EP1343886A2 - Compositions and methods for the therapy and diagnosis of lung cancer - Google Patents

Compositions and methods for the therapy and diagnosis of lung cancer

Info

Publication number
EP1343886A2
EP1343886A2 EP01984164A EP01984164A EP1343886A2 EP 1343886 A2 EP1343886 A2 EP 1343886A2 EP 01984164 A EP01984164 A EP 01984164A EP 01984164 A EP01984164 A EP 01984164A EP 1343886 A2 EP1343886 A2 EP 1343886A2
Authority
EP
European Patent Office
Prior art keywords
seq
clone
cdna sequence
determined cdna
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01984164A
Other languages
German (de)
English (en)
French (fr)
Inventor
Tongtong Wang
Yoshihiro Watanabe
Robert A. Henderson
Jeffrey C. Johnson
Marc W. Retter
Margarita Durham
Darrick Carter
Gary Richard Fanger
Thomas S. Vedvick
Chaitanya S. Bangur
Andria Mcnabb
Aijun Wang
Neil Fanger
Ann Switzer
Patricia D. Mcneill
Jonathan D. Clapper
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Corixa Corp
Original Assignee
Corixa Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/614,124 external-priority patent/US6630574B1/en
Priority claimed from US09/651,563 external-priority patent/US6914132B1/en
Priority claimed from US09/658,824 external-priority patent/US6746846B1/en
Priority claimed from US09/671,325 external-priority patent/US6667154B1/en
Priority claimed from US09/702,705 external-priority patent/US6504010B1/en
Priority claimed from US09/736,457 external-priority patent/US6509448B2/en
Priority claimed from US09/849,626 external-priority patent/US20020197669A1/en
Application filed by Corixa Corp filed Critical Corixa Corp
Publication of EP1343886A2 publication Critical patent/EP1343886A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to therapy and diagnosis of cancer, such as lung cancer.
  • the invention is more specifically related to polypeptides, comprising at least a portion of a lung tumor protein, and to polynucleotides encoding such polypeptides.
  • polypeptides and polynucleotides are useful in pharmaceutical compositions, e.g., vaccines, and other compositions for the diagnosis and treatment of lung cancer.
  • CD-ROM No. 1 is labeled "COPY 1 - SEQUENCE LISTING PART”
  • CD-ROM No.2 is labeled "COPY 2 - SEQUENCE LISTING”
  • CD-ROM No. 1 contains the file 47802pc.app.txt which is 1.4 MB and created on July 10, 2001
  • CD-ROM No.2 contains the file 47802pc.app.txt which is 1.4 MB and created on July 10, 2001
  • COORDY 3 - SEQUENCE LISTING PART contains the file 47802pc.app.txt which is 1.4 MB and created on July 10, 2001;
  • CD-ROM No. 4 is labeled “CRF,” contains the file 47802pc.app.txt which is 1.4 Mb and created on July 10, 2001.
  • Cancer is a significant health problem throughout the world. Although advances have been made in detection and therapy of cancer, no vaccine or other universally successful method for prevention or treatment is currently available.
  • Lung cancer is the primary cause of cancer death among both men and women in the U.S., with an estimated 172,000 new cases being reported in 1994.
  • the five-year survival rate among all lung cancer patients, regardless of the stage of disease at diagnosis, is only 13%. This contrasts with a five-year survival rate of 46% among cases detected while the disease is still localized.
  • Only 16% of lung cancers are discovered before the disease has spread.
  • Early detection is difficult since clinical symptoms are often not seen until the disease has reached an advanced stage.
  • diagnosis is aided by the use of chest x-rays, analysis of the type of cells contained in sputum and fiberoptic examination of the bronchial passages.
  • Treatment regimens are determined by the type and stage ofthe cancer, and include surgery, radiation therapy and/or chemotherapy. In spite of considerable research into therapies for this and other cancers, lung cancer remains difficult to diagnose and treat effectively. Accordingly, there is a need in the art for improved methods for detecting and treating such cancers.
  • the present invention fulfills these needs and
  • the present invention provides polynucleotide compositions comprising a sequence selected from the group consisting of:
  • sequences consisting of at least 20, 25, 30, 35, 40, 45, 50, 75 and 100 contiguous residues of a sequence provided in SEQ ID NOs: 1-57, 59-323, 341- 782, 784-785, 788, 790, 792, 794, 796, 800-804, 807, 808, 810-826, 828-1664, 1668, 1669, 1676, 1680-1805, 1824, 1826-1829, 1865-1868, 1873, 1875, 1877, 1879, 1881, 1883, 1891-1900, 1910, 1914, 1918, 1922-1924, 1931, 1933, 1938, 1941 and 1974- 2002;
  • the polynucleotide compositions of the invention are expressed in at least about 20%, more preferably in at least about 30%, and most preferably in at least about 50% of lung tumors samples tested, at a level that is at least about 2-fold, preferably at least about 5 -fold, and most preferably at least about 10-fold higher than that for normal tissues.
  • the present invention in another aspect, provides polypeptide compositions comprising an amino acid sequence that is encoded by a polynucleotide sequence described above.
  • the present invention further provides polypeptide compositions comprising an amino acid sequence selected from the group consisting of sequences recited in SEQ ID NOs:324-340, 783, 786, 787, 789, 791, 793, 795, 797-799, 805, 806, 809, 827, 1667, 1670-1675, 1677-1679, 1806-1822, 1825, 1830-1833, 1834-1856, 1863, 1864, 1869-1872, 1874, 1876, 1878, 1880, 1882, 1884-1890, 1901-1909, 1913, 1917, 1921, 1925-1930, 1932, 1934, 1937, 1940 and 1942-1973.
  • the polypeptides and/or polynucleotides of the present invention are immunogenic, i.e., they are capable of eliciting an immune response, particularly a humoral and/or cellular immune response, as further described herein.
  • the present invention further provides fragments, variants and/or derivatives of the disclosed polypeptide and or polynucleotide sequences, wherein the fragments, variants and/or derivatives preferably have a level of immunogenic activity of at least about 50%, preferably at least about 70% and more preferably at least about 90% ofthe level of immunogenic activity of a polypeptide sequence set forth in SEQ ID NOs:324-340, 783, 786, 787, 789, 791, 793, 795, 797-799, 805, 806, 809, 827, 1667, 1670-1675, 1677-1679, 1806-1822, 1825, 1830-1833, 1834-1856, 1863, 1864, 1869- 1872, 1874, 1876, 1878, 1880, 1882, 1884-1890, 1901-1909, 1913, 1917, 1921, 1925- 1930, 1932, 1934, 1937, 1940 and 1942-1973 or a polypeptide sequence encoded by a polynucleotide sequence set forth in
  • compositions comprising a polypeptide or polynucleotide as described above and a physiologically acceptable carrier.
  • compositions for prophylactic or therapeutic applications.
  • Such compositions generally comprise an immunogenic polypeptide or polynucleotide ofthe invention and an immunostimulant, such as an adjuvant.
  • the present invention further provides pharmaceutical compositions that comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide of the present invention, or a fragment thereof; and (b) a physiologically acceptable carrier.
  • compositions comprising: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) a pharmaceutically acceptable carrier or excipient.
  • antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B cells.
  • compositions comprise: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) an immunostimulant.
  • the present invention further provides, in other aspects, fusion proteins that comprise at least one polypeptide as described above, as well as polynucleotides encoding such fusion proteins, typically in the form of pharmaceutical compositions, e.g., vaccine compositions, comprising a physiologically acceptable carrier and/or an immunostimulant.
  • the fusions proteins may comprise multiple immunogenic polypeptides or portions/variants thereof, as described herein, and may further comprise one or more polypeptide segments for facilitating the expression, purification and/or immunogenicity ofthe polypeptide(s).
  • the present invention provides methods for stimulating an immune response in a patient, preferably a T cell response in a human patient, comprising administering a pharmaceutical composition described herein.
  • the patient may be afflicted with lung cancer, in which case the methods provide treatment for the disease, or patient considered at risk for such a disease may be treated prophylactically.
  • the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a pharmaceutical composition as recited above.
  • the patient may be afflicted with lung cancer, in which case the methods provide treatment for the disease, or patient considered at risk for such a disease may be treated prophylactically.
  • the present invention further provides, within other aspects, methods for removing tumor cells from a biological sample, comprising contacting a biological sample with T cells that specifically react with a polypeptide of the present invention, wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of cells expressing the protein from the sample.
  • methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a biological sample treated as described above.
  • Methods are further provided, within other aspects, for stimulating and/or expanding T cells specific for a polypeptide ofthe present invention, comprising contacting T cells with one or more of: (i) a polypeptide as described above; (ii) a polynucleotide encoding such a polypeptide; and/or (iii) an antigen presenting cell that expresses such a polypeptide; under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells.
  • Isolated T cell populations comprising T cells prepared as described above are also provided.
  • the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a T cell population as described above.
  • the present invention further provides methods for inhibiting the development of a cancer in a patient, comprising the steps of: (a) incubating CD4 + and/or CD8 + T cells isolated from a patient with one or more of: (i) a polypeptide comprising at least an immunogenic portion of polypeptide disclosed herein; (ii) a polynucleotide encoding such a polypeptide; and (iii) an antigen-presenting cell that expressed such a polypeptide; and (b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development of a cancer in the patient.
  • Proliferated cells may, but need not, be cloned prior to administration to the patient.
  • the present invention provides methods for determining the presence or absence of a cancer, preferably a lung cancer, in a patient comprising: (a) contacting a biological sample obtained from a patient with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; and (c) comparing the amount of polypeptide with a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient.
  • the binding agent is an antibody, more preferably a monoclonal antibody.
  • the present invention also provides, within other aspects, methods for monitoring the progression of a cancer in a patient.
  • Such methods comprise the steps of: (a) contacting a biological sample obtained from a patient at a first point in time with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.
  • the present invention further provides, within other aspects, methods for determining the presence or absence of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a polypeptide of the present invention; (b) detecting in the sample a level of a polynucleotidej preferably mRNA, that hybridizes to the oligonucleotide; and (c) comparing the level of polynucleotide that hybridizes to the oligonucleotide with a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient.
  • the amount of mRNA is detected via polymerase chain reaction using, for example, at least one oligonucleotide primer that hybridizes to a polynucleotide encoding a polypeptide as recited above, or a complement of such a polynucleotide.
  • the amount of mRNA is detected using a hybridization technique, employing an oligonucleotide probe that hybridizes to a polynucleotide that encodes a polypeptide as recited above, or a complement of such a polynucleotide.
  • methods for monitoring the progression of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a polypeptide ofthe present invention; (b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polynucleotide detected in step (c) with the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.
  • the present invention provides antibodies, such as monoclonal antibodies, that bind to a polypeptide as described above, as well as diagnostic kits comprising such antibodies. Diagnostic kits comprising one or more oligonucleotide probes or primers as described above are also provided.
  • SEQ ID NO:l is the determined cDNA sequence for clone #19038, also referred to as
  • SEQ ID NO:2 is the determined cDNA sequence for clone #19036.
  • SEQ ID NO:3 is the determined cDNA sequence for clone #19034.
  • SEQ ID NO:4 is the determined cDNA sequence for clone #19033.
  • SEQ ID NO:5 is the determined cDNA sequence for clone #19032.
  • SEQ ID NO: 6 is the determined cDNA sequence for clone #19030, also referred to as L559S.
  • SEQ ID NO:7 is the determined cDNA sequence for clone #19029.
  • SEQ ID NO:8 is the determined cDNA sequence for clone #19025.
  • SEQ ID NO:9 is the determined cDNA sequence for clone #19023.
  • SEQ ID NO: 10 is the determined cDNA sequence for clone #18929.
  • SEQ ID NO: 11 is the determined cDNA sequence for clone #19010.
  • SEQ ID NO: 12 is the determined cDNA sequence for clone #19009.
  • SEQ ID NO: 13 is the determined cDNA sequence for clones #19005, 19007, 19016 and
  • SEQ ID NO:14 is the determined cDNA sequence for clone #19004.
  • SEQ ID NO:15 is the determined cDNA sequence for clones #19002 and 18965.
  • SEQ ID NO: 16 is the determined cDNA sequence for clone #18998.
  • SEQ ID NO:17 is the determined cDNA sequence for clone #18997.
  • SEQ ID NO: 18 is the determined cDNA sequence for clone #18996.
  • SEQ ID NO: 19 is the determined cDNA sequence for clone #18995.
  • SEQ ID NO:20 is the determined cDNA sequence for clone #18994, also known as
  • SEQ ID NO.-21 is the determined cDNA sequence for clone #18992.
  • SEQ ID NO:22 is the determined cDNA sequence for clone #18991.
  • SEQ ID NO:23 is the determined cDNA sequence for clone #18990, also referred to as clone #20111.
  • SEQ ID NO:24 is the determined cDNA sequence for clone #18987.
  • SEQ ID NO:25 is the determined cDNA sequence for clone #18985, also referred as
  • SEQ ID NO:26 is the determined cDNA sequence for clone #18984, also referred to as L847P.
  • SEQ ID NO:27 is the determined cDNA sequence for clone #18983.
  • SEQ ID NO:28 is the determined cDNA sequence for clones #18976 and 18980.
  • SEQ ID NO:29 is the determined cDNA sequence for clone #18975.
  • SEQ ID NO:30 is the determined cDNA sequence for clone #18974.
  • SEQ ID NO:31 is the determined cDNA sequence for clone #18973.
  • SEQ ID NO:32 is the determined cDNA sequence for clone #18972.
  • SEQ ID NO:33 is the determined cDNA sequence for clone #18971, also referred to as
  • SEQ ID NO:34 is the determined cDNA sequence for clone #18970.
  • SEQ ID NO:35 is the determined cDNA sequence for clone #18966.
  • SEQ ID NO:36 is the determined cDNA sequence for clones #18964, 18968 and 19039.
  • SEQ ID NO:37 is the determined cDNA sequence for clone #18960.
  • SEQ ID NO:38 is the determined cDNA sequence for clone #18959.
  • SEQ ID NO:39 is the determined cDNA sequence for clones #18958 and 18982.
  • SEQ ID NO:40 is the determined cDNA sequence for clones #18956 and 19015.
  • SEQ ID NO:41 is the determined cDNA sequence for clone #18954, also referred to
  • SEQ ID NO:42 is the determined cDNA sequence for clone #18951.
  • SEQ ID NO:43 is the determined cDNA sequence for clone #18950.
  • SEQ ID NO:44 is the determined cDNA sequence for clones #18949 and 19024, also referred to as L844P.
  • SEQ ID NO:45 is the determined cDNA sequence for clone #18948.
  • SEQ ID NO:46 is the determined cDNA sequence for clone #18947, also referred to as
  • SEQ ID NO:47 is the determined cDNA sequence for clones #18946, 18953, 18969 and
  • SEQ ID NO:48 is the determined cDNA sequence for clone #18942.
  • SEQ ID NO:49 is the determined cDNA sequence for clone #18940, 18962, 18963,
  • SEQ ID NO:50 is the determined cDNA sequence for clone #18939.
  • SEQ ID NO:51 is the determined cDNA sequence for clones #18938 and 18952.
