EP1230364A2 - Zusammensetzungen und verfahren zur behandlung und diagnose von prostatakrebs - Google Patents

Zusammensetzungen und verfahren zur behandlung und diagnose von prostatakrebs

Info

Publication number
EP1230364A2
EP1230364A2 EP00979155A EP00979155A EP1230364A2 EP 1230364 A2 EP1230364 A2 EP 1230364A2 EP 00979155 A EP00979155 A EP 00979155A EP 00979155 A EP00979155 A EP 00979155A EP 1230364 A2 EP1230364 A2 EP 1230364A2
Authority
EP
European Patent Office
Prior art keywords
seq
prostate
cdna sequence
polynucleotide
cells
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Withdrawn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
EP00979155A
Other languages
English (en)
French (fr)
Inventor
Jiangchun Xu
Davin C. Dillon
Jennifer L. Mitcham
Susan L. Harlocker
Yuqiu Jiang
Steven G. Reed
Michael D. Kalos
Marc W. Retter
John A. Stolk
Craig H. Day
Yasir A.W. Skeiky
Aijun Wang
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 US09/439,313 external-priority patent/US6329505B1/en
Application filed by Corixa Corp filed Critical Corixa Corp
Publication of EP1230364A2 publication Critical patent/EP1230364A2/de
Withdrawn legal-status Critical Current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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464493—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
    • A61K39/464494—Prostate specific antigen [PSA]
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates generally to therapy and diagnosis of cancer, such as prostate cancer.
  • the invention is more specifically related to polypeptides comprising at least a portion of a prostate-specific protein, and to polynucleotides encoding such polypeptides.
  • polypeptides and polynucleotides may be used in vaccines and pharmaceutical compositions for prevention and treatment of prostate cancer, and for the diagnosis and monitoring of such cancers.
  • Prostate cancer is the most common form of cancer among males, with an estimated incidence of 30% in men over the age of 50. Overwhelming clinical evidence shows that human prostate cancer has the propensity to metastasize to bone, and the disease appears to progress inevitably from androgen dependent to androgen refractory status, leading to increased patient mortality. This prevalent disease is currently the second leading cause of cancer death among men in the U.S.
  • PSA prostate specific antigen
  • PAP prostatic acid phosphatase
  • the present invention provides compositions and methods for the diagnosis and therapy of cancer, such as prostate cancer.
  • the present invention provides polypeptides comprising at least a portion of a prostate-specific protein, or a variant thereof. Certain portions and other variants are immunogenic, such that the ability of the variant to react with antigen-specific antisera is not substantially diminished.
  • the polypeptide comprises at least an immunogenic portion of a prostate-specific protein, or a variant thereof, wherein the protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (a) sequences recited in any one of SEQ ID NOs:l-l l l, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382,384-476, 524, 526, 530, 531, 533, 535 and 536; (b) sequences that hybridize to any of the foregoing sequences under moderately stringent conditions; and (c) complements of any of the sequence of (a) or (b).
  • a polynucleotide sequence selected from the group consisting of: (a) sequences recited in any one of SEQ ID NOs:l-l l l, 115-171, 173-175, 177,
  • such a polypeptide comprises at least a portion, or variant thereof, of a protein that includes an amino acid sequence selected from the group consisting of sequences recited in any one of SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-550.
  • the present invention further provides polynucleotides that encode a polypeptide as described above, or a portion thereof (such as a portion encoding at least 15 amino acid residues of a prostate-specific protein), expression vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.
  • compositions comprising a polypeptide or polynucleotide as described above and a physiologically acceptable carrier.
  • vaccines for prophylactic or therapeutic use comprise a polypeptide or polynucleotide as described above and an immunostimulant.
  • present invention further provides pharmaceutical compositions that comprise:
  • the present invention provides monoclonal antibodies that specifically bind to an amino acid sequence selected from the group consisting of SEQ ID NO: 496, 504, 505, 509-517, 522 and 541-550, together with monoclonal antibodies comprising a complementarity determining region selected from the group consisting of SEQ ID NO: 502, 503 and 506-508.
  • the present invention provides pharmaceutical 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.
  • vaccines 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.
  • fusion proteins that comprise at least one polypeptide as described above, as well as polynucleotides encoding such fusion proteins.
  • pharmaceutical compositions comprising a fusion protein, or a polynucleotide encoding a fusion protein, in combination with a physiologically acceptable carrier are provided.
  • Vaccines are further provided, within other aspects, that comprise a fusion protein, or a polynucleotide encoding a fusion protein, in combination with an immunostimulant.
  • the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as recited above.
  • 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 prostate-specific protein, 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 prostate-specific protein, 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 a prostate-specific protein; (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 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 cancer may be prostate cancer.
  • 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 prostate-specific protein; (b) detecting in the sample a level of a polynucleotide, 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 prostate-specific protein; (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.
  • Figure 1 illustrates the ability of T cells to kill fibroblasts expressing the representative prostate-specific polypeptide P502S, as compared to control fibroblasts. The percentage lysis is shown as a series of effector:target ratios, as indicated.
  • Figures 2A and 2B illustrate the ability of T cells to recognize cells expressing the representative prostate-specific polypeptide P502S. In each case, the number of ⁇ -interferon spots is shown for different numbers of responders.
  • Figure 2A data is presented for fibroblasts pulsed with the P2S-12 peptide, as compared to fibroblasts pulsed with a control E75 peptide.
  • Figure 2B data is presented for fibroblasts expressing P502S, as compared to fibroblasts expressing HER- 2/neu.
  • Figure 3 represents a peptide competition binding assay showing that the P1S#10 peptide, derived from P501S, binds HLA-A2.
  • Peptide P1S#10 inhibits HLA-A2 restricted presentation of fluM58 peptide to CTL clone D150M58 in TNF release bioassay.
  • D150M58 CTL is specific for the HLA-A2 binding influenza matrix peptide fluM58.
  • Figure 4 illustrates the ability of T cell lines generated from P1S#10 immunized mice to specifically lyse PlS#10-pulsed Jurkat A2Kb targets and P501S-transduced Jurkat A2Kb targets, as compared to EGFP-transduced Jurkat A2Kb. The percent lysis is shown as a series of effector to target ratios, as indicated.
  • Figure 5 illustrates the ability of a T cell clone to recognize and specifically lyse Jurkat A2Kb cells expressing the representative prostate-specific polypeptide P501S, thereby demonstrating that the P1S#10 peptide may be a naturally processed epitope of the P501S polypeptide.
  • Figures 6A and 6B are graphs illustrating the specificity of a CD8 + cell line (3A-1) for a representative prostate-specific antigen (P501S).
  • Figure 6A shows the results of a 51 Cr release assay. The percent specific lysis is shown as a series of effector :target ratios, as indicated.
  • Figure 6A shows the results of a 51 Cr release assay. The percent specific lysis is shown as a series of effector :target ratios, as indicated.
  • 6B shows the production of interferon-gamma by 3A-1 cells stimulated with autologous B-LCL transduced with P501S, at varying effector:target rations as indicated.
  • Figure 7 is a Western blot showing the expression of P501S in baculovirus.
  • Figure 8 illustrates the results of epitope mapping studies on P501S.
  • Figure 9 is a schematic representation of the P501S protein showing the location of transmembrane domains and predicted intracellular and extracellular domains.
  • Figure 10 is a genomic map showing the location of the prostate genes P775P, P704P, B305D, P712P and P774P within the Cat Eye Syndrome region of chromosome 22ql 1.2
  • Figure 11 shows the results of an ELISA assay of antibody specificity to P501S peptides.
