined by T test as 4.6E 03.
IS Threshold of 5 fold overexpression was found to differentiate between cancer and normal samples with P value of 1.78E-02 as checked by exact fisher test. The above values demonstrate statistical signif cance of the results. Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair:
Z21368juncl 7-21 F forward primer; Z21368junc17-~ 1 R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: ~21368junc17-~1 22I368junc17-2IF (SEQ ID N0:845)- GGACGGATACAGCAGGAACG
Z21368junc17-21R (SEQ ID N0:846}- TATTTTCCP~AAAAAGGCCAGCTC
Z21368junc17-21 (SEQ ID NO:847}-GGACGGATACAGCAGGAACGAAAAAACATCCGACCCAACATTATTCTTGTGCTTAC
CGATGATCAAGATGTGGAGCTGGCCTTTTTTGGAAAATA
Expression of SUL1 HUMAN - Extracellu.lar sulfatase Sulf 1 221368 transcripts which are detectable by amplicon .us depicted in sequence name Z21368juncl7-21 in different normal tissues Expression of SUL1 HLTIvIAN - Extracellular sulfatase Sulf 1 221368 transcripts detectable by or according to amplicon Z21368junc17-21 was measured by real time PCR. In parallel the expression of four housekeeping genes -RPL19 (GenBanl: Accession No.
NM-000981; RPL19 amplicon), TATA box (GenBank Accession No. NM_003194; TATA
amplicon); UBC (GenBank Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA -(GenBanl: Accession No. NM 004168; amplicon - SDHA-amplicon) was measured similarly. For each RT sample, the expression of the above amplicon was norn~alized to the geometric mean of the quantities of the housekeeping genes, as above. The normalized quantity of each RT sample was then divided by the median of the quantities of the breast samples (Sample Nos. - 33-35 Table 2 above, 'Tissue samples on normal panel"), to obtain a value of relative expression of each sample relative to median of the breast samples.
Primers and amplicon are as above.
The results are presented in Figure 16, demonstrating the expression of SUL 1 HUMAN
- Extracellular sulfatase Sulf 1 221368 transcripts, which are detectable by amplicon as depicted in sequence name Z21368junc17-21, in different normal tissues.
''S Cluster T59832 features 6 transcripts) and 33 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3 Table 1 - Transcripts of interest Transcript Name ' ' Sequence ID No ,.
Table 2 - Segments of interest Segment.Nam~e i ~e,~uenc,~ I17 No T59832node1 109 T59832node22 110 T59832node23 111 T59832node24 112 T59832node29 113 T59832_node39 114 T59832_node7 115 T59832_node_10 116 T59832node_11 117 T59832_node_12 118 T59832_node_14 119 T59832_node_16 120 T59832_node_19 121 T59832node_2 122 T59832_node20 123 T59832_node25 124 T59832_node26 125 T59832_node27 126 T59832_node_28 127 T59832_node3 128 T59832_node30 129 T59832node31 130 T59832_node32 131 T59832_node34 132 T59832node_35 133 T59832node36 134 T59S32_node37 135 T59832node_38 136 T59832node_4 137 T5983'?_node5 13S
T59832_node6 139 T59832node8 140 T59832node9 141 Table s - Proteins of interest Protein Nataie ! Sequence LD No T59832_PS 143 T59832 P18 ~ 147 These sequences are variants of the known protein Gamma-interferon inducible lysosomal thiol reductase precursor (SwissProt accession identifier GILT HUIvIAN; known also according to the synonyms Gamma-interferon-inducible protein IP-30), SEQ ID NU: 142, referred to herein as the previously known protein.
Protein Garruna-interferon inducible lysosomal thiol reductase precursor is known or believed to have the following function(s): Cleaves disulfide bonds in proteins by reduction.
May facilitate the complet unfolding of proteins destined for lysosomal degradation. May be involved in MHC class II-restricted antigen processing. The sequence for protein Gamma-interferon inducible lysosomal thiol reductase precursor is given at the end of the application, as "Gamma-interferon inducible lysosomal thiol reductase precursor amino acid sequence". known polymorphisms for this sequence are as shown in Table 4.
Table d - Arnirro acid rnrctations far Known Protein ~,~ ~~sitic~n(s)Comment.
on amino acid sequence 109 L -> S
130 H -> L
Protein Gamma-interferon inducible lysosomal thiol reductase precursor localization is believed to be Lysosomal.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: extracellular; lysosome, wlvch are annotations) 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/>.