  • SEQ ID NO:52 is the determined cDNA sequence for clone #18938.
  • SEQ ID NO:53 is the determined cDNA sequence for clone #18937.
  • SEQ ID NO:54 is the determined cDNA sequence for clones #18934, 18935, 18993 and 19022, also referred to as L548S.
  • SEQ ID NO:55 is the determined cDNA sequence for clone #18932.
  • SEQ ID NO:56 is the determined cDNA sequence for clones #18931 and 18936.
  • SEQ ID NO:57 is the determined cDNA sequence for clone #18930.
  • SEQ ID NO: 58 is the determined cDNA sequence for clone #19014 (this sequence has homology to clone L773P, which is also described in co-pending U.S. application
  • SEQ ID NO:59 is the determined cDNA sequence for clone #19127.
  • SEQ ID NO:60 is the determined cDNA sequence for clones #19057 and 19064.
  • SEQ ID NO:61 is the determined cDNA sequence for clone #19122.
  • SEQ ID NO:62 is the determined cDNA sequence for clones #19120 and 18121.
  • SEQ ID NO:63 is the determined cDNA sequence for clone #19118.
  • SEQ ID NO: 64 is the determined cDNA sequence for clone #19117.
  • SEQ ID NO:65 is the determined cDNA sequence for clone #19116.
  • SEQ ID NO: 66 is the determined cDNA sequence for clone #19114.
  • SEQ ID NO: 67 is the determined cDNA sequence for clone #19112, also known as L561S.
  • SEQ ID NO:68 is the determined cDNA sequence for clone #19110.
  • SEQ ID NO: 69 is the determined cDNA sequence for clone #19107, also referred to as
  • SEQ ID NO:70 is the determined cDNA sequence for clone #19106, also referred to as L547S.
  • SEQ ID NO:71 is the determined cDNA sequence for clones #19105 and 19111.
  • SEQ ID NO:72 is the determined cDNA sequence for clone #19099.
  • SEQ ID NO:73 is the determined cDNA sequence for clones #19095, 19104 and 19125, also referred to as L549S.
  • SEQ ID NO:74 is the determined cDNA sequence for clone #19094.
  • SEQ ID NO:75 is the determined cDNA sequence for clones #19089 and 19101.
  • SEQ ID NO:76 is the determined cDNA sequence for clone #19088.
  • SEQ ID NO:77 is the determined cDNA sequence for clones #19087, 19092, 19096,
  • SEQ ID NO:78 is the determined cDNA sequence for clone #19086.
  • SEQ ID NO:79 is the determined cDNA sequence for clone #19085, also referred to as
  • SEQ ID NO:80 is the determined cDNA sequence for clone #19084, also referred to as clone #19079.
  • SEQ ID NO:81 is the determined cDNA sequence for clone #19082.
  • SEQ ID NO:82 is the determined cDNA sequence for clone #19080.
  • SEQ ID NO:83 is the determined cDNA sequence for clone #19077.
  • SEQ ID NO:84 is the determined cDNA sequence for clone #19076, also referred to as
  • SEQ ID NO:85 is the determined cDNA sequence for clone #19074, also referred to as clone #20102.
  • SEQ ID NO:86 is the determined cDNA sequence for clone #19073, also referred to as
  • SEQ ID NO:87 is the determined cDNA sequence for clones #19072 and 19115.
  • SEQ ID NO:88 is the determined cDNA sequence for clone #19071.
  • SEQ ID NO:89 is the determined cDNA sequence for clone #19070.
  • SEQ ID NO:90 is the determined cDNA sequence for clone #19069.
  • SEQ ID NO:91 is the determined cDNA sequence for clone #19068, also referred to
  • SEQ ID NO:92 is the determined cDNA sequence for clone #19066.
  • SEQ ID NO:93 is the determined cDNA sequence for clone #19065.
  • SEQ ID NO:94 is the determined cDNA sequence for clone #19063.
  • SEQ ID NO:95 is the determined cDNA sequence for clones #19061, 19081, 19108 and
  • SEQ ID NO:96 is the determined cDNA sequence for clones #19060, 19067 and 19083, also referred to as L548S.
  • SEQ ID NO:97 is the determined cDNA sequence for clones #19059 and 19062.
  • SEQ ID NO:98 is the determined cDNA sequence for clone #19058.
  • SEQ ID NO:99 is the determined cDNA sequence for clone #19124.
  • SEQ ID NO: 100 is the determined cDNA sequence for clone #18929.
  • SEQ ID NO:101 is the determined cDNA sequence for clone #18422.
  • SEQ ID NO.T02 is the determined cDNA sequence for clone #18425.
  • SEQ ID NO: 103 is the determined cDNA sequence for clone #18431.
  • SEQ ID NO: 104 is the determined cDNA sequence for clone #18433.
  • SEQ ID NO: 105 is the determined cDNA sequence for clone #18444.
  • SEQ ID NO: 106 is the determined cDNA sequence for clone #18449.
  • SEQ ID NO: 107 is the determined cDNA sequence for clone #18451.
  • SEQ ID NO: 108 is the determined cDNA sequence for clone #18452.
  • SEQ ID NO: 109 is the determined cDNA sequence for clone #18455.
  • SEQ ID NO:l 10 is the determined cDNA sequence for clone #18457.
  • SEQ ID NO: 111 is the determined cDNA sequence for clone #18466.
  • SEQ ID NO: 112 is the determined cDNA sequence for clone #18468.
  • SEQ ID NO:l 13 is the determined cDNA sequence for clone #18471.
  • SEQ ID NO: 114 is the determined cDNA sequence for clone #18475.
  • SEQ ID NO:l 15 is the determined cDNA sequence for clone #18476.
  • SEQ ID NO:l 16 is the determined cDNA sequence for clone #18477.
  • SEQ ID NO : 117 is the determined cDNA sequence for clone #20631.
  • SEQ ID NO:l 18 is the determined cDNA sequence for clone #20634.
  • SEQ ID NO:l 19 is the determined cDNA sequence for clone #20635.
  • SEQ ID NO: 120 is the determined cDNA sequence for clone #20637.
  • SEQ ID NO: 121 is the determined cDNA sequence for clone #20638.
  • SEQ ID NO: 122 is the determined cDNA sequence for clone #20643.
  • SEQ ID NO: 123 is the determined cDNA sequence for clone #20652.
  • SEQ ID NO:124 is the determined cDNA sequence for clone #20653.
  • SEQ ID NO: 125 is the determined cDNA sequence for clone #20657.
  • SEQ ID NO: 126 is the determined cDNA sequence for clone #20658.
  • SEQ ID NO: 127 is the determined cDNA sequence for clone #20660.
  • SEQ ID NO: 128 is the determined cDNA sequence for clone #20661.
  • SEQ ID NO: 129 is the determined cDNA sequence for clone #20663.
  • SEQ ID NO:130 is the determined cDNA sequence for clone #20665.
  • SEQ ID NO: 131 is the determined cDNA sequence for clone #20670.
  • SEQ ID NO.-132 is the determined cDNA sequence for clone #20671.
  • SEQ ID NO:133 is the determined cDNA sequence for clone #20672.
  • SEQ ID NO:134 is the determined cDNA sequence for clone #20675.
  • SEQ ID NO: 135 is the determined cDNA sequence for clone #20679.
  • SEQ ID NO:136 is the determined cDNA sequence for clone #20681.
  • SEQ ID NO:137 is the determined cDNA sequence for clone #20682.
  • SEQ ID NO:138 is the determined cDNA sequence for clone #20684.
  • SEQ ID NO:139 is the determined cDNA sequence for clone #20685.
  • SEQ ID NO: 140 is the determined cDNA sequence for clone #20689.
  • SEQ ID NO: 141 is the determined cDNA sequence for clone #20699.
  • SEQ ID NO:142 is the determined cDNA sequence for clone #20701.
  • SEQ ID NO: 143 is the determined cDNA sequence for clone #20702.
  • SEQ ID NO: 144 is the determined cDNA sequence for clone #20708.
  • SEQ ID NO: 145 is the determined cDNA sequence for clone #20715.
  • SEQ ID NO:146 is the determined cDNA sequence for clone #20716.
  • SEQ ID NO:147 is the determined cDNA sequence for clone #20719.
  • SEQ ID NO: 148 is the determined cDNA sequence for clone #19129.
  • SEQ ID NO:149 is the determined cDNA sequence for clone #19131.1.
  • SEQ ID NO: 150 is the determined cDNA sequence for clone #19132.2.
  • SEQ ID NO:151 is the determined cDNA sequence for clone #19133.
  • SEQ ID NO:152 is the determined cDNA sequence for clone #19134.2.
  • SEQ ID NO:153 is the determined cDNA sequence for clone #19135.2.
  • SEQ ID NO:154 is the determined cDNA sequence for clone #19137.
  • SEQ ID NO:155 is a first determined cDNA sequence for clone #19138.1.
  • SEQ ID NO:156 is a second determined cDNA sequence for clone #19138.2.
  • SEQ ID NO:157 is the determined cDNA sequence for clone #19139.
  • SEQ ID NO:158 is a first determined cDNA sequence for clone #19140.1.
  • SEQ ID NO:159 is a second determined cDNA sequence for clone #19140.2.
  • SEQ ID NO:160 is the determined cDNA sequence for clone #19141.
  • SEQ ID NO:161 is the determined cDNA sequence for clone #19143.
  • SEQ ID NO:162 is the determined cDNA sequence for clone #19144.
  • SEQ ID NO:163 is a first determined cDNA sequence for clone #19145.1.
  • SEQ ID NO:164 is a second determined cDNA sequence for clone #19145.2.
  • SEQ ID NO:165 is the determined cDNA sequence for clone #19146.
  • SEQ ID NO:166 is the determined cDNA sequence for clone #19149.1.
  • SEQ ID NO: 167 is the determined cDNA sequence for clone #19152.
  • SEQ ID NO:168 is a first determined cDNA sequence for clone #19153.1.
  • SEQ ID NO:169 is a second determined cDNA sequence for clone #19153.2.
  • SEQ ID NO:170 is the determined cDNA sequence for clone #19155.
  • SEQ ID NO:171 is the determined cDNA sequence for clone #19157.
  • SEQ ID NO: 172 is the determined cDNA sequence for clone #19159.
  • SEQ ID NO:173 is the determined cDNA sequence for clone #19160.
  • SEQ ID NO:174 is a first determined cDNA sequence for clone #19161.1.
  • SEQ ID NO:175 is a second determined cDNA sequence for clone #19161.2.
  • SEQ ID NO:176 is the determined cDNA sequence for clone #19162.1.
  • SEQ ID NO: 177 is the determined cDNA sequence for clone #19166.
  • SEQ ID NO:178 is the determined cDNA sequence for clone #19169.
  • SEQ ID NO:179 is the determined cDNA sequence for clone #19171.
  • SEQ ID NO:180 is a first determined cDNA sequence for clone #19173.1.
  • SEQ ID NO:181 is a second determined cDNA sequence for clone #19173.2.
  • SEQ ID NO: 182 is the determined cDNA sequence for clone #19174.1.
  • SEQ ID NO:183 is the determined cDNA sequence for clone #19175.
  • SEQ ID NO:184 is the determined cDNA sequence for clone #19177.
  • SEQ ID NO:185 is the determined cDNA sequence for clone #19178.
  • SEQ ID NO:186 is the determined cDNA sequence for clone #19179.1.
  • SEQ ID NO: 187 is the determined cDNA sequence for clone #19179.2.
  • SEQ ID NO: 188 is the determined cDNA sequence for clone #19180.
  • SEQ ID NO:189 is a first determined cDNA sequence for clone #19182.1.
  • SEQ ID NO:190 is a second determined cDNA sequence for clone #19182.2.
  • SEQ ID NO:191 is the determined cDNA sequence for clone #19183.1.
  • SEQ ID NO:192 is the determined cDNA sequence for clone #19185.1.
  • SEQ ID NO:193 is the determined cDNA sequence for clone #19187.
  • SEQ ID NO: 194 is the determined cDNA sequence for clone #19188.
  • SEQ ID NO: 195 is the determined cDNA sequence for clone #19190.
  • SEQ ID NO:196 is the determined cDNA sequence for clone #19191.
  • SEQ ID NO:197 is the determined cDNA sequence for clone #19192.
  • SEQ ID NO:198 is the determined cDNA sequence for clone #19193.
  • SEQ ID NO.-199 is a first determined cDNA sequence for clone #19194.1.
  • SEQ ID NO:200 is a second determined cDNA sequence for clone #19194.2.
  • SEQ ID NO:201 is the determined cDNA sequence for clone #19197.
  • SEQ ID NO:202 is a first determined cDNA sequence for clone #19200.1.
  • SEQ ID NO:203 is a second determined cDNA sequence for clone #19200.2.
  • SEQ ID NO:204 is the determined cDNA sequence for clone # 19202.
  • SEQ ID NO:205 is a first determined cDNA sequence for clone #19204.1.
  • SEQ ID NO:206 is a second determined cDNA sequence for clone #19204.2.
  • SEQ ID NO:207 is the determined cDNA sequence for clone #19205.
  • SEQ ID NO:208 is a first determined cDNA sequence for clone #19206.1.
  • SEQ ID NO:209 is a second determined cDNA sequence for clone #19206.2.
  • SEQ ID NO:210 is the determined cDNA sequence for clone #19207.
  • SEQ ID NO:211 is the determined cDNA sequence for clone #19208.
  • SEQ ID NO :212 is a first determined cDNA sequence for clone #19211.1.
  • SEQ ID NO:213 is a second determined cDNA sequence for clone #19211.2.
  • SEQ ID NO:214 is a first determined cDNA sequence for clone #19214.1.
  • SEQ ID NO:215 is a second determined cDNA sequence for clone #19214.2.
  • SEQ ID NO:216 is the determined cDNA sequence for clone #19215.
  • SEQ ID NO:217 is a first determined cDNA sequence for clone #19217. 2.
  • SEQ ID NO:218 is a second determined cDNA sequence for clone #19217.2.
  • SEQ ID NO:219 is a first determined cDNA sequence for clone #19218.1.
  • SEQ ID NO:220 is a second determined cDNA sequence for clone #19218.2.
  • SEQ ID NO:221 is a first determined cDNA sequence for clone #19220.1.
  • SEQ ID NO:222 is a second determined cDNA sequence for clone #19220.2.
  • SEQ ID NO:223 is the determined cDNA sequence for clone #22015.
  • SEQ ID NO.-224 is the determined cDNA sequence for clone #22017.
  • SEQ ID NO :225 is the determined cDNA sequence for clone #22019.
  • SEQ ID NO:226 is the determined cDNA sequence for clone #22020.
  • SEQ ID NO:227 is the determined cDNA sequence for clone #22023.
  • SEQ ID NO:228 is the determined cDNA sequence for clone #22026.
  • SEQ ID NO:229 is the determined cDNA sequence for clone #22027.
  • SEQ ID NO.-230 is the determined cDNA sequence for clone #22028.
  • SEQ ID NO:231 is the determined cDNA sequence for clone #22032.
  • SEQ ID NO:232 is the determined cDNA sequence for clone #22037.