  • SEQ ID NO: 1 is the determined cDNA sequence for Fl-13 SEQ ID NO: 2 is the determined 3' cDNA sequence for Fl-12 SEQ ID NO: 3 is the determined 5' cDNA sequence for Fl-12 SEQ ID NO: 4 is the determined 3' cDNA sequence for F 1-16 SEQ ID NO : 5 is the determined 3 ' cDNA sequence for H 1 - 1 SEQ ID NO: 6 is the determined 3' cDNA sequence for HI -9 SEQ ID NO: 7 is the determined 3' cDNA sequence for HI -4 SEQ ID NO 8 is the determined 3' cDNA sequence for Jl-17 SEQ ID NO 9 is the determined 5' cDNA sequence for Jl-17 SEQ ID NO 10 is the determined 3 cDNA sequence for Ll-12 SEQ ID NO 11 is the determined 5 cDNA sequence for Ll-12 SEQ ID NO 12 is the determined 3 cDNA sequence for Nl-1862 SEQ ID NO 13 is the determined 5 cDNA sequence for Nl-1862 SEQ ID NO 14 is the determined 3 cDNA sequence for
  • SEQ ID NO 105 iis the determined cDNA sequence for 1D-4296
  • SEQ ID NO 106 i is the determined cDNA sequence for 1D-4280
  • SEQ ID NO 107 i is the determined full length cDNA sequence for Fl-12 (also referred to as P504S)
  • SEQ ID NO 108 i is the predicted amino acid sequence for Fl-12
  • SEQ ID NO 109 i is the determined full length cDNA sequence for Jl-17
  • SEQ ID NO 110 iis the determined full length cDNA sequence for Ll-12 (also referred to as P501S)
  • SEQ ID NO l l l i is the determined full length cDNA sequence for Nl-1862 (also referred to as P503S)
  • SEQ ID NO 112 i is the predicted amino acid sequence for Jl-17
  • SEQ ID NO 113 iis the predicted amino acid sequence for Ll-12 (also referred to as P501S)
  • SEQ ID NO 114 iis the predicted amino acid sequence for Nl-1862 (also referred to as P503S)
  • SEQ ID NO 115 i is the determined cDNA sequence for P89
  • SEQ ID NO 116 i is the determined cDNA sequence for P90
  • SEQ ID NO 117 i is the determined cDNA sequence for P92
  • SEQ ID NO 118 ⁇ is the determined cDNA sequence for P95
  • SEQ ID NO 119 iis the determined cDNA sequence for P98
  • SEQ ID NO 120 i is the determined cDNA sequence for PI 02
  • SEQ ID NO 121 i is the determined cDNA sequence for PI 10
  • SEQ ID NO 122 i is the determined cDNA sequence for PI 11
  • SEQ ID NO 123 i is the determined cDNA sequence for PI 14
  • SEQ ID NO 124 i is the determined cDNA sequence for PI 15
  • SEQ ID NO 125 i is the determined cDNA sequence for PI 16
  • SEQ ID NO 126 i is the determined cDNA sequence for PI 24
  • SEQ ID NO 127 i is the determined cDNA sequence for PI 26
  • SEQ ID NO 128 i is the determined cDNA sequence for PI 30
  • SEQ ID NO 129 i is the determined cDNA sequence for PI 33
  • SEQ ID NO 130 i is the determined cDNA sequence for PI 38
  • SEQ ID NO 131 i is the determined cDNA sequence for PI 43
  • SEQ ID NO 132 i is the determined cDNA sequence for P151
  • SEQ ID NO 133 i is the determined cDNA sequence for PI 56
  • SEQ ID NO 134 i is the determined cDNA sequence for PI 57
  • SEQ ID NO 135 i is the determined cDNA sequence for PI 66
  • SEQ ID NO 136 i is the determined cDNA sequence for PI 76
  • SEQ ID NO 137 i is the determined cDNA sequence for PI 78
  • SEQ ID NO 138 i is the determined cDNA sequence for PI 79
  • SEQ ID NO 139 i is the determined cDNA sequence for PI 85
  • SEQ ID NO 140 i is the determined cDNA sequence for PI 92
  • SEQ ID NO 141 i is the determined cDNA sequence for P201
  • SEQ ID NO 142 i is the determined cDNA sequence for P204
  • SEQ ID NO 143 i is the determined cDNA sequence for P208
  • SEQ ID NO 144 i is the determined cDNA sequence for P211
  • SEQ ID NO 145 i is the determined cDNA sequence for P213
  • SEQ ID NO 146 i is the determined cDNA sequence for P219
  • SEQ ID NO 147 iis the determined cDNA sequence for P237
  • SEQ ID NO 148 iis the determined cDNA sequence for P239
  • SEQ ID NO 149 iis the determined cDNA sequence for P248
  • SEQ ID NO 150 iis the determined cDNA sequence for P251
  • SEQ ID NO 151 i is the determined cDNA sequence for P255
  • SEQ ID NO 152 iis the determined cDNA sequence for P256
  • SEQ ID NO 153 iis the determined cDNA sequence for P259
  • SEQ ID NO 154 iis the determined cDNA sequence for P260
  • SEQ ID NO 155 i is the determined cDNA sequence for P263
  • SEQ ID NO 156 iis the determined cDNA sequence for P264
  • SEQ ID NO 157 i is the determined cDNA sequence for P266
  • SEQ ID NO 158 i is the determined cDNA sequence for P270
  • SEQ ID NO 159 i is the determined cDNA sequence for P272
  • SEQ ID NO 160 i is the determined cDNA sequence for P278
  • SEQ ID NO 161 i is the determined cDNA sequence for PI 05
  • SEQ ID NO 162 i is the determined cDNA sequence for PI 07
  • SEQ ID NO 163 i is the determined cDNA sequence for PI 37
  • SEQ ID NO 164 i is the determined cDNA sequence for PI 94
  • SEQ ID NO 165 iis the determined cDNA sequence for PI 95 SEQ ID NO e determined cDNA sequence for PI 96 SEQ ID NO e determined cDNA sequence for P220 SEQ ID NO e determined cDNA sequence for P234 SEQ ID NO e determined cDNA sequence for P235 SEQ ID NO e determined cDNA sequence for P243 SEQ ID NO e determined cDNA sequence for P703P-DE1 SEQ ID NO e predicted amino acid sequence for P703P-DE1 SEQ ID NO e determined cDNA sequence for P703P-DE2 SEQ ID NO e determined cDNA sequence for P703P-DE6 SEQ ID NO e determined cDNA sequence for P703P-DE13 SEQ ID NO e predicted amino acid sequence for P703P-DE13 SEQ ID NO e determined cDNA sequence for P703P-DE14 SEQ ID NO e predicted amino acid sequence for P703P-DE14 SEQ ID NO e determined extended cDNA sequence
  • SEQ ID NO: 199 is the determined extended cDNA sequence for 1H-4772
  • SEQ ID NO: 200 is the determined extended cDNA sequence for 1D-4309
  • SEQ ID NO: 201 is the determined extended cDNA sequence for ID.1-4278
  • SEQ ID NO: 202 is the determined extended cDNA sequence for 1D-4288
  • SEQ ID NO: 203 is the determined extended cDNA sequence for 1D-4283
  • SEQ ID NO: 204 is the determined extended cDNA sequence for 1D-4304
  • SEQ ID NO: 205 is the determined extended cDNA sequence for 1D-4296
  • SEQ ID NO: 206 is the determined extended cDNA sequence for 1D-4280
  • SEQ ID NO: 207 is the determined cDNA sequence for 10-d8fwd
  • SEQ ID NO: 208 is the determined cDNA sequence for 10-HlOcon
  • SEQ ID NO: 209 is the determined cDNA sequence for 11-C8rev
  • SEQ ID NO: 210 is the determined cDNA sequence for 7.g6fwd
  • SEQ ID NO: 211 is the determined cDNA sequence for 7.g6rev
  • SEQ ID NO: 212 is the determined cDNA sequence for 8-b5fwd
  • SEQ ID NO: 213 is the determined cDNA sequence for 8-b5rev
  • SEQ ID NO: 214 is the determined cDNA sequence for 8-b6fwd
  • SEQ ID NO: 215 is the determined cDNA sequence for 8-b6 rev
  • SEQ ID NO: 216 is the determined cDNA sequence for 8-d4fwd
  • SEQ ID NO: 217 is the determined cDNA sequence for 8-d9rev
  • SEQ ID NO: 218 is the determined cDNA sequence for 8-g3fwd
  • SEQ ID NO: 219 is the determined cDNA sequence for 8-g3rev
  • SEQ ID NO: 220 is the determined cDNA sequence for 8-hl lrev
  • SEQ ID NO: 221 is the determined cDNA sequence for g-fl2fwd
  • SEQ ID NO: 222 is the determined cDNA sequence for g-f3rev
  • SEQ ID NO: 223 is the determined cDNA sequence for P509S
  • SEQ ID NO: 224 is the determined cDNA sequence for P510S
  • SEQ ID NO: 225 is the determined cDNA sequence for P703DE5
  • SEQ ID NO: 226 is the determined cDNA sequence for 9-A11
  • SEQ ID NO: 227 is the determined cDNA sequence for 8-C6
  • SEQ ID NO: 228 is the determined cDNA sequence for 8-H7
  • SEQ ID NO: 229 is the determined cDNA sequence for JPTPN13
  • SEQ ID NO: 231 iis the determined cDNA sequence for JPTPN23
  • SEQ ID NO: 232 i is the determined cDNA sequence for JPTPN24
  • SEQ ID NO: 233 iis the determined cDNA sequence for JPTPN25
  • SEQ ID NO: 234 iis the determined cDNA sequence for JPTPN30
  • SEQ ID NO: 235 iis the determined cDNA sequence for JPTPN34
  • SEQ ID NO: 236 iis the determined cDNA sequence for PTPN35
  • SEQ ID NO: 237 iis the determined cDNA sequence for JPTPN36
  • SEQ ID NO: 238 i is the determined cDNA sequence for JPTPN38
  • SEQ ID NO: 239 iis the determined cDNA sequence for JPTPN39
  • SEQ ID NO: 240 i is the determined cDNA sequence for JPTPN40
  • SEQ ID NO: 241 i is the determined cDNA sequence for JPTPN41
  • SEQ ID NO: 242 i is the determined cDNA sequence for JPTPN42
  • SEQ ID NO: 243 iis the determined cDNA sequence for JPTPN45
  • SEQ ID NO: 244 iis the determined cDNA sequence for JPTPN46
  • SEQ ID NO: 245 iis the determined cDNA sequence for JPTPN51
  • SEQ ID NO: 246 iis the determined cDNA sequence for JPTPN56
  • SEQ ID NO: 247 iis the determined cDNA sequence for PTPN64
  • SEQ ID NO: 248 iis the determined cDNA sequence for JPTPN65
  • SEQ ID NO: 249 iis the determined cDNA sequence for JPTPN67
  • SEQ ID NO: 250 iis the determined cDNA sequence for JPTPN76
  • SEQ ID NO: 251 i is the determined cDNA sequence for JPTPN84
  • SEQ ID NO: 252 i is the determined cDNA sequence for JPTPN85
  • SEQ ID NO: 253 iis the determined cDNA sequence for JPTPN86
  • SEQ ID NO: 254 iis the determined cDNA sequence for JPTPN87
  • SEQ ID NO: 255 iis the determined cDNA sequence for JPTPN88
  • SEQ ID NO: 256 iis the determined cDNA sequence for JP1F1
  • SEQ ID NO: 257 iis the determined cDNA sequence for JP1F2
  • SEQ ID NO: 258 iis the determined cDNA sequence for JP1C2
  • SEQ ID NO: 259 iis the determined cDNA sequence for JP1B1