Cluster T59832 can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term "number" in the left hand column of the table and the numbers on the y axis of Figure 17 refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histograms in Figure 17 and Table 5. This cluster is overexpressed (at least at a minimum level) in the following pathological conditions: brain malignant tumors, breast malignant tumors, ovarian carcinoma and pancreas carcinoma.
Table S - Norrrzal tissue distribcrtiorr Name cif Tissue Dumber Adrenal 208 Bladder SOS
Bone X00 Brain 18 Colon 236 Epithelial 143 General 280 head and neck 192 Kidney 71 Liver 53 Lung 459 lymph nodes X48 Breast 0 bone marrow 94 Ovary 0 Pancreas ~0 Prostate 86 Skin 2 Stomach 109 T cells 557 Thyroid 0 Uterus 63 Table 6 - F values and ratios for expression in cancerous tissa~e Narne: o~'TissueP1 ~ P2 SP1 ~ SPA R4 adrenal 4.9e-015.9e-014.7e-031.1 2.9e-020.8 bladder 3.7e-015.6e-013.7e-021.3 2.5e-010.9 Bone 6.6e-0 6.7e-0 3.4e-0 0.6 9.1 0.4 1 1 1 e-O
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Claims (52)
1. An isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: R11723_PEA_1_T15, R11723_PEA_1_T17, R11723_PEA_1_T19, R11723_PEA_1_T20, R11723_PEA_1_T5, or R11723_PEA_1_T6.
2. An isolated polynucleotide comprising a node having a sequence selected from the group consisting of: R11723_PEA_1_node_13, R11723_PEA_I_node_16, R11723_PEA_1_node_19, R11723_PEA_1_node_2, R11723_PEA_1_node_22, R11723_PEA_1_node 31, R11723_PEA_1_node_10, R11723_PEA_1_node_11, R11723_PEA_1_node_15, R11723_PEA_1_node_18, R11723_PEA_1_node_20, R11723_PEA_1_node_21, R11723_PEA_1_node_23, R11723_PEA_1_node_24, R11723_PEA_1_node_25, R11723_PEA_1_node_26, R11723_PEA_1_node_27, R11723_PEA_1_node_28, R11723_PEA_1_node_29, R11723_PEA_1_node_3, R11723_PEA_1_node_30, R11723_PEA_1_node_4, R11723_PEA_1_node_5, R11723_PEA_1_node_6, R11723_PEA_1_node_7 or R11723_PEA_1_node_8.
3. An isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of: R11723_PEA_1_P2, R11723_PEA_1_P6, R11723_PEA_1_P7, R11723_PEA_1_P13, or R11723_PEA_1_P10.
4. An isolated chimeric polypeptide encoding for R11723_PEA_1_P6, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRKSCASSAACLIASAGSPCRGLAPGREEQRALHIKAGAVGGGVR
corresponding to amino acids 1 - 110 of R11723_PEA_1_P6, and a second amino acid sequence being at least 90 % homologous to MYAQALLVVGVLQRQAAAQHLHEHPPKLLRGHRVQERVDDRAEVEKRLREGEEDHV
RPEVGPRPVVLGFGRSHDPPNLVGHPAYGQCHNNQPWADTSRRERQRKEKHSMRTQ
corresponding to amino acids 1 - 112 of Q8IXM0, which also corresponds to amino acids 111 -222 of R11723_PEA_1_P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSAGIMYRKSCASSAACLIASAGSPCRGLAPGREEQRALHIKAGAVGGGVR
corresponding to amino acids 1 - 110 of R11723_PEA_1_P6, and a second amino acid sequence being at least 90 % homologous to MYAQALLVVGVLQRQAAAQHLHEHPPKLLRGHRVQERVDDRAEVEKRLREGEEDHV
RPEVGPRPVVLGFGRSHDPPNLVGHPAYGQCHNNQPWADTSRRERQRKEKHSMRTQ
corresponding to amino acids 1 - 112 of Q8IXM0, which also corresponds to amino acids 111 -222 of R11723_PEA_1_P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
5. An isolated polypeptide encoding for a head of R11723_PEA_1_P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRIKSCASSAACLIASAGSPCRGLAPGREEQRALHKAGAVVGGGVR of R11723_PEA_1_F6.
homologous to the sequence MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRIKSCASSAACLIASAGSPCRGLAPGREEQRALHKAGAVVGGGVR of R11723_PEA_1_F6.