  • SEQ ID NO:233 is the determined cDNA sequence for clone #22045.
  • SEQ ID NO:234 is the determined cDNA sequence for clone #22048.
  • SEQ ID NO:235 is the determined cDNA sequence for clone #22050.
  • SEQ ID NO:236 is the determined cDNA sequence for clone #22052.
  • SEQ ID NO:237 is the determined cDNA sequence for clone #22053.
  • SEQ ID NO:238 is the determined cDNA sequence for clone #22057.
  • SEQ ID NO:239 is the determined cDNA sequence for clone #22066.
  • SEQ ID NO:240 is the determined cDNA sequence for clone #22077.
  • SEQ ID NO:241 is the determined cDNA sequence for clone #22085.
  • SEQ ID NO:242 is the determined cDNA sequence for clone #22105.
  • SEQ ID NO:243 is the determined cDNA sequence for clone #22108.
  • SEQ ID NO:244 is the determined cDNA sequence for clone #22109.
  • SEQ ID NO:245 is the determined cDNA sequence for clone #24842.
  • SEQ ID NO:246 is the determined cDNA sequence for clone #24843.
  • SEQ ID NO:247 is the determined cDNA sequence for clone #24845.
  • SEQ ID NO:248 is the determined cDNA sequence for clone #24851.
  • SEQ ID NO:249 is the determined cDNA sequence for clone #24852.
  • SEQ ID NO:250 is the determined cDNA sequence for clone #24853.
  • SEQ ID NO:251 is the determined cDNA sequence for clone #24854.
  • SEQ ID NO:252 is the determined cDNA sequence for clone #24855.
  • SEQ ID NO:253 is the determined cDNA sequence for clone #24860.
  • SEQ ID NO:254 is the determined cDNA sequence for clone #24864.
  • SEQ ID NO:255 is the determined cDNA sequence for clone #24866.
  • SEQ ID NO:256 is the determined cDNA sequence for clone #24867.
  • SEQ ID NO:257 is the determined cDNA sequence for clone #24868.
  • SEQ ID NO:258 is the determined cDNA sequence for clone #24869.
  • SEQ ID NO:259 is the determined cDNA sequence for clone #24870.
  • SEQ ID NO:260 is the determined cDNA sequence for clone #24872.
  • SEQ ID NO:261 is the determined cDNA sequence for clone #24873.
  • SEQ ID NO:262 is the determined cDNA sequence for clone #24875.
  • SEQ ID NO:263 is the determined cDNA sequence for clone #24882.
  • SEQ ID NO:264 is the determined cDNA sequence for clone #24885.
  • SEQ ID NO:265 is the determined cDNA sequence for clone #24886.
  • SEQ ID NO:266 is the determined cDNA sequence for clone #24887.
  • SEQ ID NO:267 is the determined cDNA sequence for clone #24888.
  • SEQ ID NO:268 is the determined cDNA sequence for clone #24890.
  • SEQ ID NO:269 is the determined cDNA sequence for clone #24896.
  • SEQ ID NO:270 is the determined cDNA sequence for clone #24897.
  • SEQ ID NO:271 is the determined cDNA sequence for clone #24899.
  • SEQ ID NO:272 is the determined cDNA sequence for clone #24901.
  • SEQ ID NO.-273 is the determined cDNA sequence for clone #24902.
  • SEQ ID NO:274 is the determined cDNA sequence for clone #24906.
  • SEQ ID NO:275 is the determined cDNA sequence for clone #24912.
  • SEQ ID NO:276 is the determined cDNA sequence for clone #24913.
  • SEQ ID NO:277 is the determined cDNA sequence for clone #24920.
  • SEQ ID NO:278 is the determined cDNA sequence for clone #24927.
  • SEQ ID NO.-279 is the determined cDNA sequence for clone #24930.
  • SEQ ID NO:280 is the determined cDNA sequence for clone #26938.
  • SEQ ID NO:281 is the determined cDNA sequence for clone #26939.
  • SEQ ID NO:282 is the determined cDNA sequence for clone #26943.
  • SEQ ID O:283 is the determined cDNA sequence for clone #26948.
  • SEQ ID NO:284 is the determined cDNA sequence for clone #26951.
  • SEQ ID NO:285 is the determined cDNA sequence for clone #26955.
  • SEQ ID NO:286 is the determined cDNA sequence for clone #26956.
  • SEQ ID NO:287 is the determined cDNA sequence for clone #26959.
  • SEQ ID NO:288 is the determined cDNA sequence for clone #26961.
  • SEQ ID NO:289 is the determined cDNA sequence for clone #26962.
  • SEQ ID NO:290 is the determined cDNA sequence for clone #26964.
  • SEQ ID NO:291 is the determined cDNA sequence for clone #26966.
  • SEQ ID NO:292 is the determined cDNA sequence for clone #26968.
  • SEQ ID NO:293 is the determined cDNA sequence for clone #26972.
  • SEQ ID NO:294 is the determined cDNA sequence for clone #26973.
  • SEQ ID NO:295 is the determined cDNA sequence for clone #26974.
  • SEQ ID NO:296 is the determined cDNA sequence for clone #26976.
  • SEQ ID NO:297 is the determined cDNA sequence for clone #26977.
  • SEQ ID NO:298 is the determined cDNA sequence for clone #26979.
  • SEQ ID NO:299 is the determined cDNA sequence for clone #26980.
  • SEQ ID NO:300 is the determined cDNA sequence for clone #26981.
  • SEQ ID NO:301 is the determined cDNA sequence for clone #26984.
  • SEQ ID NO:302 is the determined cDNA sequence for clone #26985.
  • SEQ ID NO:303 is the determined cDNA sequence for clone #26986.
  • SEQ ID NO:304 is the determined cDNA sequence for clone #26993.
  • SEQ ID NO:305 is the determined cDNA sequence for clone #26994.
  • SEQ ID NO:306 is the determined cDNA sequence for clone #26995.
  • SEQ ID NO:307 is the determined cDNA sequence for clone #27003.
  • SEQ ID NO:308 is the determined cDNA sequence for clone #27005.
  • SEQ ID NO:309 is the determined cDNA sequence for clone #27010.
  • SEQ ID NO:310 is the determined cDNA sequence for clone #27011.
  • SEQ ID NO:311 is the determined cDNA sequence for clone #27013.
  • SEQ ID NO:312 is the determined cDNA sequence for clone #27016
  • SEQ ID NO:313 is the determined cDNA sequence for clone #27017.
  • SEQ ID NO:314 is the determined cDNA sequence for clone #27019.
  • SEQ ID NO:315 is the determined cDNA sequence for clone #27028.
  • SEQ ID NO:316 is the full-length cDNA sequence for clone #19060.
  • SEQ-ID NO:317 is the full-length cDNA sequence for clone #18964.
  • SEQ ID NO:318 is the full-length cDNA sequence for clone #18929.
  • SEQ ID NO:319 is the full-length cDNA sequence for clone #18991.
  • SEQ ID NO:320 is the full-length cDNA sequence for clone #18996.
  • SEQ ID NO:321 is the full-length cDNA sequence for clone #18966.
  • SEQ ID NO:322 is the full-length cDNA sequence for clone #18951.
  • SEQ ID NO.-323 is the full-length cDNA sequence for clone #18973 (also known as
  • SEQ ID NO: 324 is the amino acid sequence for clone #19060.
  • SEQ ID NO:325 is the amino acid sequence for clone #19063.
  • SEQ ID NO:326 is the amino acid sequence for clone #19077.
  • SEQ ID NO:327 is the amino acid sequence for clone #19110.
  • SEQ ID NO:328 is the amino acid sequence for clone #19122.
  • SEQ ID NO:329 is the amino acid sequence for clone #19118.
  • SEQ ID NO:330 is the amino acid sequence for clone #19080.
  • SEQ ID NO:331 is the amino acid sequence for clone #19127.
  • SEQ ID NO:332 is the amino acid sequence for clone #19117.
  • SEQ ID NO:333 is the amino acid sequence for clone #19095, also referred to L549S.
  • SEQ ID NO:334 is the amino acid sequence for clone #18964.
  • SEQ ID NO:335 is the amino acid sequence for clone #18929.
  • SEQ ID NO:336 is the amino acid sequence for clone #18991.
  • SEQ ID NO:337 is the amino acid sequence for clone #18996.
  • SEQ ID NO:338 is the amino acid sequence for clone #18966.
  • SEQ ID NO:339 is the amino acid sequence for clone #18951.
  • SEQ ID NO:340 is the amino acid sequence for clone #18973.
  • SEQ ID NO:341 is the determined cDNA sequence for clone 26461.
  • SEQ ID NO:342 is the determined cDNA sequence for clone 26462.
  • SEQ ID NO:343 is the determined cDNA sequence for clone 26463.
  • SEQ ID NO:344 is the determined cDNA sequence for clone 26464.
  • SEQ ID NO:345 is the determined cDNA sequence for clone 26465.
  • SEQ ID NO:346 is the determined cDNA sequence for clone 26466.
  • SEQ ID NO:347 is the determined cDNA sequence for clone 26467.
  • SEQ ID NO:348 is the determined cDNA sequence for clone 26468.
  • SEQ ID NO:349 is the determined cDNA sequence for clone 26469.
  • SEQ ID NO:350 is the determined cDNA sequence for clone 26470.
  • SEQ ID NO.-351 is the determined cDNA sequence for clone 26471.
  • SEQ ID NO:352 is the determined cDNA sequence for clone 26472.
  • SEQ ID NO:353 is the determined cDNA sequence for clone 26474.
  • SEQ ID NO:354 is the determined cDNA sequence for clone 26475.
  • SEQ ID NO:355 is the determined cDNA sequence for clone 26476.
  • SEQ ID O:356 is the determined cDNA sequence for clone 26477.
  • SEQ ID NO:357 is the determined cDNA sequence for clone 26478.
  • SEQ ID NO:358 is the determined cDNA sequence for clone 26479.
  • SEQ ID NO:359 is the determined cDNA sequence for clone 26480.
  • SEQ ID NO:360 is the determined cDNA sequence for clone 26481.
  • SEQ ID NO:361 is the determined cDNA sequence for clone 26482
  • SEQ ID NO:362 is the determined cDNA sequence for clone 26483.
  • SEQ ID NO:363 is the determined cDNA sequence for clone 26484.
  • SEQ ID NO:364 is the determined cDNA sequence for clone 26485.
  • SEQ ID NO:365 is the determined cDNA sequence for clone 26486.
  • SEQ ID NO:366 is the determined cDNA sequence for clone 26487.
  • SEQ ID NO:367 is the determined cDNA sequence for clone 26488.
  • SEQ ID NO:368 is the determined cDNA sequence for clone 26489.
  • SEQ ID NO:369 is the determined cDNA sequence for clone 26490.
  • SEQ ID NO:370 is the determined cDNA sequence for clone 26491.
  • SEQ ID NO:371 is the determined cDNA sequence for clone 26492.
  • SEQ ID NO.-372 is the determined cDNA sequence for clone 26493.
  • SEQ ID NO:373 is the determined cDNA sequence for clone 26494.
  • SEQ ID NO:374 is the determined cDNA sequence for clone 26495.
  • SEQ ID NO:375 is the determined cDNA sequence for clone 26496.
  • SEQ ID NO:376 is the determined cDNA sequence for clone 26497.
  • SEQ ID NO:377 is the determined cDNA sequence for clone 26498.
  • SEQ ID NO:378 is the determined cDNA sequence for clone 26499.
  • SEQ ID NO:379 is the determined cDNA sequence for clone 26500.
  • SEQ ID NO:380 is the determined cDNA sequence for clone 26501.
  • SEQ ID NO:381 is the determined cDNA sequence for clone 26502.
  • SEQ ID NO:382 is the determined cDNA sequence for clone 26503.
  • SEQ ID NO:383 is the determined cDNA sequence for clone 26504.
  • SEQ ID NO:384 is the determined cDNA sequence for clone 26505.
  • SEQ ID NO:385 is the determined cDNA sequence for clone 26506.
  • SEQ ID NO:386 is the determined cDNA sequence for clone 26507.
  • SEQ ID NO:387 is the determined cDNA sequence for clone 26508.
  • SEQ ID NO:388 is the determined cDNA sequence for clone 26509.
  • SEQ ID NO:389 is the determined cDNA sequence for clone 26511.
  • SEQ ID NO:390 is the determined cDNA sequence for clone 26513.
  • SEQIDNO:391 is the determined cDNA sequence for clone 26514.
  • SEQIDNO:392 is the determined cDNA sequence for clone 26515.
  • SEQ ID NO:393 is the determined cDNA sequence for clone 26516.
  • SEQIDNO:394 is the determined cDNA sequence for clone 26517.
  • SEQIDNO:395 is the determined cDNA sequence for clone 26518.
  • SEQID O:396 is the determined cDNA sequence for clone 26519.
  • SEQIDNO.-397 is the determined cDNA sequence for clone 26520.
  • SEQIDNO:398 is the determined cDNA sequence for clone 26521.
  • SEQ ID NO:399 is the determined cDNA sequence for clone 26522.
  • SEQIDNO:400 is the determined cDNA sequence for clone 26523.
  • SEQ ID NO:401 is the determined cDNA sequence for clone 26524.
  • SEQ ID NO:402 is the determined cDNA sequence for clone 26526.
  • SEQIDNO.-403 is the determined cDNA sequence for clone 26527.
  • SEQIDNO:404 is the determined cDNA sequence for clone 26528.
  • SEQIDNO.-405 is the determined cDNA sequence for clone 26529.
  • SEQIDNO:406 is the determined cDNA sequence for clone 26530.
  • SEQ ID NO:407 is the determined cDNA sequence for clone 26532.
  • SEQIDNO.-408 is the determined cDNA sequence for clone 26533.
  • SEQIDNO:409 is the determined cDNA sequence for clone 26534.
  • SEQID O:410 is the determined cDNA sequence for clone 26535.
  • SEQID O:411 is the determined cDNA sequence for clone 26536.
  • SEQIDNO:412 is the determined cDNA sequence for clone 26537.
  • SEQIDNO:413 is the determined cDNA sequence for clone 26538.
  • SEQIDNO.-414 is the determined cDNA sequence for clone 26540.
  • SEQIDNO:415 is the determined cDNA sequence for clone 26541.
  • SEQIDNO:416 is the determined cDNA sequence for clone 26542.
  • SEQIDNO:417 is the determined cDNA sequence for clone 26543.
  • SEQIDNO:418 is the determined cDNA sequence for clone 26544.
  • SEQIDNO:419 is the determined cDNA sequence for clone 26546.
  • SEQIDNO:420 is the determined cDNA sequence for clone 26547.
  • SEQIDNO:421 is the determined cDNA sequence for clone 26548.
  • SEQ ID NO:422 is the determined cDNA sequence for clone 26549.
  • SEQ ID NO:423 is the determined cDNA sequence for clone 26550.
  • SEQ ID NO:424 is the determined cDNA sequence for clone 26551.
  • SEQ ID NO:425 is the determined cDNA sequence for clone 26552.
  • SEQ ID NO:426 is the determined cDNA sequence for clone 26553.
  • SEQ ID NO:427 is the determined cDNA sequence for clone 26554.