  • SEQ ID NO: 260 i is the determined cDNA sequence for JP1B2
  • SEQ ID NO: 261 iis the determined cDNA sequence for JP1D3
  • SEQ ID NO: 262 iis the determined cDNA sequence for JP1 A4
  • SEQ ID NO: 263 i is the determined cDNA sequence for JP1F5
  • SEQ ID NO: 264 i is the determined cDNA sequence for JP1E6
  • SEQ ID NO: 265 i is the determined cDNA sequence for JP1D6
  • SEQ ID NO: 266 i is the determined cDNA sequence for JP1B5
  • SEQ ID NO: 267 iis the determined cDNA sequence for JP1 A6
  • SEQ ID NO: 268 iis the determined cDNA sequence for JP1E8
  • SEQ ID NO: 269 iis the determined cDNA sequence for JP1D7
  • SEQ ID NO: 270 i is the determined cDNA sequence for JP1D9
  • SEQ ID NO: 271 iis the determined cDNA sequence for JP1C10
  • SEQ ID NO: 272 i is the determined cDNA sequence for JP1 A9
  • SEQ ID NO: 273 iis the determined cDNA sequence for JP1F12
  • SEQ ID NO: 274 iis the determined cDNA sequence for JP1E12
  • SEQ ID NO: 275 iis the determined cDNA sequence for JP1D11
  • SEQ ID NO: 276 iis the determined cDNA sequence for JP1C11
  • SEQ ID NO: 277 iis the determined cDNA sequence for JP1C12
  • SEQ ID NO: 278 iis the determined cDNA sequence for JP1B12
  • SEQ ID NO: 279 i is the determined cDNA sequence for JP1A12
  • SEQ ID NO: 280 i is the determined cDNA sequence for JP8G2
  • SEQ ID NO: 281 i is the determined cDNA sequence for JP8H1
  • SEQ ID NO: 282 i is the determined cDNA sequence for JP8H2
  • SEQ ID NO: 283 iis the determined cDNA sequence for JP8A3
  • SEQ ID NO: 284 iis the determined cDNA sequence for JP8A4
  • SEQ ID NO: 285 iis the determined cDNA sequence for JP8C3
  • SEQ ID NO: 286 iis the determined cDNA sequence for JP8G4
  • SEQ ID NO: 287 i is the determined cDNA sequence for JP8B6
  • SEQ ID NO: 288 i is the determined cDNA sequence for JP8D6
  • SEQ ID NO: 289 iis the determined cDNA sequence for JP8F5
  • SEQ ID NO: 290 iis the determined cDNA sequence for JP8A8
  • SEQ ID NO: 291 iis the determined cDNA sequence for JP8C7
  • SEQ ID NO: 292 i is the determined cDNA sequence for JP8D7
  • SEQ ID NO: 293 iis the determined cDNA sequence for P8D8
  • SEQ ID NO: 294 iis the determined cDNA sequence for JP8E7
  • SEQ ID NO: 295 iis the determined cDNA sequence for JP8F8
  • SEQ ID NO: 296 i is the determined cDNA sequence for JP8G8
  • SEQ ID NO: 297 i is the determined cDNA sequence for JP8B10
  • SEQ ID NO: 298 i is the determined cDNA sequence for JP8C10
  • SEQ ID NO: 299 i is the determined cDNA sequence for JP8E9
  • SEQ ID NO: 300 iis the determined cDNA sequence for JP8E10
  • SEQ ID NO: 301 i is the determined cDNA sequence for JP8F9
  • SEQ ID NO: 302 iis the determined cDNA sequence for JP8H9
  • SEQ ID NO: 303 iis the determined cDNA sequence for JP8C12
  • SEQ ID NO: 304 i is the determined cDNA sequence for JP8E11
  • SEQ ID NO: 305 i is the determined cDNA sequence for JP8E12
  • SEQ ID NO: 306 i is the amino acid sequence for the peptide PS2#12
  • SEQ ID NO: 307 iis the determined cDNA sequence for P71 IP
  • SEQ ID NO: 308 iis the determined cDNA sequence for P712P
  • SEQ ID NO: 309 iis the determined cDNA sequence for CLONE23
  • SEQ ID NO: 310 iis the determined cDNA sequence for P774P
  • SEQ ID NO: 311 i is the determined cDNA sequence for P775P
  • SEQ ID NO: 312 iis the determined cDNA sequence for P715P
  • SEQ ID NO: 313 iis the determined cDNA sequence for P710P
  • SEQ ID NO: 314 iis the determined cDNA sequence for P767P
  • SEQ ID NO: 315 iis the determined cDNA sequence for P768P
  • SEQ ID NO: 316-2 325 are the determined cDNA sequences of previously isolated genes
  • SEQ ID NO: 326 iis the determined cDNA sequence for P703PDE5
  • SEQ ID NO: 327 iis the predicted amino acid sequence for P703PDE5
  • SEQ ID NO: 328 iis the determined cDNA sequence for P703P6.26
  • SEQ ID NO: 329 iis the predicted amino acid sequence for P703P6.26
  • SEQ ID NO: 330 iis the determined cDNA sequence for P703PX-23
  • SEQ ID NO: 331 iis the predicted amino acid sequence for P703PX-23
  • SEQ ID NO: 332 iis the determined full length cDNA sequence for P509S
  • SEQ ID NO: 333 i is the determined extended cDNA sequence for P707P (also referred to as 11 -C9)
  • SEQ ID NO: 334 iis the determined cDNA sequence for P714P
  • SEQ ID NO: 335 is the determined cDNA sequence for P705P (also referred to as 9-F3)
  • SEQ ID NO: 336 is the predicted amino acid sequence for P705P
  • SEQ ID NO: 337 is the amino acid sequence of the peptide P1S#10
  • SEQ ID NO: 338 is the amino acid sequence of the peptide p5 SEQ ID NO: 339 is the predicted amino acid sequence of P509S
  • SEQ ID NO: 340 is the determined cDNA sequence for P778P
  • SEQ ID NO: 341 is the determined cDNA sequence for P786P
  • SEQ ID NO: 342 is the determined cDNA sequence for P789P
  • SEQ ID NO: 343 is the determined cDNA sequence for a clone showing homology to Homo sapiens MM46 mRNA
  • SEQ ID NO: 344 is the determined cDNA sequence for a clone showing homology to Homo sapiens TNF-alpha stimulated ABC protein (ABC50) mRNA
  • SEQ ID NO: 345 is the determined cDNA sequence for a clone showing homology to Homo sapiens mRNA for E-cadherin
  • SEQ ID NO: 346 is the determined cDNA sequence for a clone showing homology to Human nuclear-encoded mitochondrial serine hydroxymethyltransferase (SHMT)
  • SEQ ID NO: 347 is the determined cDNA sequence for a clone showing homology to Homo sapiens natural resistance-associated macrophage protein2 (NRAMP2)
  • SEQ ID NO: 348 is the determined cDNA sequence for a clone showing homology to Homo sapiens phosphoglucomutase-related protein (PGMRP)
  • SEQ ID NO: 349 is the determined cDNA sequence for a clone showing homology to Human mRNA for proteosome subunit p40
  • SEQ ID NO: 350 is the determined cDNA sequence for P777P
  • SEQ ID NO: 351 is the determined cDNA sequence for P779P
  • SEQ ID NO: 352 is the determined cDNA sequence for P790P
  • SEQ ID NO: 353 is the determined cDNA sequence for P784P
  • SEQ ID NO: 354 is the determined cDNA sequence for P776P
  • SEQ ID NO: 355 is the determined cDNA sequence for P780P
  • SEQ ID NO: 356 is the determined cDNA sequence for P544S
  • SEQ ID NO: 357 is the determined cDNA sequence for P745S
  • SEQ ID NO: 358 is the determined cDNA sequence for P782P
  • SEQ ID NO: 359 is the determined cDNA sequence for P783P
  • SEQ ID NO: 360 is the determined cDNA sequence for unknown 17984
  • SEQ ID NO: 361 is the determined cDNA sequence for P787P
  • SEQ ID NO: 362 is the determined cDNA sequence for P788P
  • SEQ ID NO: 363 is the determined cDNA sequence for unknown 17994
  • SEQ ID NO: 364 is the determined cDNA sequence for P781P
  • SEQ ID NO: 365 is the determined cDNA sequence for P785P
  • SEQ ID NO: 366-375 are the determined cDNA sequences for splice variants of B305D.
  • SEQ ID NO: 376 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO:
  • SEQ ID NO: 377 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO:
  • SEQ ID NO: 378 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO:
  • SEQ ID NO: 379 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO: 374.
  • SEQ ID NO: 380 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO:
  • SEQ ID NO: 381 is the determined cDNA sequence for B716P.
  • SEQ ID NO: 382 is the determined full-length cDNA sequence for P71 IP.
  • SEQ ID NO: 383 is the predicted amino acid sequence for P71 IP.
  • SEQ ID NO: 384 is the cDNA sequence for PIOOOC.
  • SEQ ID NO: 385 is the cDNA sequence for CGI-82.
  • SEQ ID NO:386 is the cDNA sequence for 23320.
  • SEQ ID NO:387 is the cDNA sequence for CGI-69.
  • SEQ ID NO:388 is the cDNA sequence for L-iditol-2-dehydrogenase.
  • SEQ ID NO:389 is the cDNA sequence for 23379.
  • SEQ ID NO:390 is the cDNA sequence for 23381.
  • SEQ ID NO:391 is the cDNA sequence for KIAA0122.
  • SEQ ID NO:392 is the cDNA sequence for 23399.
  • SEQ ID NO:393 is the cDNA sequence for a previously identified gene.
  • SEQ ID NO:394 is the cDNA sequence for HCLBP.
  • SEQ ID NO:395 is the cDNA sequence for transglutaminase.
  • SEQ ID NO:396 is the cDNA sequence for a previously identified gene.
  • SEQ ID NO:397 is the cDNA sequence for PAP.
  • SEQ ID NO:398 is the cDNA sequence for Ets transcription factor PDEF.
  • SEQ ID NO:399 is the cDNA sequence for hTGR.
  • SEQ ID NO:400 is the cDNA sequence for KIAA0295.
  • SEQ ID NO:401 is the cDNA sequence for 22545.
  • SEQ ID NO:402 is the cDNA sequence for 22547.
  • SEQ ID NO:403 is the cDNA sequence for 22548s
  • SEQ ID NO:404 is the cDNA sequence for 22550.
  • SEQ ID NO:405 is the cDNA sequence for 22551.
  • SEQ ID NO:406 is the cDNA sequence for 22552.
  • SEQ ID NO:407 is the cDNA sequence for 22553.
  • SEQ ID NO:408 is the cDNA sequence for 22558.
  • SEQ ID NO:409 is the cDNA sequence for 22562.
  • SEQ ID NO.410 is the cDNA sequence for 22565.
  • SEQ ID NO:41 1 is the cDNA sequence for 22567.
  • SEQ ID NO:412 is the cDNA sequence for 22568.
  • SEQ ID NO:413 is the cDNA sequence for 22570.
  • SEQ ID NO.414 is the cDNA sequence for 22571.
  • SEQ ID NO:415 is the cDNA sequence for 22572.
  • SEQ ID NO:416 is the cDNA sequence for 22573.
  • SEQ ID NO:417 is the cDNA sequence for 22573.
  • SEQ ID NO.418 is the cDNA sequence for 22575.
  • SEQ ID NO :419 is the cDNA sequence for 22580.
  • SEQ ID NO:420 is the cDNA sequence for 22581.
  • SEQ ID NO:421 is the cDNA sequence for 22582.