6. An isolated chimeric polypeptide encoding for R11723_PEA_1_P6, comprising a first amino acid sequence being at least 90 % homologous to MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRKSCASSAACLIASAG corresponding to amino acids 1 - 83 of Q96AC2, which also corresponds to amino acids 1 - 83 of R11723_PEA_1_P6, 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 SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAITGQ
CHNNQPWADTSRRERQRKEKHSMRTQ corresponding to amino acids 84 - 222 of R11723_PEA_1_P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSAGIMYRKSCASSAACLIASAG corresponding to amino acids 1 - 83 of Q96AC2, which also corresponds to amino acids 1 - 83 of R11723_PEA_1_P6, 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 SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAITGQ
CHNNQPWADTSRRERQRKEKHSMRTQ corresponding to amino acids 84 - 222 of R11723_PEA_1_P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
7. An isolated polypeptide encoding for a tail of R11723_PEA_1_P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ in R11723_PEA_1_P6.
homologous to the sequence SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ in R11723_PEA_1_P6.
8. An isolated chimeric polypeptide encoding for R11723_PEA_1_P6, comprising a first amino acid sequence being at least 90 % homologous to MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRKSCASSAACLIASAG corresponding to amino acids 1 - 83 of Q8N2G4, which also corresponds to amino acids 1 - 83 of R11723_PEA_1_P6, 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 SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ corresponding to amino acids 84 - 222 of R11723_PEA_1_P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSAGIMYRKSCASSAACLIASAG corresponding to amino acids 1 - 83 of Q8N2G4, which also corresponds to amino acids 1 - 83 of R11723_PEA_1_P6, 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 SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ corresponding to amino acids 84 - 222 of R11723_PEA_1_P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
9. An isolated polypeptide encoding for a tail of R11723_PEA_1_P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ in R11723_PEA_1_P6.
homologous to the sequence SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ in R11723_PEA_1_P6.
10. An isolated chimeric polypeptide encoding for R11723_PEA_1_P6, comprising a first amino acid sequence being at least 90 % homologous to MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRKSCASSAACLIASAG corresponding to amino acids 24 - 106 of BAC85518, which also corresponds to amino acids 1 - 83 of R11723_PEA_1_P6, 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 SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ corresponding to amino acids 84 - 222 of R11723_PEA_1_P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSAGIMYRKSCASSAACLIASAG corresponding to amino acids 24 - 106 of BAC85518, which also corresponds to amino acids 1 - 83 of R11723_PEA_1_P6, 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 SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ corresponding to amino acids 84 - 222 of R11723_PEA_1_P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
11. An isolated polypeptide encoding for a tail of R11723_PEA_1_P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ in R11723_PEA_1_P6.
homologous to the sequence SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ in R11723_PEA_1_P6.
12. An isolated chimeric polypeptide encoding for R11723_PEA_1_P7, comprising a first amino acid sequence being at least 90 % homologous to MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAG corresponding to amino acids 1 - 64 of Q96AC2, which also corresponds to amino acids 1 - 64 of R11723_PEA_1_P7, 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 SHCVTRLECSGTISAHCNLCLPGSNDHPT corresponding to amino acids 65 - 93 of R11723_PEA_1_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSAG corresponding to amino acids 1 - 64 of Q96AC2, which also corresponds to amino acids 1 - 64 of R11723_PEA_1_P7, 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 SHCVTRLECSGTISAHCNLCLPGSNDHPT corresponding to amino acids 65 - 93 of R11723_PEA_1_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
13. An isolated polypeptide encoding for a tail of R11723_PEA_1_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT in R11723_PEA_1_P7.
homologous to the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT in R11723_PEA_1_P7.
14. An isolated chimeric polypeptide encoding for R11723_PEA_1_P7, comprising a first amino acid sequence being at least 90 % homologous to MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAG corresponding to amino acids 1 - 64 of Q8N2G4, which also corresponds to amino acids 1 - 64 of R11723_PEA_1_P7, 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 SHCVTRLECSGTISAHCNLCLPGSNDHPT corresponding to amino acids 65 - 93 of R11723_PEA_1_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSAG corresponding to amino acids 1 - 64 of Q8N2G4, which also corresponds to amino acids 1 - 64 of R11723_PEA_1_P7, 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 SHCVTRLECSGTISAHCNLCLPGSNDHPT corresponding to amino acids 65 - 93 of R11723_PEA_1_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
15. An isolated polypeptide encoding for a tail of R11723_PEA_1_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT in R11723_PEA_1_P7.
homologous to the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT in R11723_PEA_1_P7.