  • SEQ ID NO:428 is the determined cDNA sequence for clone 26556.
  • SEQ ID NO:429 is the determined cDNA sequence for clone 26557.
  • SEQ ID NO:430 is the determined cDNA sequence for clone 27631.
  • SEQ ID NO:431 is the determined cDNA sequence for clone 27632.
  • SEQ ID NO:432 is the determined cDNA sequence for clone 27633.
  • SEQ ID NO:433 is the determined cDNA sequence for clone 27635.
  • SEQ ID NO:434 is the determined cDNA sequence for clone 27636.
  • SEQ ID NO:435 is the determined cDNA sequence for clone 27637.
  • SEQ ID NO:436 is the determined cDNA sequence for clone 27638.
  • SEQ ID NO:437 is the determined cDNA sequence for clone 27639.
  • SEQ ID NO:438 is the determined cDNA sequence for clone 27640.
  • SEQ ID NO:439 is the determined cDNA sequence for clone 27641.
  • SEQ ID NO:440 is the determined cDNA sequence for clone 27642.
  • SEQ ID NO:441 is the determined cDNA sequence for clone 27644.
  • SEQ ID NO:442 is the determined cDNA sequence for clone 27646.
  • SEQ ID NO:443 is the determined cDNA sequence for clone 27647.
  • SEQ ID NO:444 is the determined cDNA sequence for clone 27649.
  • SEQ ID NO:445 is the determined cDNA sequence for clone 27650.
  • SEQ ID NO :446 is the determined cDNA sequence for clone 27651.
  • SEQ ID NO:447 is the determined cDNA sequence for clone 27652.
  • SEQ ID NO:448 is the determined cDNA sequence for clone 27654.
  • SEQ ID NO:449 is the determined cDNA sequence for clone 27655.
  • SEQ ID NO:450 is the determined cDNA sequence for clone 27657.
  • SEQ ID NO:451 is the determined cDNA sequence for clone 27659.
  • SEQ ID NO:452 is the determined cDNA sequence for clone 27665.
  • SEQ ID NO:453 is the determined cDNA sequence for clone 27666.
  • SEQ ID NO:454 is the determined cDNA sequence for clone 27668.
  • SEQ ID NO:455 is the determined cDNA sequence for clone 27670.
  • SEQ ID NO:456 is the determined cDNA sequence for clone 27671.
  • SEQ ID NO:457 is the determined cDNA sequence for clone 27672.
  • SEQ ID NO:458 is the determined cDNA sequence for clone 27674.
  • SEQ ID NO:459 is the determined cDNA sequence for clone 27677.
  • SEQ ID NO:460 is the determined cDNA sequence for clone 27681.
  • SEQ ID NO:461 is the determined cDNA sequence for clone 27682.
  • SEQ ID NO:462 is the determined cDNA sequence for clone 27683.
  • SEQ ID NO:463 is the determined cDNA sequence for clone 27686.
  • SEQ ID NO:464 is the determined cDNA sequence for clone 27688.
  • SEQ ID NO.-465 is the determined cDNA sequence for clone 27689.
  • SEQ ID NO:466 is the determined cDNA sequence for clone 27690.
  • SEQ ID NO :467 is the determined cDNA sequence for clone 27693.
  • SEQ ID NO:468 is the determined cDNA sequence for clone 27699.
  • SEQ ID NO:469 is the determined cDNA sequence for clone 27700.
  • SEQ ID NO:470 is the determined cDNA sequence for clone 27702.
  • SEQ ID NO:471 is the determined cDNA sequence for clone 27705.
  • SEQ ID NO:472 is the determined cDNA sequence for clone 27706.
  • SEQ ID NO:473 is the determined cDNA sequence for clone 27707.
  • SEQ ID NO:474 is the determined cDNA sequence for clone 27708.
  • SEQ ID NO:475 is the determined cDNA sequence for clone 27709.
  • SEQ ID NO:476 is the determined cDNA sequence for clone 27710.
  • SEQ ID NO :477 is the determined cDNA sequence for clone 27711.
  • SEQ ID NO:478 is the determined cDNA sequence for clone 27712.
  • SEQ ID NO:479 is the determined cDNA sequence for clone 27713.
  • SEQ ID NO:480 is the determined cDNA sequence for clone 27714.
  • SEQ ID NO:481 is the determined cDNA sequence for clone 27715.
  • SEQ ID NO:482 is the determined cDNA sequence for clone 27716.
  • SEQ ID NO:483 is the determined cDNA sequence for clone 27717.
  • SEQ ID NO:484 is the determined cDNA sequence for clone 27718.
  • SEQ ID NO:485 is the determined cDNA sequence for clone 27719.
  • SEQ ID NO:486 is the determined cDNA sequence for clone 27720.
  • SEQ ID NO:487 is the determined cDNA sequence for clone 27722.
  • SEQ ID NO:488 is the determined cDNA sequence for clone 27723.
  • SEQ ID NO:489 is the determined cDNA sequence for clone 27724.
  • SEQ ID NO:490 is the determined cDNA sequence for clone 27726.
  • SEQ ID NO:491 is the determined cDNA sequence for clone 25015.
  • SEQ ID NO:492 is the determined cDNA sequence for clone 25016.
  • SEQ ID NO:493 is the determined cDNA sequence for clone 25017.
  • SEQ ID NO:494 is the determined cDNA sequence for clone 25018
  • SEQ ID NO:495 is the determined cDNA sequence for clone 25030.
  • SEQ ID NO:496 is the determined cDNA sequence for clone 25033.
  • SEQ ID NO:497 is the determined cDNA sequence for clone 25034.
  • SEQ ID NO:498 is the determined cDNA sequence for clone 25035.
  • SEQ ID NO:499 is the determined cDNA sequence for clone 25036.
  • SEQ ID NO:500 is the determined cDNA sequence for clone 25037.
  • SEQ ID NO:501 is the determined cDNA sequence for clone 25038.
  • SEQ ID NO:502 is the determined cDNA sequence for clone 25039.
  • SEQ ID NO:503 is the determined cDNA sequence for clone 25040.
  • SEQ ID NO:504 is the determined cDNA sequence for clone 25042.
  • SEQ ID NO:505 is the determined cDNA sequence for clone 25043.
  • SEQ ID NO:506 is the determined cDNA sequence for clone 25044.
  • SEQ ID NO:507 is the determined cDNA sequence for clone 25045.
  • SEQ ID NO:508 is the determined cDNA sequence for clone 25047.
  • SEQ ID NO:509 is the determined cDNA sequence for clone 25048.
  • SEQ ID NO:510 is the determined cDNA sequence for clone 25049.
  • SEQ ID NO:511 is the determined cDNA sequence for clone 25185.
  • SEQ ID NO:512 is the determined cDNA sequence for clone 25186.
  • SEQ ID NO:513 is the determined cDNA sequence for clone 25187.
  • SEQ ID NO:514 is the determined cDNA sequence for clone 25188.
  • SEQ ID NO:515 is the determined cDNA sequence for clone 25189.
  • SEQ ID NO.-516 is the determined cDNA sequence for clone 25190.
  • SEQ ID NO:517 is the determined cDNA sequence for clone 25193.
  • SEQ ID NO:518 is the determined cDNA sequence for clone 25194.
  • SEQ ID NO:519 is the determined cDNA sequence for clone 25196.
  • SEQ ID NO:520 is the determined cDNA sequence for clone 25198.
  • SEQ ID NO:521 is the determined cDNA sequence for clone 25199.
  • SEQ ID NO:522 is the determined cDNA sequence for clone 25200.
  • SEQ ID NO:523 is the determmed cDNA sequence for clone 25202.
  • SEQ ID NO:524 is the determined cDNA sequence for clone 25364.
  • SEQ ID NO:525 is the determined cDNA sequence for clone 25366.
  • SEQ ID NO:526 is the determined cDNA sequence for clone 25367.
  • SEQ ID NO: 527 is the determined cDNA sequence for clone 25368.
  • SEQ ID NO:528 is the determined cDNA sequence for clone 25369.
  • SEQ ID NO:529 is the determined cDNA sequence for clone 25370.
  • SEQ ID NO:530 is the determined cDNA sequence for clone 25371.
  • SEQ ID NO:531 is the determined cDNA sequence for clone 25372.
  • SEQ ID NO:532 is the determined cDNA sequence for clone 25373.
  • SEQ ID NO:533 is the determined cDNA sequence for clone 25374.
  • SEQ ID NO.-534 is the determined cDNA sequence for clone 25376.
  • SEQ ID NO:535 is the determined cDNA sequence for clone 25377.
  • SEQ ID NO:536 is the determined cDNA sequence for clone 25378.
  • SEQ ID NO:537 is the determined cDNA sequence for clone 25379.
  • SEQ ID NO:538 is the determined cDNA sequence for clone 25380.
  • SEQ ID NO:539 is the determined cDNA sequence for clone 25381.
  • SEQ ID NO:540 is the determined cDNA sequence for clone 25382.
  • SEQ ID NO:541 is the determined cDNA sequence for clone 25383.
  • SEQ ID NO:542 is the determined cDNA sequence for clone 25385.
  • SEQ ID NO:543 is the determined cDNA sequence for clone 25386.
  • SEQ ID NO:544 is the determined cDNA sequence for clone 25387.
  • SEQ ID NO: 545 is the determined cDNA sequence for clone 26013.
  • SEQ ID NO:546 is the determined cDNA sequence for clone 26014.
  • SEQ ID NO:547 is the determined cDNA sequence for clone 26016.
  • SEQ ID NO:548 is the determined cDNA sequence for clone 26017.
  • SEQ ID NO:549 is the determined cDNA sequence for clone 26018.
  • SEQ ID NO:550 is the determined cDNA sequence for clone 26019.
  • SEQ ID NO:551 is the determined cDNA sequence for clone 26020.
  • SEQ ID NO:552 is the determined cDNA sequence for clone 26021.
  • SEQ ID NO:553 is the determined cDNA sequence for clone 26022.
  • SEQ ID NO:554 is the determined cDNA sequence for clone 26027.
  • SEQ ID NO.-555 is the determined cDNA sequence for clone 26197.
  • SEQ ID NO:556 is the determined cDNA sequence for clone 26199.
  • SEQ ID NO:557 is the determined cDNA sequence for clone 26201.
  • SEQ ID NO:558 is the determined cDNA sequence for clone 26202.
  • SEQ ID NO:559 is the determined cDNA sequence for clone 26203.
  • SEQ ID NO:560 is the determined cDNA sequence for clone 26204.
  • SEQ ID NO:561 is the determined cDNA sequence for clone 26205.
  • SEQ ID NO:562 is the determined cDNA sequence for clone 26206.
  • SEQ ID NO:563 is the determined cDNA sequence for clone 26208.
  • SEQ ID NO:564 is the determined cDNA sequence for clone 26211.
  • SEQ ID NO:565 is the determined cDNA sequence for clone 26212.
  • SEQ ID NO: 566 is the determined cDNA sequence for clone 26213.
  • SEQ ID NO: 567 is the determined cDNA sequence for clone 26214.
  • SEQ ID NO:568 is the determined cDNA sequence for clone 26215.
  • SEQ ID NO:569 is the determined cDNA sequence for clone 26216.
  • SEQ ID NO:570 is the determined cDNA sequence for clone 26217.
  • SEQ ID NO:571 is the determined cDNA sequence for clone 26218.
  • SEQ ID NO:572 is the determined cDNA sequence for clone 26219.
  • SEQ ID NO:573 is the determined cDNA sequence for clone 26220.
  • SEQ ID NO:574 is the determined cDNA sequence for clone 26221.
  • SEQ ID NO:575 is the determined cDNA sequence for clone 26224.
  • SEQ ID NO:576 is the determined cDNA sequence for clone 26225.
  • SEQIDNO:577 is the determined cDNA sequence for clone 26226.
  • SEQIDNO:578 is the determined cDNA sequence for clone 26227.
  • SEQ ID NO:579 is the determined cDNA sequence for clone 26228.
  • SEQIDNO:580 is the determined cDNA sequence for clone 26230.
  • SEQIDNO:581 is the determined cDNA sequence for clone 26231.
  • SEQIDNO:582 is the determined cDNA sequence for clone 26234.
  • SEQIDNO:583 is the determined cDNA sequence for clone 26236.
  • SEQIDNO:584 is the determined cDNA sequence for clone 26237.
  • SEQIDNO:585 is the determined cDNA sequence for clone 26239.
  • SEQIDNO:586 is the determined cDNA sequence for clone 26240.
  • SEQIDNO:587 is the determined cDNA sequence for clone 26241.
  • SEQIDNO:588 is the determined cDNA sequence for clone 26242.
  • SEQIDNO:589 is the determined cDNA sequence for clone 26246.
  • SEQIDNO:590 is the determined cDNA sequence for clone 26247.
  • SEQIDNO:591 is the determined cDNA sequence for clone 26248.
  • SEQIDNO:592 is the determined cDNA sequence for clone 26249.
  • SEQIDNO:593 is the determined cDNA sequence for clone 26250.
  • SEQIDNO:594 is the determined cDNA sequence for clone 26251.
  • SEQIDNO:595 is the determined cDNA sequence for clone 26252.
  • SEQIDNO:596 is the determined cDNA sequence for clone 26253.
  • SEQIDNO:597 is the determined cDNA sequence for clone 26254.
  • SEQ ID NO:598 is the determined cDNA sequence for clone 26255.
  • SEQ ID NO:599 is the determined cDNA sequence for clone 26256.
  • SEQ ID NO:600 is the determined cDNA sequence for clone 26257.
  • SEQIDNO:601 is the determined cDNA sequence for clone 26259.
  • SEQIDNO:602 is the determined cDNA sequence for clone 26260.
  • SEQIDNO:603 is the determined cDNA sequence for clone 26261.
  • SEQIDNO:604 is the determined cDNA sequence for clone 26262.
  • SEQIDNO:605 is the determined cDNA sequence for clone 26263.
  • SEQIDNO:606 is the determined cDNA sequence for clone 26264.
  • SEQIDNO.-607 is the determined cDNA sequence for clone 26265.
  • SEQIDNO:608 is the determined cDNA sequence for clone 26266
  • SEQIDNO:609 is the determined cDNA sequence for clone 26268
  • SEQIDNO:610 is the determined cDNA sequence for clone 26269
  • SEQIDNO:611 is the determined cDNA sequence for clone 26271
  • SEQIDNO:612 is the determined cDNA sequence for clone 26273
  • SEQIDNO:613 is the determined cDNA sequence for clone 26810
  • SEQIDNO:614 is the determined cDNA sequence for clone 26811
  • SEQIDNO:615 is the determined cDNA sequence for clone 26812
  • SEQIDNO:616 is the determined cDNA sequence for clone 26812
  • SEQIDNO:617 is the determined cDNA sequence for clone 26813
  • SEQIDNO:618 is the determined cDNA sequence for
  • SEQ ID NO:640 is the determined cDNA sequence for clone 26844.
  • SEQ ID NO:641 is the determined cDNA sequence for clone 26845.
  • SEQ ID NO:642 is the determined cDNA sequence for clone 26846.
  • SEQ ID NO:643 is the determined cDNA sequence for clone 26847.