  • SEQ ID NO:422 is the cDNA sequence for 22583.
  • SEQ ID NO:423 is the cDNA sequence for 22584.
  • SEQ ID NO:424 is the cDNA sequence for 22585.
  • SEQ ID NO:425 is the cDNA sequence for 22586.
  • SEQ ID NO:426 is the cDNA sequence for 22587.
  • SEQ ID NO:427 is the cDNA sequence for 22588.
  • SEQ ID NO:428 is the cDNA sequence for 22589.
  • SEQ ID NO:429 is the cDNA sequence for 22590.
  • SEQ ID NO:430 is the cDNA sequence for 22591.
  • SEQ ID NO:431 is the cDNA sequence for 22592.
  • SEQ ID NO:432 is the cDNA sequence for 22593.
  • SEQ ID NO:433 is the cDNA sequence for 22594.
  • SEQ ID NO:434 is the cDNA sequence for 22595.
  • SEQ ID NO:435 is the cDNA sequence for 22596.
  • SEQ ID NO:436 is the cDNA sequence for 22847.
  • SEQ ID NO.437 is the cDNA sequence for 22848.
  • SEQ ID NO:438 is the cDNA sequence for 22849.
  • SEQ ID NO:439 is the cDNA sequence for 22851.
  • SEQ ID NO:440 is the cDNA sequence for 22852.
  • SEQ ID NO.441 is the cDNA sequence for 22853.
  • SEQ ID NO:442 is the cDNA sequence for 22854.
  • SEQ ID NO.443 is the cDNA sequence for 22855.
  • SEQ ID NO:444 is the cDNA sequence for 22856.
  • SEQ ID NO:445 is the cDNA sequence for 22857.
  • SEQ ID NO:446 is the cDNA sequence for 23601.
  • SEQ ID NO:447 is the cDNA sequence for 23602.
  • SEQ ID NO:448 is the cDNA sequence for 23605.
  • SEQ ID NO:449 is the cDNA sequence for 23606.
  • SEQ ID NO:450 is the cDNA sequence for 23612.
  • SEQ ID NO:451 is the cDNA sequence for 23614.
  • SEQ ID NO-.452 is the cDNA sequence for 23618.
  • SEQ ID NO:453 is the cDNA sequence for 23622.
  • SEQ ID NO:454 is the cDNA sequence for folate hydrolase.
  • SEQ ID NO:455 is the cDNA sequence for LIM protein.
  • SEQ ID NO:456 is the cDNA sequence for a known gene.
  • SEQ ID NO:457 is the cDNA sequence for a known gene.
  • SEQ ID NO:458 is the cDNA sequence for a previously identified gene.
  • SEQ ID NO:459 is the cDNA sequence for 23045.
  • SEQ ID NO:460 is the cDNA sequence for 23032.
  • SEQ ID NO:461 is the cDNA sequence for 23054.
  • SEQ ID NO:462-467 are cDNA sequences for known genes.
  • SEQ ID NO:468-471 are cDNA sequences for P710P.
  • SEQ ID NO:472 is a cDNA sequence for P1001C.
  • SEQ ID NO: 473 is the determined cDNA sequence for a first splice variant of P775P (referred to as
  • SEQ ID NO: 474 is the determined cDNA sequence for a second splice variant of P775P (referred to as 19947).
  • SEQ ID NO: 475 is the determined cDNA sequence for a third splice variant of P775P (referred to as 19941).
  • SEQ ID NO: 476 is the determined cDNA sequence for a fourth splice variant of P775P (referred to as 19937).
  • SEQ ID NO: 477 is a first predicted amino acid sequence encoded by the sequence of SEQ ID NO: 474.
  • SEQ ID NO: 478 is a second predicted amino acid sequence encoded by the sequence of SEQ ID NO: 478
  • SEQ ID NO: 479 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO:
  • SEQ ID NO: 480 is a first predicted amino acid sequence encoded by the sequence of SEQ ID NO:
  • SEQ ID NO: 481 is a second predicted amino acid sequence encoded by the sequence of SEQ ID NO: 481
  • SEQ ID NO: 482 is a third predicted amino acid sequence encoded by the sequence of SEQ ID NO: 473.
  • SEQ ID NO: 483 is a fourth predicted amino acid sequence encoded by the sequence of SEQ ID NO: 483
  • SEQ ID NO: 484 is the first 30 amino acids of the M. tuberculosis antigen Ral2.
  • SEQ ID NO: 485 is the PCR primer AW025.
  • SEQ ID NO: 486 is the PCR primer AW003.
  • SEQ ID NO: 487 is the PCR primer AW027.
  • SEQ ID NO: 488 is the PCR primer AW026.
  • SEQ ID NO: 489-501 are peptides employed in epitope mapping studies.
  • SEQ ID NO: 502 is the determined cDNA sequence of the complementarity determining region for the anti-P503S monoclonal antibody 20D4.
  • SEQ ID NO: 503 is the determined cDNA sequence of the complementarity determining region for the anti-P503S monoclonal antibody JA1.
  • SEQ ID NO: 504 & 505 are peptides employed in epitope mapping studies.
  • SEQ ID NO: 506 is the determined cDNA sequence of the complementarity determining region for the anti-P703P monoclonal antibody 8H2.
  • SEQ ID NO: 507 is the determined cDNA sequence of the complementarity determining region for the anti-P703P monoclonal antibody 7H8.
  • SEQ ID NO: 508 is the determined cDNA sequence of the complementarity determining region for the anti-P703P monoclonal antibody 2D4.
  • SEQ ID NO: 509-522 are peptides employed in epitope mapping studies.
  • SEQ ID NO: 523 is a mature form of P703P used to raise antibodies against P703P.
  • SEQ ID NO: 524 is the putative full-length cDNA sequence of P703P.
  • SEQ ID NO: 525 is the predicted amino acid sequence encoded by SEQ ID NO: 524.
  • SEQ ID NO: 526 is the full-length cDNA sequence for P790P.
  • SEQ ID NO: 527 is the predicted amino acid sequence for P790P.
  • SEQ ID NO: 528 & 529 are PCR primers.
  • SEQ ID NO: 530 is the cDNA sequence of a splice variant of SEQ ID NO: 366.
  • SEQ ID NO: 531 is the cDNA sequence of the open reading frame of SEQ ID NO: 530.
  • SEQ ID NO: 532 is the predicted amino acid encoded by the sequence of SEQ ID NO: 531.
  • SEQ ID NO: 533 is the DNA sequence of a putative ORF of P775P.
  • SEQ ID NO: 534 is the predicted amino acid sequence encoded by SEQ ID NO: 533.
  • SEQ ID NO: 535 is a first full-length cDNA sequence for P51 OS.
  • SEQ ID NO: 536 is a second full-length cDNA sequence for P510S.
  • SEQ ID NO: 537 is the predicted amino acid sequence encoded by SEQ ID NO: 535.
  • SEQ ID NO: 538 is the predicted amino acid sequence encoded by SEQ ID NO: 536.
  • SEQ ID NO: 539 is the peptide P501S-370.
  • SEQ ID NO: 540 is the peptide P501S-376.
  • SEQ ID NO: 541-550 are epitopes of P501S.
  • SEQ ID NO: 551 corresponds to amino acids 543-553 of P501S. DETAILED DESCRIPTION OF THE INVENTION
  • compositions and methods for the therapy and diagnosis of cancer such as prostate cancer.
  • the compositions described herein may include prostate-specific polypeptides, polynucleotides encoding such polypeptides, binding agents such as antibodies, antigen presenting cells (APCs) and/or immune system cells (e.g., T cells).
  • Polypeptides of the present invention generally comprise at least a portion (such as an immunogenic portion) of a prostate-specific protein or a variant thereof.
  • a "prostate-specific protein” is a protein that is expressed in normal prostate and/or prostate tumor cells at a level that is at least two fold, and preferably at least five fold, greater than the level of expression in a non-prostate normal tissue, as determined using a representative assay provided herein.
  • Certain prostate-specific proteins are proteins that react detectably (within an immunoassay, such as an ELISA or Western blot) with antisera of a patient afflicted with prostate cancer.
  • Polynucleotides of the subject invention generally comprise a DNA or RNA sequence that encodes all or a portion of such a polypeptide, or that is complementary to such a sequence.
  • Antibodies are generally immune system proteins, or antigen-binding fragments thereof, that are capable of binding to a polypeptide as described above.
  • Antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B-cells that express a polypeptide as described above.
  • T cells that may be employed within such compositions are generally T cells that are specific for a polypeptide as described above.
  • the present invention is based on the discovery of human prostate-specific proteins. Sequences of polynucleotides encoding certain prostate-specific proteins, or portions thereof, are provided in SEQ ID NOs:l-l l l, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382, 384-476, 524, 526, 530, 531, 533, 535 and 536.
  • Sequences of polypeptides comprising at least a portion of a prostate-specific protein are provided in SEQ ID NOs:l 12-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534 and 537-550.
  • PROSTATE-SPECIFIC PROTEIN POLYNUCLEOTIDES Any polynucleotide that encodes a prostate-specific protein or a portion or other variant thereof as described herein is encompassed by the present invention.
  • Preferred polynucleotides comprise at least 15 consecutive nucleotides, preferably at least 30 consecutive nucleotides and more preferably at least 45 consecutive nucleotides, that encode a portion of a prostate-specific protein. More preferably, a polynucleotide encodes an immunogenic portion of a prostate-specific protein. Polynucleotides complementary to any such sequences are also encompassed by the present invention.
  • Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
  • RNA molecules 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 prostate-specific protein or a portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the immunogenicity of the encoded polypeptide is not diminished, relative to a native protein. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein. 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 prostate-specific protein or a portion thereof. The term "variants" also encompasses homologous genes of xenogenic origin.
  • Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of nucleotides or 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 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.
  • 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 or 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 of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or 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.
  • Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof.
  • Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native prostate- specific protein (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C- 65°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.
  • 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. The resulting 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).
  • Polynucleotides may be prepared using any of a variety of techniques. For example, 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 five fold greater in a prostate-specific than in normal tissue, as determined using a representative assay provided herein). Such screens may be performed using a Synteni microarray (Palo Alto, CA) according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA i:10614-10619, 1996 and Heller et al., Proc. Natl Acad. Sci.
  • polypeptides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as prostate-specific cells.