16. An isolated chimeric polypeptide encoding for R11723_PEA_1_P7, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MWVLG corresponding to amino acids 1 - 5 of R11723_PEA_1_P7, second amino acid sequence being at least 90 % homologous to IAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEVMEQSAG
corresponding to amino acids 22 - 80 of BAC85273, which also corresponds to amino acids 6 -64 of R11723_PEA_1_P7, 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 SHCVTRLECSGTISAHCNLCLPGSNDHPT corresponding to amino acids 65 - 93 of R11723_PEA_1_P7, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
corresponding to amino acids 22 - 80 of BAC85273, which also corresponds to amino acids 6 -64 of R11723_PEA_1_P7, 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 SHCVTRLECSGTISAHCNLCLPGSNDHPT corresponding to amino acids 65 - 93 of R11723_PEA_1_P7, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
17. An isolated polypeptide encoding for a head of R11723_PEA_1_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence MWVLG of R11723_PEA_1_P7.
homologous to the sequence MWVLG of R11723_PEA_1_P7.
18. An isolated polypeptide encoding for a tail of R11723_PEA_1_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT in R11723_PEA_1_P7.
homologous to the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT in R11723_PEA_1_P7.
19. An isolated chimeric polypeptide encoding for R11723_PEA_1_P7, comprising a first amino acid sequence being at least 90 % homologous to MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAG corresponding to amino acids 24 - 87 of BAC85518, which also corresponds to amino acids 1 - 64 of R11723_PEA_1_P7, 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 SHCVTRLECSGTISAHCNLCLPGSNDHPT corresponding to amino acids 65 - 93 of R11723_PEA_1_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSAG corresponding to amino acids 24 - 87 of BAC85518, which also corresponds to amino acids 1 - 64 of R11723_PEA_1_P7, 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 SHCVTRLECSGTISAHCNLCLPGSNDHPT corresponding to amino acids 65 - 93 of R11723_PEA_1_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
20. An isolated polypeptide encoding for a tail of R11723_PEA_1_P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT in R11723_PEA_1_P7.
homologous to the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT in R11723_PEA_1_P7.
21. An isolated chimeric polypeptide encoding for R11723_PEA_1_P13, comprising a first amino acid sequence being at least 90 % homologous to MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSA corresponding to amino acids 1 - 63 of Q96AC2, which also corresponds to amino acids 1 - 63 of R11723_PEA_1_P13, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence DTKRTNTLLFEMRHFAKQLTT corresponding to amino acids 64 - 84 of R11723 PEA_1_P13, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSA corresponding to amino acids 1 - 63 of Q96AC2, which also corresponds to amino acids 1 - 63 of R11723_PEA_1_P13, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence DTKRTNTLLFEMRHFAKQLTT corresponding to amino acids 64 - 84 of R11723 PEA_1_P13, wherein said first and second amino acid sequences are contiguous and in a sequential order.
22. An isolated polypeptide _encoding for a tail of R11723_PEA_1 P13, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence DTKRTNTLLFEMRHFAKQLTT in R11723_PEA_1 P13.
homologous to the sequence DTKRTNTLLFEMRHFAKQLTT in R11723_PEA_1 P13.
23. An isolated chimeric polypeptide encoding for R11723_PEA_1_P10, comprising a first amino acid sequence being at least 90 % homologous to MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSA corresponding to amino acids 1 - 63 of Q96AC2, which also corresponds to amino acids 1 - 63 of R11723 PEA_1_P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK corresponding to amino acids 64 - 90 of R11723 PEA_1_P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSA corresponding to amino acids 1 - 63 of Q96AC2, which also corresponds to amino acids 1 - 63 of R11723 PEA_1_P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK corresponding to amino acids 64 - 90 of R11723 PEA_1_P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
24. An isolated polypeptide _ -encoding for a tail of R11723 PEA_1_P10, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK in R11723 PEA_1_P10.
homologous to the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK in R11723 PEA_1_P10.