  • SEQ ID NO:644 is the determined cDNA sequence for clone 26848.
  • SEQ ID NO:645 is the determined cDNA sequence for clone 26849.
  • SEQ ID NO:646 is the determined cDNA sequence for clone 26850.
  • SEQ ID NO:647 is the determined cDNA sequence for clone 26851.
  • SEQ ID NO:648 is the determined cDNA sequence for clone 26852.
  • SEQ ID NO:649 is the determined cDNA sequence for clone 26853.
  • SEQ ID NO:650 is the determined cDNA sequence for clone 26854.
  • SEQ ID NO:651 is the determined cDNA sequence for clone 26856.
  • SEQ ID NO:652 is the determined cDNA sequence for clone 26857.
  • SEQ ID NO:653 is the determined cDNA sequence for clone 26858.
  • SEQ ID NO:654 is the determined cDNA sequence for clone 26859.
  • SEQ ID NO:655 is the determined cDNA sequence for clone 26860.
  • SEQ ID NO:656 is the determined cDNA sequence for clone 26862.
  • SEQ ID NO:657 is the determined cDNA sequence for clone 26863.
  • SEQ ID NO:658 is the determined cDNA sequence for clone 26864.
  • SEQ ID NO:659 is the determined cDNA sequence for clone 26865.
  • SEQ ID NO:660 is the determined cDNA sequence for clone 26867.
  • SEQ ID NO:661 is the determined cDNA sequence for clone 26868.
  • SEQ ID NO:662 is the determined cDNA sequence for clone 26871.
  • SEQ ID NO:663 is the determined cDNA sequence for clone 26873.
  • SEQ ID NO:664 is the determined cDNA sequence for clone 26875.
  • SEQ ID NO:665 is the determined cDNA sequence for clone 26876.
  • SEQ ID NO:666 is the determined cDNA sequence for clone 26877.
  • SEQ ID NO:667 is the determined cDNA sequence for clone 26878.
  • SEQ ID NO:668 is the determined cDNA sequence for clone 26880.
  • SEQ ID NO:669 is the determined cDNA sequence for clone 26882.
  • SEQ ID NO:670 is the determined cDNA sequence for clone 26883.
  • SEQ ID NO:671 is the determined cDNA sequence for clone 26884.
  • SEQ ID NO:672 is the determined cDNA sequence for clone 26885.
  • SEQ ID NO:673 is the determined cDNA sequence for clone 26886.
  • SEQ ID NO:674 is the determined cDNA sequence for clone 26887.
  • SEQ ID NO:675 is the determined cDNA sequence for clone 26888.
  • SEQ ID NO:676 is the determmed cDNA sequence for clone 26889.
  • SEQ ID NO:677 is the determined cDNA sequence for clone 26890.
  • SEQ ID NO:678 is the determined cDNA sequence for clone 26892.
  • SEQ ID NO:679 is the determined cDNA sequence for clone 26894.
  • SEQ ID NO:680 is the determined cDNA sequence for clone 26895.
  • SEQ ID NO:681 is the determined cDNA sequence for clone 26897.
  • SEQ ID NO:682 is the determined cDNA sequence for clone 26898.
  • SEQ ID NO:683 is the determined cDNA sequence for clone 26899.
  • SEQ ID NO:684 is the determined cDNA sequence for clone 26900.
  • SEQ ID NO:685 is the determined cDNA sequence for clone 26901.
  • SEQ ID NO:686 is the determined cDNA sequence for clone 26903.
  • SEQ ID NO:687 is the determined cDNA sequence for clone 26905.
  • SEQ ID NO:688 is the determined cDNA sequence for clone 26906.
  • SEQ ID NO:689 is the determined cDNA sequence for clone 26708.
  • SEQ ID NO:690 is the determined cDNA sequence for clone 26709.
  • SEQ ID NO:691 is the determined cDNA sequence for clone 26710.
  • SEQ ID NO:692 is the determined cDNA sequence for clone 26711.
  • SEQ ID NO:693 is the determined cDNA sequence for clone 26712.
  • SEQ ID NO:694 is the determined cDNA sequence for clone 26713.
  • SEQ ID NO:695 is the determined cDNA sequence for clone 26714.
  • SEQ ID NO:696 is the determined cDNA sequence for clone 26715.
  • SEQ ID NO:697 is the determined cDNA sequence for clone 26716.
  • SEQ ID NO:698 is the determined cDNA sequence for clone 26717.
  • SEQ ID NO:699 is the determined cDNA sequence for clone 26718.
  • SEQ ID NO:700 is the determined cDNA sequence for clone 26719.
  • SEQIDNO:701 is the determined cDNA sequence for clone 26720.
  • SEQIDNO.-702 is the determined cDNA sequence for clone 26721.
  • SEQIDNO:703 is the determined cDNA sequence for clone 26722.
  • SEQIDNO:704 is the determined cDNA sequence for clone 26723.
  • SEQIDNO:705 is the determined cDNA sequence for clone 26724.
  • SEQIDNO:706 is the determined cDNA sequence for clone 26725.
  • SEQIDNO:707 is the determined cDNA sequence for clone 26726.
  • SEQIDNO:708 is the determined cDNA sequence for clone 26727.
  • SEQIDNO:709 is the determined cDNA sequence for clone 26728.
  • SEQIDNO:710 is the determined cDNA sequence for clone 26729.
  • SEQIDNO:711 is the determined cDNA sequence for clone 26730.
  • SEQIDNO:712 is the determined cDNA sequence for clone 26731'.
  • SEQIDNO:713 is the determined cDNA sequence for clone 26732.
  • SEQIDNO:714 is the determined cDNA sequence for clone 26733.1.
  • SEQIDNO.-715 is the determined cDNA sequence for clone 26733.2.
  • SEQIDNO:716 is the determined cDNA sequence for clone 26734.
  • SEQIDNO:717 is the determined cDNA sequence for clone 26735.
  • SEQIDNO:718 is the determined cDNA sequence for clone 26736.
  • SEQIDNO.-719 is the determined cDNA sequence for clone 26737.
  • SEQIDNO:720 is the determined cDNA sequence for clone 26738.
  • SEQIDNO:721 is the determined cDNA sequence for clone 26739.
  • SEQIDNO:722 is the determined cDNA sequence for clone 26741.
  • SEQID O:723 is the determined cDNA sequence for clone 26742.
  • SEQIDNO:724 is the determined cDNA sequence for clone 26743.
  • SEQIDNO:725 is the determined cDNA sequence for clone 26744.
  • SEQIDNO:726 is the determined cDNA sequence for clone 26745.
  • SEQ ID NO:727 is the determined cDNA sequence for clone 26746.
  • SEQIDNO:728 is the determined cDNA sequence for clone 26747.
  • SEQIDNO:729 is the determined cDNA sequence for clone 26748.
  • SEQID O:730 is the determined cDNA sequence for clone 26749.
  • SEQIDNO:731 is the determined cDNA sequence for clone 26750.
  • SEQ ID NO:732 is the determined cDNA sequence for clone 26751.
  • SEQ ID NO:733 is the determined cDNA sequence for clone 26752.
  • SEQ ID NO:734 is the determined cDNA sequence for clone 26753.
  • SEQ ID NO:735 is the determined cDNA sequence for clone 26754.
  • SEQ ID NO:736 is the determined cDNA sequence for clone 26755.
  • SEQ ID NO:737 is the determined cDNA sequence for clone 26756.
  • SEQ ID NO:738 is the determined cDNA sequence for clone 26757.
  • SEQ ID NO:739 is the determined cDNA sequence for clone 26758.
  • SEQ ID NO:740 is the determined cDNA sequence for clone 26759.
  • SEQ ID NO:741 is the determined cDNA sequence for clone 26760.
  • SEQ ID NO:742 is the determined cDNA sequence for clone 26761.
  • SEQ ID NO:743 is the determined cDNA sequence for clone 26762.
  • SEQ ID NO:744 is the determined cDNA sequence for clone 26763.
  • SEQ ID NO:745 is the determined cDNA sequence for clone 26764.
  • SEQ ID NO :746 is the determined cDNA sequence for clone 26765.
  • SEQ ID NO:747 is the determined cDNA sequence for clone 26766.
  • SEQ ID NO:748 is the determined cDNA sequence for clone 26767.
  • SEQ ID NO:749 is the determined cDNA sequence for clone 26768.
  • SEQ ID NO:750 is the determined cDNA sequence for clone 26769.
  • SEQ ID NO:751 is the determined cDNA sequence for clone 26770.
  • SEQ ID NO:752 is the determined cDNA sequence for clone 26771.
  • SEQ ID NO:753 is the determined cDNA sequence for clone 26772.
  • SEQ ID NO:754 is the determined cDNA sequence for clone 26773.
  • SEQ ID NO:755 is the determined cDNA sequence for clone 26774.
  • SEQ ID NO:756 is the determined cDNA sequence for clone 26775.
  • SEQ ID NO.-757 is the determined cDNA sequence for clone 26776.
  • SEQ ID NO.-758 is the determined cDNA sequence for clone 26777.
  • SEQ ID NO:759 is the determined cDNA sequence for clone 26778.
  • SEQ ID NO:760 is the determined cDNA sequence for clone 26779.
  • SEQ ID NO :761 is the determined cDNA sequence for clone 26781.
  • SEQ ID NO:762 is the determined cDNA sequence for clone 26782.
  • SEQ ID NO:763 is the determined cDNA sequence for clone 26783.
  • SEQ ID NO:764 is the determined cDNA sequence for clone 26784.
  • SEQ ID NO:765 is the determined cDNA sequence for clone 26785.
  • SEQ ID NO:766 is the determined cDNA sequence for clone 26786.
  • SEQ ID NO:767 is the determined cDNA sequence for clone 26787.
  • SEQ ID NO:768 is the determined cDNA sequence for clone 26788.
  • SEQ ID NO:769 is the determined cDNA sequence for clone 26790.
  • SEQ ID NO:770 is the determined cDNA sequence for clone 26791.
  • SEQ ID NO:771 is the determined cDNA sequence for clone 26792.
  • SEQ ID NO:772 is the determined cDNA sequence for clone 26793.
  • SEQ ID NO:773 is the determined cDNA sequence for clone 26794.
  • SEQ ID NO.-774 is the determined cDNA sequence for clone 26795.
  • SEQ ID NO:775 is the determined cDNA sequence for clone 26796.
  • SEQ ID NO:776 is the determined cDNA sequence for clone 26797.
  • SEQ ID NO:777 is the determined cDNA sequence for clone 26798.
  • SEQ ID NO:778 is the determined cDNA sequence for clone 26800.
  • SEQ ID NO:779 is the determined cDNA sequence for clone 26801.
  • SEQ ID NO:780 is the determined cDNA sequence for clone 26802.
  • SEQ ID NO:781 is the determined cDNA sequence for clone 26803.
  • SEQ ID NO:782 is the determined cDNA sequence for clone 26804.
  • SEQ ID NO:783 is the amino acid sequence for L773P.
  • SEQ ID NO:784 is the determined DNA sequence ofthe L773P expression construct.
  • SEQ ID NO:785 is the determined DNA sequence ofthe L773PA expression construct.
  • SEQ ID NO:786 is a predicted amino acid sequence for L552S.
  • SEQ ID NO :787 is a predicted amino acid sequence for L840P.
  • SEQ ID NO:788 is the full-length cDNA sequence for L548S.
  • SEQ ID NO:789 is the amino acid sequence encoded by SEQ ID NO:788.
  • SEQ ID NO:790 is an extended cDNA sequence for L552S.
  • SEQ ID NO: 791 is the predicted amino acid sequence encoded by the cDNA sequence of SEQ ID NO:790.
  • SEQ ID NO:792 is the determined cDNA sequence for an isoform of L552S.
  • SEQ ID NO:793 is the predicted amino acid sequence encoded by SEQ ID NO:792.
  • SEQ ID NO:794 is an extended cDNA sequence for L840P.
  • SEQ ID NO:795 is the predicted amino acid sequence encoded by SEQ Dl NO:794.
  • SEQ ID NO:796 is an extended cDNA sequence for L801P.
  • SEQ ID NO:797 is a first predicted amino acid sequence encoded by SEQ ID NO:796.
  • SEQ ID NO:798 is a second predicted amino acid sequence encoded by SEQ ID NO:798.
  • SEQ ID NO:799 is a third predicted amino acid sequence encoded by SEQ ID NO:796.
  • SEQ ID NO:800 is the determined full-length sequence for L844P.
  • SEQ ID NO:801 is the 5' consensus cDNA sequence for L551S.
  • SEQ ID NO:802 is the 3' consensus cDNA sequence for L551S.
  • SEQ ID NO:803 is the cDNA sequence for STY8.
  • SEQ ID NO:804 is an extended cDNA sequence for L551S.
  • SEQ ID NO:805 is the amino acid sequence for STY8.
  • SEQ ID NO: 806 is the extended amino acid sequence for L551 S.
  • SEQ ID NO:807 is the determined full length cDNA sequence for L773P.
  • SEQ ID NO:808 is the full-length cDNA sequence of L552S.
  • SEQ ID NO:809 is the full-length amino acid sequence of L552S.
  • SEQ ID NO:810 is the determined cDNA sequence of clone 50989.
  • SEQ ID NO:811 is the determined cDNA sequence of clone 50990.
  • SEQ ID NO:812 is the determined cDNA sequence of clone 50992.
  • SEQ ID NO: 813-824 are the determined cDNA sequences for clones isolated from lung tumor tissue.
  • SEQ ID NO: 825 is the determined cDNA sequence for the full-length L551S clone 54305.
  • SEQ ID NO: 826 is the determined cDNA sequence for the full-length L551S clone
  • SEQ ID NO:827 is the full-length amino acid sequence for L551S.
  • Tables 1-6 contain the sequence identifiers for SEQ ID NO:828-1664.
  • SEQ ID NO: 1665 and 1666 are primers used in the amplification ofthe coding region ofL548S
  • SEQ ID NO 1667 is the protein sequence of expressed recombinant L7548S.
  • SEQ ID NO 1668 is the cDNA sequence of expressed recombinant L7548S.
  • SEQ ID NO 1669 is the extended cDNA sequence of clone #18971 (L801P).
  • SEQ ID NO 1670 is the amino acid sequence of open reading frame ORF4 encoded by SEQ ID NO 1669.
  • SEQ ID NO 1671 is the amino acid sequence of open reading frame ORF5 encoded by SEQ ID NO 1669.
  • SEQ ID NO 1672 is the amino acid sequence of open reading frame ORF6 encoded by SEQ ID NO 1669.
  • SEQ ID NO 1673 is the amino acid sequence of open reading frame ORF7 encoded by SEQ ID NO 1669.
  • SEQ ID NO 1674 is the amino acid sequence of open reading frame ORF8 encoded by SEQ ID NO 1669.
  • SEQ ID NO 1675 is the amino acid sequence of open reading frame ORF9 encoded by SEQ ID NO 1669.
  • SEQ ID NO:1676 is the extended cDNA for contig 139 (SEQ ID NO:1467), also known as L985P.
  • SEQ ID NO: 1677 is the L985P amino acid sequence encoded by SEQ ID NO: 1676.