  • Such polynucleotides may be amplified via polymerase chain reaction (PCR).
  • sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesized.
  • An amplified portion may be used to isolate a full length gene from a suitable library (e.g., a prostate-specific cDNA library) using well known techniques.
  • a library cDNA or genomic
  • 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 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 are 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 for obtaining a full length coding sequence from a partial cDNA sequence.
  • amplification is generally performed via PCR. Any of a variety of commercially available kits may be used to perform the amplification step.
  • Primers may be designed using, for example, software well known in the art. Primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68°C to 72°C.
  • the amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.
  • One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res.
  • 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.
  • RACE Rapid amplification of cDNA ends
  • 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. 7P:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence. In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank.
  • EST expressed sequence tag
  • Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. Full length DNA sequences may also be obtained by analysis of genomic fragments. Certain nucleic acid sequences of cDNA molecules encoding at least a portion of a prostate-specific protein are provided in SEQ ID NO.1-111, 115-171, 173-175, 177, 179-305, 307- 315, 326, 328, 330, 332-335, 340-375, 381, 382, 384-476, 524, 526, 530, 531, 533, 535 and 536. Isolation of these polynucleotides is described below. Each of these prostate-specific proteins was overexpressed in prostate rumor tissue.
  • Polynucleotide variants may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a prostate-specific protein, or portion thereof, provided that the DNA is inco ⁇ orated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein.
  • a suitable RNA polymerase promoter such as T7 or SP6
  • a portion may be administered to a patient such that the encoded polypeptide is generated in vivo (e.g., by transfecting antigen-presenting cells, such as dendritic cells, with a cDNA construct encoding a prostate-specific polypeptide, and administering the transfected cells to the patient).
  • a portion of a sequence complementary to a coding sequence i.e., an antisense polynucleotide
  • cDNA constructs that can be transcribed into antisense RNA may also be introduced into cells of tissues to facilitate the production of antisense RNA.
  • An antisense polynucleotide may be used, as described herein, to inhibit expression of a protein.
  • Antisense technology can be used to control gene expression through triple-helix formation, which compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules (see Gee et al., In Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing Co. (Mt. Kisco, NY; 1994)).
  • an antisense molecule may be designed to hybridize with a control region of a gene (e.g., promoter, enhancer or transcription initiation site), and block transcription of the gene; or to block translation by inhibiting binding of a transcript to ribosomes.
  • a portion of a coding sequence, or of a complementary sequence, may also be designed as a probe or primer to detect gene expression.
  • Probes may be labeled with a variety of reporter groups, such as radionuclides and enzymes, and are preferably at least 10 nucleotides in length, more preferably at least 20 nucleotides in length and still more preferably at least 30 nucleotides in length.
  • Primers as noted above, are preferably 22-30 nucleotides in length.
  • Any polynucleotide may be further modified to increase stability in vivo. 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. Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques.
  • a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.
  • a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.
  • polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic pu ⁇ oses, as described below.
  • a polynucleotide may be inco ⁇ orated into a viral vector such as, but not limited to, adenovims, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus).
  • the polynucleotides may also be administered as naked plasmid vectors.
  • a retroviral vector may additionally transfer or inco ⁇ orate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.
  • colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • a preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.
  • polypeptides may comprise at least an immunogenic portion of a prostate-specific protein or a variant thereof, as described herein.
  • a "prostate-specific protein” is a protein that is expressed by normal prostate and/or prostate tumor cells. Proteins that are prostate-specific proteins also react detectably within an immunoassay (such as an ELISA) with antisera from a patient with prostate cancer.
  • Polypeptides as described herein may be of any length. Additional sequences derived from the native protein and/or heterologous sequences may be present, and such sequences may (but need not) possess further immunogenic or antigenic properties.
  • An "immunogenic portion,” as used herein is a portion of a protein that is recognized
  • Such immunogenic portions generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of a prostate-specific protein or a variant thereof.
  • Certain preferred immunogenic portions include peptides in which an N-terminal leader sequence and/or transmembrane domain have been deleted.
  • Other preferred immunogenic portions may 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.
  • 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 native prostate-specific protein is a portion that reacts with such 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). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide.
  • Such screens may generally be performed using methods well known to those of ordinary skill in the art, 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, 125 I-labeled Protein A.
  • a composition may comprise a variant of a native prostate-specific protein.
  • a polypeptide "variant,” as used herein, is a polypeptide that differs from a native prostate-specific protein in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the polypeptide is not substantially diminished.
  • the ability of a variant to react with antigen-specific antisera may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%, and preferably less than 20%, relative to the native protein.
  • Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antigen-specific antibodies or antisera as described herein.
  • Preferred variants include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed.
  • Other preferred 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 of the mature protein.
  • Polypeptide variants preferably exhibit at least about 70%, more preferably at least about 90% and most preferably at least about 95% identity (determined as described above) to the identified polypeptides.
  • a variant contains 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.
  • Amino acid substitutions may generally 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; and 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 of the 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.
  • Polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by DNA sequences as described above may be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, higher eukaryotic and plant cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.
  • Supernatants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.
  • a suitable purification matrix such as an affinity matrix or an ion exchange resin.
  • Portions and other variants having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art.
  • such polypeptides may be synthesized using any of the 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.
  • a polypeptide may be a fusion protein 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 prostate-specific 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 of the protein or to enable the protein to be targeted to desired intracellular compartments.
  • Still further fusion partners include affinity tags, which facilitate purification of the protein.
  • Fusion proteins may generally be prepared using standard techniques, including chemical conjugation.
  • a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non- fused protein, 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 protein that retains the biological activity of both component polypeptides.
  • a peptide linker sequence may be employed to separate the first and the 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 inco ⁇ orated into the fusion protein 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; Mu ⁇ hy et al., Proc. Natl. Acad. Sci. USA 53:8258-8262, 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.
  • Fusion proteins are also provided that comprise a polypeptide of the present invention together with an unrelated immunogenic protein.
  • the immunogenic protein is 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., 55(5:86-91, 1997).
  • 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 ⁇ -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 ⁇ -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.
  • fusion partners include the non-structural protein from infiuenzae virus, ⁇ S1 (hemaglutinin). Typically, the ⁇ -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 ⁇ -acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 45:265-292, 1986).
  • LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
  • the C-terminal domain of the 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 70:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be inco ⁇ orated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion inco ⁇ orates residues 188-305.
  • polypeptides including fusion proteins and polynucleotides as described herein are isolated.
  • An "isolated" polypeptide or polynucleotide is one that is removed from its original environment.
  • a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system.
  • polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
  • a polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.
  • the present invention further provides agents, such as antibodies and antigen- binding fragments thereof, that specifically bind to a prostate-specific protein.
  • an antibody, or antigen-binding fragment thereof is said to "specifically bind" to a prostate-specific protein if it reacts at a detectable level (within, for example, an ELISA) with a prostate-specific protein, and does not react detectably with unrelated proteins under similar conditions.
  • binding refers to a noncovalent association between two separate molecules such that a complex is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations.
  • binding constant may be determined using methods well known in the art.
  • Binding agents may be further capable of differentiating between patients with and without a cancer, such as prostate cancer, using the representative assays provided herein. In other words, antibodies or other binding agents that bind to a prostate-specific protein will generate a signal indicating the presence of a cancer in at least about 20% of patients with the disease, and will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without the cancer.
  • binding agent satisfies this requirement
  • biological samples e.g., blood, sera, urine and/or tumor biopsies
  • a cancer as determined using standard clinical tests
  • a statistically significant number of samples with and without the disease should be assayed.
  • Each binding agent should satisfy the above criteria; however, those of ordinary skill in the art will recognize that binding agents may be used in combination to improve sensitivity. Any agent that satisfies the above requirements may be a binding agent.
  • a binding agent may be a ribosome, with or without a peptide component, an RNA molecule or a polypeptide.
  • a binding agent is an antibody or an antigen- binding fragment thereof.
  • antibodies employed in the inventive methods have the ability to induce lysis of tumor cells by activation of complement and mediation of antibody- dependent cellular cytotoxicity (ADCC).
  • ADCC antibody- dependent cellular cytotoxicity
  • Antibodies of different classes and subclasses differ in these properties.
  • mouse antibodies of the IgG2a and IgG3 classes are capable of activating serum complement upon binding to target cells which express the antigen against which the antibodies were raised, and can mediate ADCC.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art.
  • antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.
  • an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification.
  • a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin.
  • the immunogen is injected into the animal host, preferably according to a predetermined schedule inco ⁇ orating one or more booster immunizations, and the animals are bled periodically.
  • Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
  • Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol.
  • these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest).
  • Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above.
  • the spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal.
  • a variety of fusion techniques may be employed.
  • the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells.
  • a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.
  • Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
  • Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
  • the polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.
  • mice and rabbit monoclonal antibodies that specifically bind to polypeptides of the present invention are described in detail below.
  • the antibodies of the present invention are not limited to those derived from mice.
  • Human antibodies may also be employed in the inventive methods and may prove to be preferable.
  • Such antibodies can be obtained using human hybridomas as described by Cote et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Lisa, p. 77, 1985).
  • the present invention also encompasses antibodies made by recombinant means such as chimeric antibodies, wherein the variable region and constant region are derived from different species, and CDR-grafted antibodies, wherein the complementarity determining region is derived from a different species, as described in US Patents 4,816,567 and 5,225,539.
  • Chimeric antibodies may be prepared by splicing genes for a mouse antibody molecule having a desired antigen specificity together with genes for a human antibody molecule having the desired biological activity, such as activation of human complement and mediation of ADCC (Morrison et al. Proc. Natl Acad. Sci. USA 57:6851, 1984; Neuberger et al Nature 572:604, 1984; Takeda et al. Nature 574:452, 1985).
  • antigen-binding fragments of antibodies may be preferred.
  • Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.
  • Monoclonal antibodies of the present invention may be coupled to one or more therapeutic agents.
  • Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof.
  • Preferred radionuclides include 90 Y, 123 I, 125 1, 131 I, 186 Re, 188 Re, 211 At, and 212 Bi.
  • Preferred drugs include methotrexate, and pyrimidine and purine analogs.
  • Preferred differentiation inducers include phorbol esters and butyric acid.
  • Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
  • a therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group).
  • a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other.
  • a nucleophilic group such as an amino or sulfhydryl group
  • on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
  • a linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities.