25. An isolated chimeric polypeptide encoding for R11723 PEA_1_P10, comprising a first amino acid sequence being at least 90 % homologous to MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSA corresponding to amino acids 1 - 63 of Q8N2G4, which also corresponds to amino acids 1 - 63 of R11723 PEA_1_P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 55%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK corresponding to amino acids 64 - 90 of R11723 PEA_1_P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSA corresponding to amino acids 1 - 63 of Q8N2G4, which also corresponds to amino acids 1 - 63 of R11723 PEA_1_P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 55%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK corresponding to amino acids 64 - 90 of R11723 PEA_1_P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
26. An isolated polypeptide encoding for a tail of R11723 PEA_1_P10, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK in 811723 PEA_1_P10.
homologous to the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK in 811723 PEA_1_P10.
27. An isolated chimeric polypeptide encoding for R11723 PEA_1_P10, 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 MWVLG corresponding to amino acids 1 - 5 of R11723 PEA_1_P10, second amino acid sequence being at least 90 % homologous to IAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQILEVMEQSA
corresponding to amino acids 22 - 79 of BAC85273, which also corresponds to amino acids 6 -63 of R11723 PEA_1_P10, 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 DRVSLCHEAGVQWNNFSTLQPLPPRLIK corresponding to amino acids 64 - 90 of R11723 PEA_1_P10, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
corresponding to amino acids 22 - 79 of BAC85273, which also corresponds to amino acids 6 -63 of R11723 PEA_1_P10, 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 DRVSLCHEAGVQWNNFSTLQPLPPRLIK corresponding to amino acids 64 - 90 of R11723 PEA_1_P10, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
28. An isolated polypeptide encoding for a head of R11723 PEA_1_P10, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence MWVLG of R11723 PEA_1_P10.
homologous to the sequence MWVLG of R11723 PEA_1_P10.
29. An isolated polypeptide encoding for a tail of R11723_PEA_1_P10, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK in R11723_PEA_1_P10.
homologous to the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK in R11723_PEA_1_P10.
30. An isolated chimeric -polypeptide encoding for R11723_PEA_1_P10, comprising a first amino acid sequence being at least 90 % homologous to MWLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSA corresponding to amino acids 24 - 86 of BAC85518, which also corresponds to amino acids 1 - 63 of R11723_PEA_1_P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK corresponding to amino acids 64 - 90 of R11723 PEA_1_P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
MEQSA corresponding to amino acids 24 - 86 of BAC85518, which also corresponds to amino acids 1 - 63 of R11723_PEA_1_P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK corresponding to amino acids 64 - 90 of R11723 PEA_1_P10, wherein said first and second amino acid sequences are contiguous and in a sequential order.
31. An isolated polypeptide encoding for a tail of R11723 PEA_1_P10, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK -in R11723 PEA_1_P10.
homologous to the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK -in R11723 PEA_1_P10.
32. An isolated oligonucleotide, comprising an amplicon selected from the group consisting of SEQ ID NOs: 891 or 894.
33. A primer pair, comprising a pair of isolated oligonucleotides capable of amplifying said amplicon of claim 32.
34. The primer pair of claim 33, comprising a pair of isolated oligonucleotides selected from the group consisting of: SEQ NOs 889 and 890; or 892 and 893.
35. An antibody capable of specifically binding to an epitope of an amino acid sequence of any of claims 3-31.
36. The antibody of claim 35, wherein said amino acid sequence comprises said tail of claims 4-31.
37. The antibody of claims 35 or 36, wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein PSEC.
38. A kit for detecting breast cancer, comprising a kit detecting overexpression of a splice variant according to any of the above claims.
39. The kit of claim 38, wherein said kit comprises a NAT-based technology.
40. The kit of claim 39, wherein said kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence according to claims 1 or 2.
41. The kit of claim 38, wherein said kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence according to claims 1 or 2.
42. The kit of claim 38, wherein said kit comprises an antibody according to any of claims 35-37.
43. The kit of claim 42, wherein said kit further comprises at least one reagent for performing an ELISA or a Western blot.
44. A method for detecting breast cancer, comprising detecting overexpression of a splice variant according to any of the above claims.