  • SEQ ID NO: 1678 is the amino acid sequence of open reading frame ORF5X of SEQ ID NO: 1678.
  • SEQ ID NO: 1679 is the amino acid sequence of an open reading frame for contig 139
  • SEQ ID NO:1680-1788 set forth in the Table 9, represent cDNA clones identified by microarray analysis ofthe SQL1, SCL1, SCL3 and SCL4 libraries on lung chip 5.
  • SEQ ID NO: 1789 is the cDNA sequence of clone #47988 (L972P).
  • SEQ ID NO: 1790 is the cDNA sequence of clone #48005 (L979P).
  • SEQ ID NO: 1791 is an extended cDNA sequence for clone #48005 (L979P).
  • SEQ ID NO: 1792 is an extended cDNA sequence for clone #49826 (SEQ ID NO: 1279; L980P).
  • SEQ ID NO: 1793 is an extended cDNA sequence for clone #20631 (SEQ ID NO:l 17; L973P).
  • SEQ ID NO:1794 is an extended cDNA sequence for clone #20661 (SEQ ID NO:128; L974P).
  • SEQ ID NO:1795 is an extended cDNA sequence for clone #50430 (SEQ ID NO:1442; L996P).
  • SEQ ID NO: 1796 is an extended cDNA sequence for clone #26961 (SEQ ID NO:288;
  • SEQ ID NO: 1797 is an extended cDNA sequence for clone #24928 (SEQ ID NO:1339;
  • SEQ ID NO: 1798 is an extended cDNA sequence for clone #50507 (SEQ ID NO: 1446;
  • SEQ ID NO: 1799 is an extended cDNA sequence for clone #50645 (SEQ ID NO:1531;
  • SEQ ID NO :1800 is an extended cDNA sequence for clone #50628 (SEQ ID NO:1533; L1423P).
  • SEQ ID NO 1801 is an extended cDNA sequence for clone #50560 (SEQ ID NO:1527;
  • SEQ ID NO 1802 is an extended cDNA sequence for clone #27699 (SEQ ID NO:468;
  • SEQ ID NO 1803 is an extended cDNA sequence for clone #59303 (SEQ ID NO:949;
  • SEQ ID NO 1804 is an extended cDNA sequence for clone #59314 (SEQ ID NO: 1156;
  • SEQ ID NO 1.805 is an extended cDNA sequence for clone #59298 (SEQ ID NO:921; L1427P).
  • SEQ ID NO 1806 is an amino acid sequence encoded by SEQ ID NO:1791.
  • SEQ ID NO 1807 is an amino acid sequence encoded by SEQ ID NO:1792.
  • SEQ ID NO 1808 is an amino acid sequence encoded by SEQ ID NO: 1793.
  • SEQ ID NO 1809 is an amino acid sequence encoded by SEQ ID NO:1794.
  • SEQ ID NO 1810 is an amino acid sequence encoded by SEQ ID NO: 1795.
  • SEQ ID NO 1811 is an amino acid sequence encoded by SEQ ID NO:1796.
  • SEQ ID NO 1812 is an amino acid sequence encoded by SEQ ID NO: 1797.
  • SEQ ID NO 1813 is an amino acid sequence encoded by SEQ ID NO-.1798.
  • SEQ ID NO 1814 is an amino acid sequence encoded by SEQ ID NO:1799.
  • SEQ ID NO 1815 is an amino acid sequence encoded by SEQ ID NO: 1800.
  • SEQ ID NO 1816 is an amino acid sequence encoded by SEQ ID NO:1527 (L987P).
  • SEQ ID NO 1817 is an amino acid sequence encoded by SEQ ID NO:1823.
  • SEQ ID NO:1818 is an amino acid sequence encoded by SEQ ID NO:1801.
  • SEQ ID NO: 1819 is an amino acid sequence encoded by SEQ ID NO: 1802.
  • SEQ ID NO: 1820 is an amino acid sequence encoded by SEQ ID NO: 1803.
  • SEQ ID NO: 1821 is an amino acid sequence encoded by SEQ ID NO: 1804.
  • SEQ ID NO: 1822 is an amino acid sequence encoded by SEQ ID NO: 1805.
  • SEQ ID NO: 1823 is an extended cDNA sequence for clone #50560 (SEQ ID NO: 1527;
  • SEQ ID NO: 1824 is a full length cDNA sequence for clone L872P (SEQ ID NO:34).
  • SEQ ID NO: 1825 is the amino acid sequence encoded by SEQ ID NO: 1824.
  • SEQ ID NO: 1826 is the cDNA sequence encoding the N-terminal portion of L552S.
  • SEQ ID NO: 1827-1829 are cDNA sequences of portions of L552S.
  • SEQ ID NO:1830 is the N-terminal portion of L552S.
  • SEQ ID NO:1831-1833 are the amino acid sequences encoded by SEQ ID NO:1827-
  • SEQ ID NO.T 834-1856 are the amino acid sequences of peptides of L548S.
  • SEQ ID NO: 1857-1860 are PCR primers.
  • SEQ ID NO:1861 is the deteraiined DNA sequence for a fusion of Ral2 and ORF4 of
  • SEQ ID NO:1862 is the determined DNA sequence for a fusion of Ral2 and ORF5 of P801P.
  • SEQ ID NO:1863 is the amino acid sequence ofthe fusion of Ral2 and ORF4 of
  • SEQ ID NO:1864 is the amino acid sequence ofthe fusion of Ral2 and ORF5 of
  • SEQ ID NO: 1865 is the determined cDNA sequence for clone L984PJ573 A).
  • SEQ ID NO: 1866 is the determined cDNA sequence for clone L984P 512A).
  • SEQ ID NO.T 867 is the determined cDNA sequence for clone L984P_(NCI-H128).
  • SEQ ID NO: 1868 is the determined cDNA sequence for clone L984P DMS-79).
  • SEQ ID NO:1869 is the amino acid sequence encoded by SEQ ID NO:1865.
  • SEQ ID NO: 1870 is the amino acid sequence encoded by SEQ ID NO: 1866.
  • SEQ ID NO:1871 is the amino acid sequence encoded by SEQ ID NO:1867.
  • SEQ ID NO: 1872 is the amino acid sequence encoded by SEQ ID NO: 1868.
  • SEQ ID NO: 1873 is a full length cDNA sequence for clone L985P (partial sequence given in SEQ ID NO: 1467).
  • SEQ ID NO:1874 is the amino acid sequence for L985P encoded by SEQ ID NO:1873.
  • SEQ ID NO: 1875 is the predicted and determined cDNA sequence for a fusion of Ral2 and L985P.
  • SEQ ID NO: 1876 is the predicted amino acid sequence of a fusion of Ral2 and L985P encoded by SEQ ID NO: 1875.
  • SEQ ID NO: 1877 is the predicted cDNA sequence for a fusion of Ral2S and L985P.
  • SEQ ID NO:1878 is the predicted amino acid sequence of a fusion of Ral2S and L985P encoded by SEQ ID NO: 1877.
  • SEQ ID NO: 1879 is the predicted cDNA sequence for a fusion of Ral2S and L985PEx.
  • SEQ ID NO: 1880 is the predicted amino acid sequence of a fusion of Ral2S and
  • SEQ ID NO: 1881 is the predicted cDNA sequence the extracellular loop 2 peptide of L985P.
  • SEQ ID NO: 1882 is the predicted amino acid sequence for the extracellular loop 2 peptide of L985P encoded by SEQ ID NO: 1875.
  • SEQ ID NO: 1883 is an extended cDNA sequence for clone #59316 (SEQ ID NO: 1180;
  • SEQ ID NO: 1884 is a first predicted amino acid sequence encoded by SEQ ID NO: 1883 and designated L1428P-ORF1.
  • SEQ ID NO: 1885 is a second predicted amino acid sequence encoded by SEQ ID NO: 1885
  • SEQ ID NO: 1886 is a third predicted amino acid sequence encoded by SEQ ID NO: 1883 and designated L 1428P-ORF3.
  • SEQ ID NO: 1887 is a fourth predicted amino acid sequence encoded by SEQ ID NO:
  • SEQ ID NO: 1888 is a fifth predicted amino acid sequence encoded by SEQ ID NO:
  • SEQ ID NO: 1883 is a sixth predicted amino acid sequence encoded by SEQ ID NO: 1883 and designated L1428P-ORF5.
  • SEQ ID NO: 1889 is a sixth predicted amino acid sequence encoded by SEQ ID NO: 1889
  • SEQ ID NO: 1883 is a seventh predicted amino acid sequence encoded by SEQ ID NO: 1883 and designated L1428P-ORF7.
  • SEQ ID NO:1891-1900 are the nucleotide sequences for the database hits described in Table 17.
  • SEQ ID NO:1901-1909 are the deduced amino acid sequences encoded by the nucleotide sequences described in Table 17.
  • SEQ ID NO: 1910 is the full-length cDNA for clone L1437P (partial sequence given in SEQ ID NO: 1896).
  • SEQ ID NO: 1911 is the forward primer PDM-433 for the coding region of clone L548S.
  • SEQ ID NO:1912 is the reverse primer PDM-438 for the coding region of clone L548S.
  • SEQ ID NO: 1913 is the amino acid sequence for the expressed recombinant L548S.
  • SEQ ID NO:1914 is the DNA coding sequence for the recombinant L548S.
  • SEQ ID NO:1915 is the forward primer PDM-498 for the coding region of clone L551S
  • SEQ ID NO:1916 is the reverse primer PDM-499 for the coding region of clone L551S
  • SEQ ID NO: 1917 is the amino acid sequence for the expressed recombinant L55 IS.
  • SEQ ID NO:1918 is the DNA coding sequence for the recombinant L551S.
  • SEQ ID NO:1919 is the forward primer PDM-479 for the coding region of clone L552S
  • SEQ ID NO: 1920 is the reverse primer PDM-480 for the coding region of clone L552S
  • SEQ ID NO:1921 is the amino acid sequence for the expressed recombinant L552S.
  • SEQ ID NO: 1922 is the DNA coding sequence for the recombinant L552S.
  • SEQ ID NO: 1923 is the predicted full-length cDNA sequence for clone #19069 (partial sequence given in SEQ ID NO:90).
  • SEQ ID NO: 1924 is the predicted full-length cDNA sequence for clone #18965 or #19002 (partial sequence given in SEQ ID NO: 15).
  • SEQ ID NO: 1925 is the deduced amino acid sequence encoded by SEQ ID NO: 1923.
  • SEQ ID NO: 1926 is the deduced amino acid sequence encoded by SEQ ID NO: 1924.
  • SEQ ID NO.T 927 is the determined amino acid sequence of a first L552S epitope.
  • SEQ ID NO: 1928 is the determined amino acid sequence of a second L552S epitope.
  • SEQ ID NO: 1929 is the determined amino acid sequence of a third L552S epitope.
  • SEQ ID NO: 1930 is the amino acid sequence for L985P peptide #3482.
  • SEQ ID NO:1931 is an extended cDNA sequence for clone #61144 (SEQ ID NO:1761,
  • SEQ ID NO: 1932 is the deduced amino acid sequence encoded by SEQ ID NO: 1931.
  • SEQ ID NO: 1933 is the full-length cDNA of the NUF2R gene to which SEQ ID NO: 1931 shows some sequence similarity.
  • SEQ ID NO.T 934 is the deduced amino acid sequence encoded by SEQ ID NO: 1933.
  • SEQ ID NO: 1935 is a forward primer PDM-737 for the coding region of clone L552S.
  • SEQ ID NO: 1936 is a reverse primer PDM-738 for the coding region of clone L552S.
  • SEQ ID NO: 1937 is the amino acid sequence for the expressed recombinant L552S.
  • SEQ ID NO:1938 is the DNA coding sequence for the recombinant L552S.
  • SEQ ID NO: 1939 is another forward primer PDM-736 for the coding region of clone
  • SEQ ID NO: 1940 is the amino acid sequence for a second expressed recombinant
  • SEQ ID NO: 1941 is the DNA coding sequence for a second recombinant L552S.
  • SEQ ID NO: 1942 is the determined amino acid sequence of a fourth L552S epitope.
  • SEQ ID NO: 1943 is the determined amino acid sequence of a first XAGE-1 epitope.
  • SEQ ID NO: 1944 is the determined amino acid sequence of a second XAGE-1 epitope.
  • SEQ ID NO: 1945 is the determined amino acid sequence of a first 20-mer peptide corresponding to amino acid residues 1-20 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1946 is the determined amino acid sequence of a second 20-mer peptide corresponding to amino acid residues 6-25 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1947 is the determined amino acid sequence of a third 20-mer peptide corresponding to amino acid residues 11-30 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1948 is the determined amino acid sequence of a fourth 20-mer peptide corresponding to amino acid residues 16-35 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1949 is the determined amino acid sequence of a fifth 20-mer peptide corresponding to amino acid residues 21-40 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1950 is the determined amino acid sequence of a sixth 20-mer peptide corresponding to amino acid residues 26-45 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1951 is the determined amino acid sequence of a seventh 20-mer peptide corresponding to amino acid residues 31-50 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1952 is the determined amino acid sequence of a eigth 20-mer peptide corresponding to amino acid residues 36-55 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1953 is the determined amino acid sequence of a ninth 20-mer peptide corresponding to amino acid residues 41-60 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1954 is the determined amino acid sequence of a tenth 20-mer peptide corresponding to amino acid residues 46-65 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1955 is the determined amino acid sequence of a eleventh 20-mer peptide corresponding to amino acid residues 51 -70 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1955 is the determined amino acid sequence of a twelveth 20-mer peptide corresponding to amino acid residues 56-75 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1956 is the determined amino acid sequence of a thirth 20-mer peptide corresponding to amino acid residues 61-80 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1957 is the determmed amino acid sequence of a fourteenth 20-mer peptide corresponding to amino acid residues 66-85 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1958 is the determined amino acid sequence of a fifteenth 20-mer peptide corresponding to amino acid residues 71-90 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1959 is the determined amino acid sequence of a sixteenth 20-mer peptide corresponding to amino acid residues 76-95 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1961 is the determined amino acid sequence of a seventeen 20-mer peptide corresponding to amino acid residues 81-100 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1962 is the determined amino acid sequence of a eighthth 20-mer peptide corresponding to amino acid residues 86-105 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1963 is the determined amino acid sequence of a nineteenth 20-mer peptide corresponding to amino acid residues 91-110 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1964 is the determined amino acid sequence of a twentieth 20-mer peptide corresponding to amino acid residues 96-115 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1965 is the determined amino acid sequence of a twenty-first 20-mer peptide corresponding to amino acid residues 101-120 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1966 is the determined amino acid sequence of a twenty-second 20-mer peptide corresponding to amino acid residues 106-125 of full-length L552S (SEQ ID NO:
  • SEQ ID NO: 1967 is the determined amino acid sequence of a twenty-third 20-mer peptide corresponding to amino acid residues 111-130 of full-length L552S (SEQ ID NO: 1967).
  • SEQ ID NO: 1968 is the determined amino acid sequence of a twenty-fourth 20-mer peptide corresponding to amino acid residues 116-135 of full-length L552S (SEQ ID NO: 1968).