  • a linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.
  • a linker group which is cleavable during or upon intemalization into a cell.
  • a number of different cleavable linker groups have been described.
  • the mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Patent No. 4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S. Patent No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S.
  • Patent No. 4,638,045 to Kohn et al.
  • serum complement-mediated hydrolysis e.g., U.S. Patent No. 4,671,958, to Rodwell et al.
  • acid-catalyzed hydrolysis e.g., U.S. Patent No. 4,569,789, to Blattler et al.
  • immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used.
  • a carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group.
  • Suitable carriers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.).
  • a carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088).
  • Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds.
  • U.S. Patent No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis.
  • a radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
  • U.S. Patent No. 4,673,562 to Davison et al. discloses representative chelating compounds and their synthesis.
  • a variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody.
  • T CELLS Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for a prostate-specific protein.
  • T cells may generally be prepared in vitro or ex vivo, using standard procedures.
  • T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the ISOLEXTM system, available from Nexell Therapeutics Inc., Irvine, CA (see also U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243).
  • T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
  • T cells may be stimulated with a prostate-specific polypeptide, polynucleotide encoding a prostate-specific polypeptide and or an antigen presenting cell (APC) that expresses such a polypeptide.
  • APC antigen presenting cell
  • Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide.
  • a prostate-specific polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.
  • T cells are considered to be specific for a prostate-specific polypeptide if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide.
  • T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques.
  • T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse- labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine inco ⁇ orated into DNA).
  • a prostate-specific polypeptide 100 ng/ml - 100 ⁇ g/ml, preferably 200 ng/ml - 25 ⁇ g/ml
  • contact with a prostate-specific polypeptide 100 ng/ml - 100 ⁇ g/ml, preferably 200 ng/ml - 25 ⁇ g/ml
  • T cells that have been activated in response to a prostate-specific polypeptide, polynucleotide or polypeptide-expressing APC may be CD4 + and/or CD80
  • Prostate-specific protein-specific T cells may be expanded using standard techniques.
  • the T cells are derived from either a patient or a related, or unrelated, donor and are administered to the patient following stimulation and expansion.
  • CD4 + or CD8 + T cells that proliferate in response to a prostate-specific polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways.
  • the T cells can be re-exposed to a prostate-specific polypeptide, or a short peptide corresponding to an immunogenic portion of such a polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a prostate- specific polypeptide.
  • T cell growth factors such as interleukin-2
  • stimulator cells that synthesize a prostate- specific polypeptide.
  • one or more T cells that proliferate in the presence of a prostate-specific protein can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.
  • polypeptides, polynucleotides, T cells and/or binding agents disclosed herein may be inco ⁇ orated into pharmaceutical compositions or immunogenic compositions (i.e., vaccines).
  • Pharmaceutical compositions comprise one or more such compounds and a physiologically acceptable carrier.
  • Vaccines may comprise one or more such compounds and an immunostimulant.
  • An immunostimulant may be any substance that enhances an immune response to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is inco ⁇ orated; see e.g., Fullerton, U.S. Patent No. 4,235,877).
  • compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.
  • one or more immunogenic portions of other tumor antigens may be present, either inco ⁇ orated into a fusion polypeptide or as a separate compound, within the composition or vaccine.
  • a pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 75:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerri ⁇ ) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.
  • the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox vims, retrovirus, or adenovims), which may involve the use of a non-pathogenic (defective), replication competent vims.
  • vaccinia or other pox vims, retrovirus, or adenovims e.g., vaccinia or other pox vims, retrovirus, or adenovims
  • Suitable systems are disclosed, for example, in Fisher- Hoch et al., Proc. Natl. Acad. Sci. USA 560317-321, 1989; Flexner et al., Ann. N. Y. Acad. Sci.
  • compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.
  • parenteral administration such as subcutaneous injection
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
  • Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol e.g., proteins, polypeptides or amino acids
  • antioxidants e.g
  • immunostimulants may be employed in the vaccines of this invention.
  • an adjuvant may be included.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
  • Cytokines such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
  • the adjuvant composition is preferably designed to induce an immune response predominantly of the Thl type.
  • High levels of Thl -type cytokines e.g., IFN- ⁇ , TNF , IL-2 and IL-12
  • Th2-type cytokines e.g., IL-4, IL-5, IL-6 and IL-10
  • a patient will support an immune response that includes Thl- and Th2- type responses.
  • Thl -type cytokines in which a response is predominantly Thl -type, the level of Thl -type cytokines will increase to a greater extent than the level of Th2-type cytokines.
  • the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.
  • Preferred adjuvants for use in eliciting a predominantly Thl -type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt.
  • MPL adjuvants are available from Ribi ImmunoChem Research Inc.
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated also induce a predominantly Thl response.
  • Such oligonucleotides are well known and are described, for example, in WO 96/02555.
  • Another preferred adjuvant is a saponin, preferably QS21, which may be used alone or in combination with other adjuvants.
  • an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • Other preferred formulations comprises an oil-in-water emulsion and tocopherol.
  • a particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
  • Any vaccine provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient.
  • compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides for example) that effects a slow release of compound following administration).
  • sustained release formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
  • Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
  • Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release.
  • APCs antigen presenting cells
  • APCs antigen presenting cells
  • Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and or to be immunologically compatible with the receiver (i.e., matched HLA haplotype).
  • APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
  • Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 592:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:501-529, 1999).
  • dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take-up, process and present antigens with high efficiency, and their ability to activate na ⁇ ' ve T cell responses.
  • Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention.
  • secreted vesicles antigen-loaded dendritic cells called exosomes
  • exosomes antigen-loaded dendritic cells
  • Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid.
  • dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF ⁇ to cultures of monocytes harvested from peripheral blood.
  • CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF ⁇ , CD40 ligand, LPS, flt3 ligand and or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
  • Dendritic cells are conveniently categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be constmed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fc ⁇ receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CDl 1) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-lBB).
  • cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CDl 1) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-lBB).
  • APCs may generally be transfected with a polynucleotide encoding a prostate-specific protein (or portion or other variant thereof) such that the prostate-specific polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells may then be used for therapeutic pu ⁇ oses, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo.
  • In vivo and ex vivo transfection of dendritic cells may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.
  • Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the prostate-specific polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or vimses (e.g., vaccinia, fowlpox, adenovims or lentivims vectors).
  • the polypeptide Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule).
  • an immunological partner that provides T cell help e.g., a carrier molecule.
  • a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
  • compositions described herein may be used for immunotherapy of cancer, such as prostate cancer.
  • pharmaceutical compositions and vaccines are typically administered to a patient.
  • a patient refers to any warm-blooded animal, preferably a human.
  • a patient may or may not be afflicted with cancer.
  • the above pharmaceutical compositions and vaccines may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer.
  • a cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.
  • Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs.
  • immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as polypeptides and polynucleotides disclosed herein).
  • immune response-modifying agents such as polypeptides and polynucleotides disclosed herein.
  • immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system.
  • agents with established tumor-immune reactivity such as effector cells or antibodies
  • effector cells include T cells as discussed above, T lymphocytes (such as CD8 + cytotoxic T lymphocytes and CD4 + T-helper tumor- infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein.
  • T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy.
  • the polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Patent No. 4,918,164) for passive immunotherapy.
  • Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein.
  • Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art.
  • Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells.
  • cytokines such as IL-2
  • immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy.
  • antigen-presenting cells such as dendritic, macrophage, monocyte, fibroblast or B cells
  • antigen-presenting cells may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art.
  • antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant vims or other expression system.
  • Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo.
  • a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient.
  • Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration.
  • the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally.
  • injection e.g., intracutaneous, intramuscular, intravenous or subcutaneous
  • intranasally e.g., by aspiration
  • between 1 and 10 doses may be administered over a 52 week period.
  • 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter.
  • Alternate protocols may be appropriate for individual patients.
  • a suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti -tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level.
  • Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro.
  • Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non- vaccinated patients.
  • the amount of each polypeptide present in a dose ranges from about 25 ⁇ g to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients.
  • Increases in preexisting immune responses to a prostate-specific protein generally correlate with an improved clinical outcome.
  • Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.
  • a cancer may be detected in a patient based on the presence of one or more prostate-specific proteins and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, urine and/or tumor biopsies) obtained from the patient.
  • a biological sample for example, blood, sera, urine and/or tumor biopsies
  • proteins may be used as markers to indicate the presence or absence of a cancer such as prostate cancer.
  • proteins may be useful for the detection of other cancers.
  • the binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample.
  • Polynucleotide primers and probes may be used to detect the level of mRNA encoding a tumor protein, which is also indicative of the presence or absence of a cancer.
  • a prostate tumor sequence should be present at a level that is at least three fold higher in tumor tissue than in normal tissue
  • the presence or absence of a cancer in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.
  • the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample.
  • the bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex.
  • detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin.
  • a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample.
  • the extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent.
  • Suitable polypeptides for use within such assays include full length prostate-specific proteins and portions thereof to which the binding agent binds, as described above.
  • the solid support may be any material known to those of ordinary skill in the art to which the protein may be attached.
  • the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
  • the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
  • the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
  • the binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature.
  • immobilization refers to both noncovalent association, such as adso ⁇ tion, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adso ⁇ tion to a well in a microtiter plate or to a membrane is preferred. In such cases, adso ⁇ tion may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day.
  • contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 ⁇ g, and preferably about 100 ng to about 1 ⁇ g, is sufficient to immobilize an adequate amount of binding agent.
  • a plastic microtiter plate such as polystyrene or polyvinylchloride
  • Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
  • a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
  • the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
  • the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
  • a detection reagent preferably a second antibody capable of binding to a different site on the polypeptide
  • the immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody.
  • the sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation.
  • PBS phosphate-buffered saline
  • an appropriate contact time is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with prostate cancer.
  • the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
  • a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
  • the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
  • Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM.
  • the second antibody which contains a reporter group, may then be added to the solid support.
  • Preferred reporter groups include those groups recited above.
  • the detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide.
  • An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time.
  • Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group.
  • the method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
  • the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value.
  • the cut-off value for the detection of a cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer.
  • a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the cancer.
  • the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7.
  • the cut-off value may be determined from a plot of pairs of tme positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result.