45. The method of claim 44, wherein said detecting overexpression is performed with a NAT-based technology.
46. The method of claim 44, wherein said detecting overexpression is performed with an immunoassay.
47. The method of claim 46, wherein said immunoassay comprises an antibody according to any of the above claims.
48. A biomarker capable of detecting breast cancer, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
49. A method for screening for breast cancer, comprising detecting breastcancer cells with a biomarker or an antibody or a method or assay according to any of the above claims.
50. A method for diagnosing breast cancer, comprising detecting breast cancer cells with a biomarker or an antibody or a method or assay according to any of the above claims.
51. A method for monitoring disease progression and/or treatment efficacy and/or relapse of breast cancer, comprising detecting breast cancer cells with a biomarker or an antibody or a method or assay according to any of the above claims.
52. A method of selecting a therapy for breast cancer, comprising detecting breast cancer cells with a biomarker or an antibody or a method or assay according to any of the above claims and selecting a therapy according to said detection.
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| US53912804P | 2004-01-27 | 2004-01-27 | |
| US60/539,129 | 2004-01-27 | ||
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| US62091704P | 2004-10-22 | 2004-10-22 | |
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| US62065604P | 2004-10-22 | 2004-10-22 | |
| US62085304P | 2004-10-22 | 2004-10-22 | |
| US62100404P | 2004-10-22 | 2004-10-22 | |
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| US62087404P | 2004-10-22 | 2004-10-22 | |
| US60/620,853 | 2004-10-22 | ||
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| US60/621,004 | 2004-10-22 | ||
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| US60/620,656 | 2004-10-22 | ||
| US60/620,974 | 2004-10-22 | ||
| US62113104P | 2004-10-25 | 2004-10-25 | |
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| US62012304P | 2004-11-17 | 2004-11-17 | |
| US62813404P | 2004-11-17 | 2004-11-17 | |
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| US62817804P | 2004-11-17 | 2004-11-17 | |
| US62815604P | 2004-11-17 | 2004-11-17 | |
| US62825104P | 2004-11-17 | 2004-11-17 | |
| US62816704P | 2004-11-17 | 2004-11-17 | |
| US62810104P | 2004-11-17 | 2004-11-17 | |
| US62823104P | 2004-11-17 | 2004-11-17 | |
| US62811204P | 2004-11-17 | 2004-11-17 | |
| US60/628,134 | 2004-11-17 | ||
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| US60/628,156 | 2004-11-17 | ||
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| US60/628,111 | 2004-11-17 | ||
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| US60/628,231 | 2004-11-17 | ||
| US11/043,842 US20060183131A1 (en) | 2004-01-27 | 2005-01-27 | Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of breast cancer |
| PCT/IB2005/000433 WO2005072050A2 (en) | 2004-01-27 | 2005-01-27 | Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of breast cancer |
| US11/043,842 | 2005-01-27 |
Publications (1)
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|---|---|
| CA2554440A1 true CA2554440A1 (en) | 2005-08-11 |
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| CA002554440A Abandoned CA2554440A1 (en) | 2004-01-27 | 2005-01-27 | Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of breast cancer |
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| AU (1) | AU2005207882A1 (en) |
| CA (1) | CA2554440A1 (en) |
| WO (1) | WO2005072050A2 (en) |
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| US20090215042A1 (en) * | 2005-06-08 | 2009-08-27 | Compugen Ltd. | Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis |
| JP5299885B2 (en) * | 2005-08-12 | 2013-09-25 | 学校法人日本大学 | Genes related to carcinogenesis / recurrence of HCV positive hepatocellular carcinoma |
| CN104479018B (en) | 2008-12-09 | 2018-09-21 | 霍夫曼-拉罗奇有限公司 | Anti- PD-L1 antibody and they be used to enhance the purposes of T cell function |
| GB201004551D0 (en) | 2010-03-19 | 2010-05-05 | Immatics Biotechnologies Gmbh | NOvel immunotherapy against several tumors including gastrointestinal and gastric cancer |
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- 2005-01-27 WO PCT/IB2005/000433 patent/WO2005072050A2/en active Application Filing
- 2005-01-27 CA CA002554440A patent/CA2554440A1/en not_active Abandoned
- 2005-01-27 AU AU2005207882A patent/AU2005207882A1/en not_active Abandoned
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| AU2005207882A1 (en) | 2005-08-11 |
| WO2005072050A2 (en) | 2005-08-11 |
| WO2005072050A9 (en) | 2010-01-14 |
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