  • SEQ ID NO:809 is the determined amino acid sequence of a twenty-fifth 20-mer peptide corresponding to amino acid residues 121-140 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1969 is the determined amino acid sequence of a twenty-fifth 20-mer peptide corresponding to amino acid residues 121-140 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1969 is the determined amino acid sequence of a twenty-fifth 20-mer peptide corresponding to amino acid residues 121-140 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1969 is the determined amino acid sequence of a twenty-fifth 20-mer peptide corresponding to amino acid residues 121-140 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1970 is the determined amino acid sequence of a twenty-sixth 20-mer peptide corresponding to amino acid residues 126-145 of full-length L552S (SEQ ID NO:809).
  • SEQ ID NO: 1971 is the determined amino acid sequence of a twenth-seventh 20-mer peptide corresponding to amino acid residues 131-150 of full-length L552S (SEQ ID NO: 1971).
  • SEQ ID NO: 1972 is the determined amino acid sequence of a twenty-eigth 20-mer peptide corresponding to amino acid residues 136-155 of full-length L552S (SEQ ID NO: 1972).
  • SEQ ID NO: 1973 is the determined amino acid sequence of a twenty-ninth 20-mer peptide corresponding to amino acid residues 141-160 of full-length L552S (SEQ ID NO: 1973).
  • SEQ ID NOT974 is the DNA sequence which encodes the 20-mer of SEQ ID NOT945.
  • SEQ ID NO.T 975 is the DNA sequence which encodes the 20-mer of SEQ ID NO.T 946.
  • SEQ ID NOT976 is the DNA sequence which encodes the 20-mer of SEQ ID NOT947.
  • SEQ ID NO: 1977 is the DNA sequence which encodes the 20-mer of SEQ ID NO: 1948.
  • SEQ ID NO: 1978 is the DNA sequence which encodes the 20-mer of SEQ ID NOT949.
  • SEQ ID NO: 1979 is the DNA sequence which encodes the 20-mer of SEQ ID NO: 1950.
  • SEQ ID NOT980 is the DNA sequence which encodes the 20-mer of SEQ ID NOT951.
  • SEQIDNOT981 s the DNA sequence which encodes the 20- mer of SEQ ID NO: 1952.
  • SEQ ID NO.T 982 s the DNA sequence which encodes the 20- merofSEQIDNO:1953.
  • SEQ ID NO: 1983 s the DNA sequence which encodes the 20- merofSEQIDNO:1954.
  • SEQ ID NO: 1984 s the DNA sequence which encodes the 20- merofSEQIDNO:1955.
  • SEQ ID NO: 1985 s the DNA sequence which encodes the 20- merofSEQIDNO:1956.
  • SEQ ID NO: 1986 s the DNA sequence which encodes the 20- merofSEQIDNO:1957.
  • SEQ ID NO: 1987 s the DNA sequence which encodes the 20- merofSEQIDNO:1958.
  • SEQ ID NO: 1988 s the DNA sequence which encodes the 20- ⁇ merofSEQIDNO:1959.
  • SEQ ID NO: 1989 s the DNA sequence which encodes the 20- ⁇ merofSEQIDNO:1960.
  • SEQ ID NO: 1990 s the DNA sequence which encodes the 20- merofSEQIDNO:1961.
  • SEQ ID NO: 1991 s the DNA sequence which encodes the 20- merofSEQIDNO:1962.
  • SEQ ID NO: 1992 s the DNA sequence which encodes the 20- ⁇ merofSEQIDNO:1963.
  • SEQ ID NO: 1993 s the DNA sequence which encodes the 20- ⁇ merofSEQIDNO:1964.
  • SEQ ID NO: 1994 s the DNA sequence which encodes the 20- ⁇ merofSEQIDNO:1965.
  • SEQ ID NO: 1995 s the DNA sequence which encodes the 20- •merofSEQIDNO:1966.
  • SEQ ID NO: 1996 s the DNA sequence which encodes the 20- •merofSEQIDNO:1967.
  • SEQ ID NO: 1997 s the DNA sequence which encodes the 20- •mer of SEQ ID NO: 1968.
  • SEQ ID NO: 1998 s the DNA sequence which encodes the 20- ⁇ merofSEQIDNO:1969.
  • SEQ ID NO: 1999 s the DNA sequence which encodes the 20- ⁇ merofSEQIDNO:1970.
  • SEQIDNO:2000 s the DNA sequence which encodes the 20- ⁇ merofSEQIDNO:1971.
  • SEQIDNO.-2001 s the DNA sequence which encodes the 20- ⁇ mer of SEQ ID NO.T 972.
  • SEQIDNO:2002 s the DNA sequence which encodes the 20- -merofSEQIDNO:1973.
  • compositions of the present invention include, but are not restricted to, polypeptides, particularly immunogenic polypeptides, polynucleotides encoding such polypeptides, antibodies and other binding agents, antigen presenting cells (APCs) and immune system cells (e.g., T cells).
  • APCs antigen presenting cells
  • T cells immune system cells
  • polypeptide is used in its conventional meaning, i.e., as a sequence of amino acids.
  • the polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • This term also does not refer to or exclude post- expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a polypeptide may be an entire protein, or a subsequence thereof.
  • polypeptides of interest in the context of this invention are amino acid subsequences comprising epitopes, i.e., antigenic determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response.
  • polypeptides of the present invention comprise those encoded by a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-57, 59-323, 341-782, 784-785, 788, 790, 792, 794, 796, 800-804, 807, 808, 810-826, 828- 1664, 1668, 1669, 1676, 1680-1805, 1824, 1826-1829, 1865-1868, 1873, 1875, 1877, 1879, 1881, 1883, 1891-1900, 1910, 1914, 1918, 1922-1924, 1931, 1933, 1938, 1941 and 1974-2002, or a sequence that hybridizes under moderately stringent conditions, or, alternatively, under highly stringent conditions, to a polynucleotide sequence set forth in any one of SEQ ID NOs:l-57, 59-323, 341-782, 784-785, 788, 790, 792, 794, 796, 800- 804, 8
  • polypeptides of the invention comprise amino acid sequences as set forth in any one of SEQ ID NOs:324-340, 786, 787, 789, 791, 793, 795, 797-799, 805, 806, 809, 827, 1667, 1670- 1675, 1677-1679, 1806-1822, 1825, 1830-1833, 1834-1856, 1863, 1864, 1869-1872, 1874, 1876, 1878, 1880, 1882, 1884-1890, 1901-1909, 1913, 1917, 1921, 1925-1930, 1932, 1934, 1937, 1940 and 1942-1973.
  • lung tumor polypeptide or "lung tumor protein,” refers generally to a polypeptide sequence of the present invention, or a polynucleotide sequence encoding such a polypeptide, that is expressed in a substantial proportion of lung tumor samples, for example preferably greater than about 20%, more preferably greater than about 30%, and most preferably greater than about 50% or more of lung tumor samples tested, at a level that is at least two fold, and preferably at least five fold, greater than the level of expression in normal tissues, as determined using a representative assay provided herein.
  • a lung tumor polypeptide sequence of the invention based upon its increased level of expression in tumor cells, has particular utility both as a diagnostic marker as well as a therapeutic target, as further described below.
  • the polypeptides of the invention are immunogenic, i.e., they react detectably within an immunoassay (such as an ELISA or T-cell stimulation assay) with antisera and/or T-cells from a patient with lung cancer. Screening for immunogenic activity can be performed using techniques well known to the skilled artisan. For example, such screens can be performed using methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, I-labeled Protein A.
  • immunogenic portions of the polypeptides disclosed herein are also encompassed by the present invention.
  • An "immunogenic portion,” as used herein, is a fragment of an immunogenic polypeptide of the invention that itself is immunologically reactive (i.e., specifically binds) with the B-cells and/or T-cell surface antigen receptors that recognize the polypeptide.
  • Immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones.
  • antisera and antibodies are "antigen-specific” if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins).
  • antisera and antibodies may be prepared as described herein, and using well-known techniques.
  • an immunogenic portion of a polypeptide ofthe present invention is a portion that reacts with antisera and/or T-cells at a level that is not substantially less than the reactivity of the full-length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay).
  • the level of immunogenic activity of the immunogenic portion is at least about 50%, preferably at least about 70% and most preferably greater than about 90% of the immunogenicity for the full-length polypeptide.
  • preferred immunogenic portions will be identified that have a level of immunogenic activity greater than that of the corresponding full-length polypeptide, e.g., having greater than about 100% or 150% or more immunogenic activity.
  • illustrative immunogenic portions may include peptides in which an N-terminal leader sequence and/or transmembrane domain have been deleted.
  • Other illustrative immunogenic portions will contain a small N- and/or C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to the mature protein.
  • a polypeptide composition of the invention may also comprise one or more polypeptides that are immunologically reactive with T cells and/or antibodies generated against a polypeptide of the invention, particularly a polypeptide having an amino acid sequence disclosed herein, or to an immunogenic fragment or variant thereof.
  • polypeptides comprise one or more polypeptides that are capable of eliciting T cells and/or antibodies that are immunologically reactive with one or more polypeptides described herein, or one or more polypeptides encoded by contiguous nucleic acid sequences contained in the polynucleotide sequences disclosed herein, or immunogenic fragments or variants thereof, or to one or more nucleic acid sequences which hybridize to one or more of these sequences under conditions of moderate to high stringency.
  • the present invention in another aspect, provides polypeptide fragments comprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide compositions set forth herein, such as those set forth in SEQ ID NOs:324-340, 786, 787, 789, 791, 793, 795, 797-799, 805, 806, 809, 827, 1667, 1670-1675, 1677-1679, 1806-1822, 1825, 1830-1833, 1834-1856, 1863, 1864, 1869-1872, 1874, 1876, 1878, 1880, 1882, 1884-1890, 1901-1909, 1913, 1917, 1921, 1925-1930, 1932, 1934, 1937, 1940 and 1942-1973, or those encoded by a polynucleotide sequence set forth in a sequence of SEQ ID NOs:l-57, 59-323, 341-782, 784-785, 788, 790, 792, 794
  • the present invention provides variants of the polypeptide compositions described herein.
  • Polypeptide variants generally encompassed by the present invention will typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below), along its length, to a polypeptide sequences set forth herein.
  • the polypeptide fragments and variants provided by the present invention are immunologically reactive with an antibody and/or T-cell that reacts with a full-length polypeptide specifically set for the herein.
  • the polypeptide fragments and variants provided by the present invention exhibit a level of immunogenic activity of at least about 50%, preferably at least about 70%, and most preferably at least about 90% or more of that exhibited by a full-length polypeptide sequence specifically set forth herein.
  • a polypeptide "variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating their immunogenic activity as described herein and/or using any of a number of techniques well known in the art.
  • certain illustrative variants of the polypeptides of the invention include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed.
  • Other illustrative variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminal ofthe mature protein.
  • a variant will contain conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics, e.g., with immunogenic characteristics.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
  • Patent 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property ofthe protein. As detailed in U. S.
  • Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (- 2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within +2 is preferred, those within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • any polynucleotide may be further modified to increase stability in vivo.
  • flanking sequences at the 5' and/or 3' ends Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature ofthe polypeptide.
  • polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein.
  • the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
  • a polypeptide may be conjugated to an immunoglobulin Fc region.
  • two sequences are said to be “identical” if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
  • a “comparison window” as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters.
  • This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol.
  • optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
  • BLAST and BLAST 2.0 are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.
  • BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • a scoring matrix can be used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed ofthe alignment.
  • the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment ofthe two sequences.
  • the percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
  • a polypeptide may be a fusion polypeptide that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein.
  • a fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein.
  • Certain preferred fusion partners are both immunological and expression enhancing fusion partners.
  • Other fusion partners may be selected so as to increase the solubility ofthe polypeptide or to enable the polypeptide to be targeted to desired intracellular compartments.
  • Still further fusion partners include affinity tags, which facilitate purification ofthe polypeptide.
  • Fusion polypeptides may generally be prepared using standard techniques, including chemical conjugation.
  • a fusion polypeptide is expressed as a recombinant polypeptide, allowing the production of increased levels, relative to a non-fused polypeptide, in an expression system.
  • DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector.
  • the 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.
  • a peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
  • Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques well known in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • Preferred peptide linker sequences contain Gly, Asn and Ser residues.
  • linker sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl Acad. Sci. USA 55:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.
  • the linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • the ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements.
  • the regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides.
  • stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.
  • the fusion polypeptide can comprise a polypeptide as described herein together with an unrelated immunogenic protein, such as an immunogenic protein capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl. J. Med., 550:86-91, 1997).
  • the immunological fusion partner is derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-derived Ral2 fragment.
  • a Mycobacterium sp. such as a Mycobacterium tuberculosis-derived Ral2 fragment.
  • Ral2 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences is described in U.S. Patent Application 60/158,585, the disclosure of which is incorporated herein by reference in its entirety. Briefly, Ral2 refers to a polynucleotide region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid.
  • MTB32A is a serine protease of 32 KD molecular weight encoded by a gene in virulent and avirulent strains of M. tuberculosis.
  • the nucleotide sequence and amino acid sequence of MTB32A have been described (for example, U.S. Patent Application 60/158,585; see also, Skeiky et al.,- Infection and Immun. (1999) 67:3998-4007, incorporated herein by reference).
  • a 14KD C- terminal fragment of the MTB32A coding sequence expresses at high levels on its own and remains as a soluble polypeptide throughout the purification process.
  • this fragment may enhance the immunogenicity of heterologous antigenic polypeptides with which it is fused.
  • This 14 KD C-terminal fragment is referred to herein as Ral2 and represents a fragment comprising some or all of amino acid residues 192 to 323 of MTB32A.
  • Ral2 polynucleotides generally comprise at least about 15 consecutive nucleotides, at least about 30 nucleotides, at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, or at least about 300 nucleotides that encode a portion of a Ral2 polypeptide.
  • Ral2 polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Ral2 polypeptide or a portion thereof) or may comprise a variant of such a sequence.
  • Ral2 polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ral2 polypeptide.
  • Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native Ral2 polypeptide or a portion thereof.
  • an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926).
  • a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated.
  • the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer).
  • the lipid tail ensures optimal presentation of the antigen to antigen presenting cells.
  • Other fusion partners include the non-structural protein from influenzae virus, NS1 (hemaglutinin).
  • the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
  • the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion).
  • LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986).
  • LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
  • the C-terminal domain ofthe LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E.
  • coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798, 1992).
  • a repeat portion of LYTA may be incorporated into a fusion polypeptide. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.
  • Yet another illustrative embodiment involves fusion polypeptides, and the polynucleotides encoding them, wherein the fusion partner comprises a targeting signal capable of directing a polypeptide to the endosomal/lysosomal compartment, as described in U.S. Patent No. 5,633,234.
  • a targeting signal capable of directing a polypeptide to the endosomal/lysosomal compartment, as described in U.S. Patent No. 5,633,234.
  • An immunogenic polypeptide ofthe invention when fused with this targeting signal, will associate more efficiently with MHC class II molecules and thereby provide enhanced in vivo stimulation of CD4 + T-cells specific for the polypeptide.