  • the cut-off value on the plot that is the closest to the upper left-hand corner i.e., the value that encloses the largest area
  • a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive.
  • the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate.
  • a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer.
  • the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose.
  • a membrane such as nitrocellulose.
  • polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane.
  • a second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane.
  • the detection of bound second binding agent may then be performed as described above.
  • the strip test format one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent.
  • Concentration of second binding agent at the area of immobilized antibody indicates the presence of a cancer.
  • concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result.
  • the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above.
  • Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof.
  • the amount of antibody immobilized on the membrane ranges from about 25 ng to about l ⁇ g, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.
  • a cancer may also, or alternatively, be detected based on the presence of T cells that specifically react with a prostate-specific protein in a biological sample.
  • a biological sample comprising CD4 + and/or CD8 + T cells isolated from a patient is incubated with a prostate-specific polypeptide, a polynucleotide encoding such a polypeptide and/or an APC that expresses at least an immunogenic portion of such a polypeptide, and the presence or absence of specific activation of the T cells is detected.
  • Suitable biological samples include, but are not limited to, isolated T cells.
  • T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes).
  • T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37°C with prostate-specific polypeptide (e.g., 5 - 25 ⁇ g/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of prostate-specific polypeptide to serve as a control.
  • activation is preferably detected by evaluating proliferation of the T cells.
  • activation is preferably detected by evaluating cytolytic activity.
  • a level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a cancer in the patient.
  • a cancer may also, or alternatively, be detected based on the level of mRNA encoding a prostate-specific protein in a biological sample.
  • at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a prostate-specific cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the prostate-specific protein.
  • PCR polymerase chain reaction
  • the amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis.
  • oligonucleotide probes that specifically hybridize to a polynucleotide encoding a prostate-specific protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the protein in a biological sample.
  • oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a prostate-specific protein that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length.
  • oligonucleotide primers and/or probes will hybridize to a polynucleotide encoding a polypeptide disclosed herein under moderately stringent conditions, as defined above.
  • Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length.
  • the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule having a sequence recited in SEQ ID NO: 1-111, 115- 171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381 , 382, 384-476, 524, 526, 530, 531, 533, 535 and 536.
  • RNA is extracted from a biological sample, such as biopsy tissue, and is reverse transcribed to produce cDNA molecules.
  • PCR amplification using at least one specific primer generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis.
  • Amplification may be performed on biological samples taken from a test patient and from an individual who is not afflicted with a cancer. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the test patient sample as compared to the same dilutions of the non-cancerous sample is typically considered positive.
  • the disclosed compositions may be used as markers for the progression of cancer.
  • assays as described above for the diagnosis of a cancer may be performed over time, and the change in the level of reactive polypeptide(s) or polynucleotide evaluated.
  • the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed.
  • a cancer is progressing in those patients in whom the level of polypeptide or polynucleotide detected increases over time.
  • the cancer is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time.
  • Certain in vivo diagnostic assays may be performed directly on a tumor.
  • One such assay involves contacting tumor cells with a binding agent.
  • the bound binding agent may then be detected directly or indirectly via a reporter group.
  • binding agents may also be used in histological applications.
  • polynucleotide probes may be used within such applications.
  • multiple prostate-specific protein markers may be assayed within a given sample. It will be apparent that binding agents specific for different proteins provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of protein markers may be based on routine experiments to determine combinations that results in optimal sensitivity.
  • assays for proteins provided herein may be combined with assays for other known tumor antigens. DIAGNOSTIC KITS
  • kits for use within any of the above diagnostic methods.
  • Such kits typically comprise two or more components necessary for performing a diagnostic assay.
  • Components may be compounds, reagents, containers and/or equipment.
  • one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a prostate-specific protein.
  • Such antibodies or fragments may be provided attached to a support material, as described above.
  • One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
  • Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.
  • kits may be designed to detect the level of mRNA encoding a prostate-specific protein in a biological sample.
  • kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a prostate-specific protein.
  • Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a prostate-specific protein.
  • This Example describes the isolation of certain prostate-specific polypeptides from a prostate tumor cDNA library.
  • a human prostate tumor cDNA expression library was constructed from prostate tumor poly A + RNA using a Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning kit (BRL Life Technologies, Gaithersburg, MD 20897) following the manufacturer's protocol. Specifically, prostate tumor tissues were homogenized with polytron (Kinematica, Switzerland) and total RNA was extracted using Trizol reagent (BRL Life Technologies) as directed by the manufacturer. The poly A + RNA was then purified using a Qiagen oligotex spin column mRNA purification kit (Qiagen, Santa Clarita, CA 91355) according to the manufacturer's protocol. First- strand cDNA was synthesized using the NotI/Oligo-dT18 primer.
  • Double-stranded cDNA was synthesized, ligated with EcoRI/BAXI adaptors (Invitrogen, San Diego, CA) and digested with Notl. Following size fractionation with Chroma Spin- 1000 columns (Clontech, Palo Alto, CA), the cDNA was ligated into the EcoRI/Notl site of pCDNA3.1 (Invitrogen) and transformed into ElectroMax E. coli DH10B cells (BRL Life Technologies) by electroporation.
  • a normal human pancreas cDNA expression library was prepared from a pool of six tissue specimens (Clontech).
  • the cDNA libraries were characterized by determining the number of independent colonies, the percentage of clones that carried insert, the average insert size and by sequence analysis.
  • the prostate tumor library contained 1.64 x 10 7 independent colonies, with 70%) of clones having an insert and the average insert size being 1745 base pairs.
  • the normal pancreas cDNA library contained 3.3 x 10 6 independent colonies, with 69% of clones having inserts and the average insert size being 1120 base pairs.
  • sequence analysis showed that the majority of clones had a full length cDNA sequence and were synthesized from mRNA, with minimal rRNA and mitochondrial DNA contamination.
  • cDNA library subtraction was performed using the above prostate tumor and normal pancreas cDNA libraries, as described by Hara et al. (Blood, 54:189-199, 1994) with some modifications. Specifically, a prostate tumor-specific subtracted cDNA library was generated as follows. Normal pancreas cDNA library (70 ⁇ g) was digested with EcoRI, Notl, and Sful, followed by a filling-in reaction with DNA polymerase Klenow fragment. After phenol-chloroform extraction and ethanol precipitation, the DNA was dissolved in 100 ⁇ l of H 2 O, heat-denatured and mixed with 100 ⁇ l (100 ⁇ g) of Photoprobe biotin (Vector Laboratories, Burlingame, CA).
  • the resulting mixture was irradiated with a 270 W sunlamp on ice for 20 minutes. Additional Photoprobe biotin (50 ⁇ l) was added and the biotinylation reaction was repeated. After extraction with butanol five times, the DNA was ethanol-precipitated and dissolved in 23 ⁇ l H 2 O to form the driver DNA.
  • ⁇ g prostate tumor cDNA library was digested with BamHI and Xhol, phenol chloroform extracted and passed through Chroma spin-400 columns (Clontech). Following ethanol precipitation, the tracer DNA was dissolved in 5 ⁇ l H 2 O. Tracer DNA was mixed with 15 ⁇ l driver DNA and 20 ⁇ l of 2 x hybridization buffer (1.5 M NaCl/10 mM EDTA/50 mM HEPES pH 7.5/0.2% sodium dodecyl sulfate), overlaid with mineral oil, and heat- denatured completely. The sample was immediately transferred into a 68 °C water bath and incubated for 20 hours (long hybridization [LH]).
  • 2 x hybridization buffer 1.5 M NaCl/10 mM EDTA/50 mM HEPES pH 7.5/0.2% sodium dodecyl sulfate
  • plasmid DNA was prepared from 100 independent clones, randomly picked from the subtracted prostate tumor specific library and grouped based on insert size. Representative cDNA clones were further characterized by DNA sequencing with a Perkin Elmer/ Applied Biosystems Division Automated Sequencer Model 373A (Foster City, CA). Six cDNA clones, hereinafter referred to as Fl-13, Fl-12, Fl-16, Hl-1, Hl-9 and HI -4, were shown to be abundant in the subtracted prostate-specific cDNA library.
  • the determined 3' and 5' cDNA sequences for Fl-12 are provided in SEQ ID NO: 2 and 3, respectively, with determined 3 ' cDNA sequences for Fl-13, Fl-16, Hl-1, Hl-9 and H 1 -4 being provided in SEQ ID NO: 1 and 4-7, respectively.
  • the cDNA sequences for the isolated clones were compared to known sequences in the gene bank using the EMBL and GenBank databases (release 96).
  • Four of the prostate tumor cDNA clones, Fl-13, Fl-16, Hl-1, and HI -4 were determined to encode the following previously identified proteins: prostate specific antigen (PSA), human glandular kallikrein, human tumor expression enhanced gene, and mitochondria cytochrome C oxidase subunit II.
  • PSA prostate specific antigen
  • Hl-9 was found to be identical to a previously identified human autonomously replicating sequence. No significant homologies to the cDNA sequence for Fl-12 were found.
  • cDNA library subtraction was performed by subtracting the prostate tumor cDNA library described above with the normal pancreas cDNA library and with the three most abundant genes in the previously subtracted prostate tumor specific cDNA library: human glandular kallikrein, prostate specific antigen (PSA), and mitochondria cytochrome C oxidase subunit II. Specifically, 1 ⁇ g each of human glandular kallikrein, PSA and mitochondria cytochrome C oxidase subunit II cDNAs in pCDNA3.1 were added to the driver DNA and subtraction was performed as described above to provide a second subtracted cDNA library hereinafter referred to as the "subtracted prostate tumor specific cDNA library with spike".
  • cDNA clones were isolated from the subtracted prostate tumor specific cDNA library with spike.
  • the determined 3' and 5' cDNA sequences for the clones referred to as Jl-17, Ll-12, Nl-1862, Jl-13, Jl-19, Jl-25, Jl-24, Kl-58, Kl-63, Ll-4 and Ll-14 are provided in SEQ ID NOS: 8-9, 10-11, 12-13, 14-15, 16-17, 18-19, 20-21, 22-23, 24-25, 26-27 and 28-29, respectively.