  • Polypeptides of the invention are prepared using any of a variety of well known synthetic and/or recombinant techniques, the latter of which are further described below. Polypeptides, portions and other variants generally less than about 150 amino acids can be generated by synthetic means, using techniques well known to those of ordinary skill in the art. In one illustrative example, such polypeptides are synthesized using any ofthe commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 55:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.
  • polypeptide compositions (including fusion polypeptides) of the invention are isolated.
  • An "isolated" polypeptide is one that is removed from its original environment.
  • a naturally-occurring protein or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system.
  • polypeptides are also purified, e.g., are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
  • Polynucleotide Compositions are also purified, e.g., are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
  • the present invention provides polynucleotide compositions.
  • DNA and “polynucleotide” are used essentially interchangeably herein to refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species.
  • isolated means that a polynucleotide is substantially away from other coding sequences, and that the DNA molecule does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA molecule as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • polynucleotide compositions of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.
  • polynucleotides of the invention may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
  • RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one- to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
  • Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a polypeptide/protein of the invention or a portion thereof) or may comprise a sequence that encodes a variant or derivative, preferably and immunogenic variant or derivative, of such a sequence.
  • polynucleotide compositions comprise some or all of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-57, 59-323, 341-782, 784-785, 788, 790, 792, 794, 796, 800-804, 807, 808, 810-826, 828-1664, 1668, 1669, 1676, 1680- 1805, 1824, 1826-1829, 1865-1868, 1873, 1875, 1877, 1879, 1881, 1883, 1891-1900, 1910, 1914, 1918, 1922-1924, 1931, 1933, 1938, 1941 and 1974-2002, complements of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-57, 59-323, 341-782, 784-785, 788, 790, 792, 794, 796, 800-804, 807, 808, 810-826, 828-1664, 1668,
  • the present invention provides polynucleotide variants having substantial identity to the sequences disclosed herein in SEQ ID NOs:l-57, 59-323, 341-782, 784-785, 788, 790, 792, 794, 796, 800-804, 807, 808, 810-826, 828-1664, 1668, 1669, 1676, 1680-1805, 1824, 1826-1829, 1865-1868, 1873, 1875, 1877, 1879, 1881, 1883, 1891-1900, 1910, 1914, 1918, 1922-1924, 1931, 1933, 1938, 1941 and 1974-2002, for example those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below).
  • BLAST analysis using standard parameters, as
  • polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably such that the immunogenicity of the polypeptide encoded by the variant polynucleotide is not substantially diminished relative to a polypeptide encoded by a polynucleotide sequence specifically set forth herein).
  • variants should also be understood to encompasses homologous genes of xenogenic origin.
  • the present invention provides polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein.
  • polynucleotides are provided by this invention that comprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between.
  • intermediate lengths means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like.
  • polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof.
  • Hybridization techniques are well known in the art of molecular biology.
  • suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-60°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.
  • suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65°C or 65- 70°C.
  • polynucleotides described above e.g., polynucleotide variants, fragments and hybridizing sequences, encode polypeptides that are immunologically cross-reactive with a polypeptide sequence specifically set forth herein.
  • such polynucleotides encode polypeptides that have a level of immunogenic activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of that for a polypeptide sequence specifically set forth herein.
  • polynucleotides of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention.
  • two sequences are said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
  • a “comparison window” as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters.
  • This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol.
  • optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
  • BLAST and BLAST 2.0 are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.
  • BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • additions or deletions i.e., gaps
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
  • nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides.
  • mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison). Therefore, in another embodiment of the invention, a mutagenesis approach, such as site-specific mutagenesis, is employed for the preparation of immunogenic variants and/or derivatives of the polypeptides described herein. By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques provides a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence ofthe encoded polypeptide.
  • the inventors contemplate the mutagenesis of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide, such as the immunogenicity of a polypeptide vaccine.
  • the techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides.
  • site-specific mutagenesis is often used to alter a specific portion of a DNA molecule.
  • a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction ofthe sequence being altered.
  • site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art.
  • Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • mutagenic agents such as hydroxylamine
  • oligonucleotide directed mutagenesis procedure refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification.
  • oligonucleotide directed mutagenesis procedure is intended to refer to a process that involves the template-dependent extension of a primer molecule.
  • template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987).
  • vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U. S. Patent No. 4,237,224, specifically incorporated herein by reference in its entirety.
  • recursive sequence recombination as described in U.S. Patent No. 5,837,458, may be employed. In this approach, iterative cycles of recombination and screening or selection are performed to "evolve" individual polynucleotide variants of the invention having, for example, enhanced immunogenic activity.
  • the polynucleotide sequences provided herein can be advantageously used as probes or primers for nucleic acid hybridization.
  • nucleic acid segments that comprise a sequence region of at least about 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein will find particular utility.
  • Longer contiguous identical or complementary sequences e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.
  • nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample.
  • sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
  • Polynucleotide molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to a polynucleotide sequence disclosed herein, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow a gene product, or fragment thereof, to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment.
  • Smaller fragments will generally find use in hybridization embodiments, wherein the length ofthe contiguous complementary region may be varied, such as between about 15 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
  • the use of a hybridization probe of about 15-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 15 bases in length are generally preferred, though, in order to increase stability and selectivity ofthe hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequences set forth herein, or to any continuous portion of the sequences, from about 15-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer.
  • the choice of probe and primer sequences may be governed by various factors. For example, one may wish to employ primers from towards the termini ofthe total sequence.
  • Small polynucleotide segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U. S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • the nucleotide sequences ofthe invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire gene or gene fragments of interest.
  • relatively stringent conditions e.g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50°C to about 70°C.
  • Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating related sequences.
  • polynucleotide compositions comprising antisense oligonucleotides are provided.
  • Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and, consequently, provide a therapeutic approach by which a disease can be treated by inhibiting the synthesis of proteins that contribute to the disease.
  • the efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U. S. Patent 5,739,119 and U. S.
  • Patent 5,759,829) examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA A receptor and human EGF (Jaskulski et al., Science. 1988 Jun 10;240(4858): 1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225- 32; Peris et al, Brain Res Mol Brain Res. 1998 Jun 15;57(2):310-20; U. S. Patent 5,801,154; U.S. Patent 5,789,573; U. S. Patent 5,718,709 and U.S. Patent 5,610,288).
  • MDG1 multiple drug resistance gene
  • Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g. cancer (U. S. Patent 5,747,470; U. S. Patent 5,591,317 and U. S. Patent 5,783,683).
  • the present invention provides oligonucleotide sequences that comprise all, or a portion of, any sequence that is capable of specifically binding to polynucleotide sequence described herein, or a complement thereof.
  • the antisense oligonucleotides comprise DNA or derivatives thereof.
  • the oligonucleotides comprise RNA or derivatives thereof.
  • the oligonucleotides are modified DNAs comprising a phosphorothioated modified backbone.
  • the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof.
  • compositions comprise a sequence region that is complementary, and more preferably substantially-complementary, and even more preferably, completely complementary to one or more portions of polynucleotides disclosed herein.
  • Selection of antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence and determination of secondary structure, T m , binding energy, and relative stability.
  • Antisense compositions may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell.
  • Highly preferred target regions of the mRNA are those which are at or near the AUG translation initiation codon, and those sequences which are substantially complementary to 5' regions ofthe mRNA.
  • MPG short peptide vector
  • the MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain from the nuclear localization sequence of SV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 Jul 15;25(14):2730-6).
  • the polynucleotide compositions described herein are used in the design and preparation of ribozyme molecules for inhibiting expression of the tumor polypeptides and proteins of the present invention in tumor cells.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci U S A. 1987 Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):211-20).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, Cell. 1981 Dec;27(3 Pt 2):487-96; Michel and Westhof, J Mol Biol. 1990 Dec 5;216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14;357(6374):173-6).
  • This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") ofthe ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
  • RNA Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • ribozyme The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide.
  • This advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.
  • the enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis ⁇ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif
  • hammerhead motifs are described by Rossi et al. Nucleic Acids Res. 1992 Sep 11;20(17):4559-65.
  • hairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun 13;28(12):4929-33; Hampel et al, Nucleic Acids Res. 1990 Jan 25;18(2):299-304 and U. S.
  • Patent 5,631,359 An example of the hepatitis ⁇ virus motif is described by Perrotta and Been, Biochemistry. 1992 Dec 1;31(47): 11843-52; an example of the RNaseP motif is described by Guerrier-Takada et al, Cell. 1983 Dec;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins, Proc Natl Acad Sci U S A. 1991 Oct l;88(19):8826-30; Collins and Olive, Biochemistry.
  • WO 94/02595 each specifically incorporated herein by reference
  • Such ribozymes can also be optimized for delivery. While specific examples are provided, those in the art will recognize that equivalent RNA targets in other species can be utilized when necessary.
  • Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U. S. Patent 5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.
  • Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
  • the RNA vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent.
  • routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically incorporated herein by reference.
  • RNA polymerase I RNA polymerase I
  • RNA polymerase II RNA polymerase II
  • RNA polymerase III RNA polymerase III
  • Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells Ribozymes expressed from such promoters have been shown to function in mammalian cells.
  • Such transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors (such as refroviral, semliki forest virus, Sindbis virus vectors).
  • peptide nucleic acids (PNAs) compositions are provided.
  • PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to be utilized in a number methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. A review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (Trends Biotechnol 1997 Jun;15(6):224-9).
  • PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al, Science 1991 Dec 6;254(5037):1497- 500; Hanvey et al, Science. 1992 Nov 27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 Jan;4(l):5-23).
  • PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used.
  • PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, MA). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al, Bioorg Med Chem. 1995 Apr;3(4):437-45). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.
  • PNAs can incorporate any combination of nucleotide bases
  • the presence of adjacent purines can lead to deletions of one or more residues in the product.
  • Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine.
  • PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements.
  • the identity of PNAs and their derivatives can be confirmed by mass spectrometry.
  • Several studies have made and utilized modifications of PNAs (for example, Norton et al, Bioorg Med Chem. 1995 Apr;3(4):437-45; Petersen et al, J Pept Sci.
  • U.S. Patent No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.
  • PNAs include use in DNA strand invasion, antisense inhibition, mutational analysis, enhancers of transcription, nucleic acid purification, isolation of transcriptionally active genes, blocking of transcription factor binding, genome cleavage, biosensors, in situ hybridization, and the like.
  • compositions ofthe present invention may be identified, prepared and/or manipulated using any of a variety of well established techniques (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989, and other like references).
  • a polynucleotide may be identified, as described in more detail below, by screening a microarray of cDNAs for tumor-associated expression (i.e., expression that is at least two fold greater in a tumor than in normal tissue, as determined using a representative assay provided herein). Such screens may be performed, for example, using the microarray technology of Affymetrix, Inc.
  • polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as tumor cells.
  • PCRTM polymerase chain reaction
  • the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides.
  • the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated.
  • reverse transcription and PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS) (PCT Intl. Pat. Appl. Publ. No. WO 88/10315), including nucleic acid sequence based amplification (NASBA) and 3SR.
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR nucleic acid sequence based amplification
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • WO 89/06700 describes a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence.
  • Other amplification methods such as “RACE” (Frohman, 1990), and “one-sided PCR” (Ohara, 1989) are also well-known to those of skill in the art.
  • An amplified portion of a polynucleotide ofthe present invention may be used to isolate a full length gene from a suitable library (e.g., a tumor cDNA library) using well known techniques.
  • a library cDNA or genomic
  • a library is screened using one or more polynucleotide probes or primers suitable for amplification.
  • a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequences.
  • a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32 P) using well known techniques.
  • a bacterial or bacteriophage library is then generally screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis.
  • cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector.
  • Restriction maps and partial sequences may be generated to identify one or more overlapping clones.
  • the complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones.
  • the resulting overlapping sequences can then assembled into a single contiguous sequence.
  • a full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
  • amplification techniques can be useful for obtaining a full length coding sequence from a partial cDNA sequence.
  • One such amplification technique is inverse PCR (see Triglia et al., Nucl Acids Res. 16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular Iigation and used as a template for PCR with divergent primers derived from the known region.
  • sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region.
  • the amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region.
  • a variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591.
  • Another such technique is known as "rapid amplification of cDNA ends" or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 7:111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res.
  • RNA sequences may also be obtained by analysis of genomic fragments.
  • EST expressed sequence tag
  • Other methods employing amplification may also be employed to obtain a full length cDNA sequence.
  • Full length DNA sequences may also be obtained by analysis of genomic fragments.
  • polynucleotide sequences or fragments thereof which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half- life which is longer than that of a transcript generated from the naturally occurring sequence.
  • polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.
  • natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence to encode a fusion protein.
  • a fusion protein may also be engineered to contain a , cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.
  • Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).
  • the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof.
  • peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al.
  • a newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.) or other comparable techniques available in the art.
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • the nucleotide sequences encoding the polypeptide, or functional equivalents may be inserted into appropriate expression vector, i.e., a vector which contains the necerney elements for the transcription and translation of the inserted coding sequence.
  • appropriate expression vector i.e., a vector which contains the necerney elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, J. et al.
  • a variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • plant cell systems transformed with virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
  • control elements or "regulatory sequences” present in an expression vector are those non-translated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions— which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) and the like may be used.
  • inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) and the like may
  • promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies ofthe sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.
  • any of a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide.
  • vectors which direct high level expression of fusion proteins that are readily purified may be used.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of .beta.- galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M.
  • pGEX Vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 5:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 77:85-105).
  • These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated fransfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).
  • An insect system may also be used to express a polypeptide of interest.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S.
  • a number of viral-based expression systems are generally available.
  • sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 57:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer
  • RSV Rous sarcoma virus
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • RSV Rous sarcoma virus
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl Cell Differ. 20:125-162).
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation. glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function.
  • Different host cells such as CHO, COS, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing ofthe foreign protein.

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US651563 1991-02-06
US614124 2000-07-11
US09/614,124 US6630574B1 (en) 1999-06-30 2000-07-11 Compositions and methods for the therapy and diagnosis of lung cancer
US09/651,563 US6914132B1 (en) 1999-06-30 2000-08-29 Compositions and methods for the therapy and diagnosis of lung cancer
US658824 2000-09-08
US09/658,824 US6746846B1 (en) 1999-06-30 2000-09-08 Methods for diagnosing lung cancer
US09/671,325 US6667154B1 (en) 1999-06-30 2000-09-26 Compositions and methods for the therapy and diagnosis of lung cancer
US671325 2000-09-26
US67741900A 2000-10-06 2000-10-06
US677419 2000-10-06
US702705 2000-10-30
US09/702,705 US6504010B1 (en) 1999-06-30 2000-10-30 Compositions and methods for the therapy and diagnosis of lung cancer
US736457 2000-12-13
US09/736,457 US6509448B2 (en) 1999-06-30 2000-12-13 Compositions and methods for the therapy and diagnosis of lung cancer
US09/849,626 US20020197669A1 (en) 2000-12-13 2001-05-03 Compositions and methods for the therapy and diagnosis of lung cancer
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