  • the determined 3' cDNA sequences for the clones referred to as Jl-12, Jl-16, Jl-21, Kl-48, Kl-55, Ll-2, Ll-6, Nl-1858, Nl-1860, Nl-1861, Nl-1864 are provided in SEQ ID NOS: 30-40, respectively. Comparison of these sequences with those in the gene bank as described above, revealed no significant homologies to three of the five most abundant DNA species, (Jl-17, Ll-12 and Nl-1862; SEQ ID NOS: 8-9, 10-11 and 12-13, respectively).
  • Jl-12 SEQ ID NO:30
  • Kl-48 SEQ ID NO:33
  • a second subtraction with spike (referred to as "prostate subtraction spike 2") was performed by subtracting a prostate tumor specific cDNA library with spike with normal pancreas cDNA library and further spiked with PSA, Jl-17, pulmonary surfactant-associated protein, mitochondrial DNA, cytochrome c oxidase subunit II, Nl-1862, autonomously replicating sequence, Ll-12 and tumor expression enhanced gene.
  • Four additional clones hereinafter referred to as Vl-3686, Rl-2330, 1B-3976 and Vl-3679, were isolated. The determined cDNA sequences for these clones are provided in SEQ ID NO: 73 -76, respectively.
  • the determined cDNA sequences for these clones are provided in SEQ ID NOS: 77-92, respectively. Comparison of these sequences with those in the gene bank as described above, revealed no significant homologies to 1G-4741, 1G-4734, 11-4807, 1J-4876 and 1K-4896 (SEQ ID NOS: 79, 81, 87, 90 and 92, respectively).
  • prostate subtraction 3 An additional subtraction was performed by subtracting a normal prostate cDNA library with normal pancreas cDNA (referred to as "prostate subtraction 3"). This led to the identification of six additional clones referred to as 1G-4761, 1G-4762, 1H-4766, 1H-4770, 1H- 4771 and 1H-4772 (SEQ ID NOS: 93-98). Comparison of these sequences with those in the gene bank revealed no significant homologies to 1G-4761 and 1H-4771 (SEQ ID NOS: 93 and 97, respectively).
  • cDNA clones isolated in prostate subtraction 1 and prostate subtraction 2 were colony PCR amplified and their mRNA expression levels in prostate tumor, normal prostate and in various other normal tissues were determined using microarray technology (Synteni, Palo Alto, CA). Briefly, the PCR amplification products were dotted onto slides in an array format, with each product occupying a unique location in the array.
  • mRNA was extracted from the tissue sample to be tested, reverse transcribed, and fluorescent-labeled cDNA probes were generated.
  • the microarrays were probed with the labeled cDNA probes, the slides scanned and fluorescence intensity was measured. This intensity correlates with the hybridization intensity.
  • Two clones (referred to as P509S and P510S) were found to be over-expressed in prostate tumor and normal prostate and expressed at low levels in all other normal tissues tested (liver, pancreas, skin, bone marrow, brain, breast, adrenal gland, bladder, testes, salivary gland, large intestine, kidney, ovary, lung, spinal cord, skeletal muscle and colon).
  • the determined cDNA sequences for P509S and P510S are provided in SEQ ID NO: 223 and 224, respectively. Comparison of these sequences with those in the gene bank as described above, revealed some homology to previously identified ESTs.
  • EXAMPLE 2 DETERMINATION OF TISSUE SPECIFICITY OF PROSTATE-SPECIFIC POLYPEPTIDES Using gene specific primers, mRNA expression levels for the representative prostate- specific polypeptides Fl-16, Hl-1, Jl-17 (also referred to as P502S), Ll-12 (also referred to as P501S), Fl-12 (also referred to as P504S) and Nl-1862 (also referred to as P503S) were examined in a variety of normal and tumor tissues using RT-PCR.
  • ⁇ -actin was used as an internal control for each of the tissues examined.
  • serial dilutions of the first strand cDNAs were prepared and RT-PCR assays were performed using ⁇ -actin specific primers.
  • ⁇ -actin levels were determined for each reverse transcription reaction from each tissue. DNA contamination was minimized by DNase treatment and by assuring a negative PCR result when using first strand cDNA that was prepared without adding reverse transcriptase.
  • mRNA Expression levels were examined in four different types of tumor tissue (prostate tumor from 2 patients, breast tumor from 3 patients, colon tumor, lung tumor), and sixteen different normal tissues, including prostate, colon, kidney, liver, lung, ovary, pancreas, skeletal muscle, skin, stomach, testes, bone marrow and brain.
  • Fl-16 was found to be expressed at high levels in prostate tumor tissue, colon tumor and normal prostate, and at lower levels in normal liver, skin and testes, with expression being undetectable in the other tissues examined.
  • Hl-1 was found to be expressed at high levels in prostate tumor, lung tumor, breast tumor, normal prostate, normal colon and normal brain, at much lower levels in normal lung, pancreas, skeletal muscle, skin, small intestine, bone marrow, and was not detected in the other tissues tested.
  • Jl-17 (P502S) and Ll-12 (P501S) appear to be specifically over-expressed in prostate, with both genes being expressed at high levels in prostate tumor and normal prostate but at low to undetectable levels in all the other tissues examined.
  • Nl-1862 (P503S) was found to be over-expressed in 60% of prostate tumors and detectable in normal colon and kidney.
  • the RT-PCR results thus indicate that Fl-16, Hl-1, Jl-17 (P502S), Nl-1862 (P503S) and Ll-12 (P501S) are either prostate specific or are expressed at significantly elevated levels in prostate.
  • Ll-12 (P501S) is over-expressed in prostate tumors and normal prostate, while being undetectable in other normal tissues tested.
  • Jl -17 (P502S) was detected in two prostate tumors and not in the other tissues tested.
  • Nl-1862 (P503S) was found to be over-expressed in three prostate tumors and to be expressed in normal prostate, colon and kidney, but not in other tissues tested.
  • Fl-12 (P504S) was found to be highly expressed in two prostate tumors and to be undetectable in all other tissues tested.
  • Ll-12 (P501S) was found to be over-expressed in normal prostate and prostate tumor, with some expression being detected in normal skeletal muscle.
  • Jl-12 and Fl-12 (P504S) were found to be over-expressed in prostate tumor, with expression being lower or undetectable in all other tissues tested.
  • Nl-1862 (P503S) was found to be expressed at high levels in prostate tumor and normal prostate, and at low levels in normal large intestine and normal colon, with expression being undetectable in all other tissues tested.
  • Rl-2330 was found to be over-expressed in prostate tumor and normal prostate, and to be expressed at lower levels in all other tissues tested.
  • 1D-4279 was found to be over-expressed in prostate tumor and normal prostate, expressed at lower levels in normal spinal cord, and to be undetectable in all other tissues tested.
  • P1000C and P1001C Two of these clones (referred to as P1000C and P1001C) were found to be over-expressed in prostate tumor and normal prostate, and expressed at low to undetectable levels in all other tissues tested (normal aorta, thymus, resting and activated PBMC, epithelial cells, spinal cord, adrenal gland, fetal tissues, skin, salivary gland, large intestine, bone marrow, liver, lung, dendritic cells, stomach, lymph nodes, brain, heart, small intestine, skeletal muscle, colon and kidney.
  • the determined cDNA sequences for P1000C and P1001C are provided in SEQ ID NO: 384 and 472, respectively.
  • P1001C The sequence of P1001C was found to show some homology to the previously isolated Human mRNA for JM27 protein. No significant homologies were found to the sequence of PIOOOC.
  • the rabbit-anti-P504S polyclonal antibody did not appear to label benign prostate cells with the same cytoplasmic granular staining, but rather with light nuclear staining. Analysis of normal tissues revealed that the encoded polypeptide was found to be expressed in some, but not all normal human tissues. Positive cytoplasmic staining with rabbit-anti-P504S polyclonal antibody was found in normal human kidney, liver, brain, colon and lung-associated macrophages, whereas heart and bone marrow were negative.
  • a cDNA subtraction library containing cDNA from normal prostate subtracted with ten other normal tissue cDNAs (brain, heart, kidney, liver, lung, ovary, placenta, skeletal muscle, spleen and thymus) and then submitted to a first round of PCR amplification, was purchased from Clontech. This library was subjected to a second round of PCR amplification, following the manufacturer's protocol. The resulting cDNA fragments were subcloned into the vector pT7 Blue T-vector (Novagen, Madison, WI) and transformed into XL-1 Blue MRF' E. coli (Stratagene).
  • DNA was isolated from independent clones and sequenced using a Perkin Elmer/Applied Biosystems Division Automated Sequencer Model 373 A. Fifty-nine positive clones were sequenced. Comparison of the DNA sequences of these clones with those in the gene bank, as described above, revealed no significant homologies to 25 of these clones, hereinafter referred to as P5, P8, P9, P18, P20, P30, P34, P36, P38, P39, P42, P49, P50, P53, P55, P60, P64, P65, P73, P75, P76, P79 and P84. The determined cDNA sequences for these clones are provided in SEQ ID NO: 41-45, 47-52 and 54-65, respectively.
  • P29, P47, P68, P80 and P82 (SEQ ID NO: 46, 53 and 66-68, respectively) were found to show some degree of homology to previously identified DNA sequences. To the best of the inventors' knowledge, none of these sequences have been previously shown to be present in prostate.
  • cDNA clones containing the P20 sequence represent splice variants of a gene referred to as P703P.
  • the determined DNA sequence for the variants referred to as DEI, DEI 3 and DEI 4 are provided in SEQ ID NOS: 171, 175 and 177, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 172, 176 and 178, respectively.
  • the determined cDNA sequence for an extended spliced form of P703 is provided in SEQ ID NO: 225.
  • the DNA sequences for the splice variants referred to as DE2 and DE6 are provided in SEQ ID NOS: 173 and 174, respectively.
  • P9 was found to be highly expressed in normal prostate and prostate tumor compared to all normal tissues tested except for normal colon which showed comparable expression.
  • P30 similar expression levels were seen in normal prostate and prostate tumor, compared to six of twelve other normal tissues tested. Increased expression was seen in breast tumors, one lung tumor sample and one colon tumor sample, and also in normal PBMC. P29 was found to be over-expressed in prostate tumor (5 of 5) and normal prostate (5 of 5) compared to the majority of normal tissues. However, substantial expression of P29 was observed in normal colon and normal lung (2 of 2). P80 was found to be over-expressed in prostate tumor (5 of 5) and normal prostate (5 of 5) compared to all other normal tissues tested, with increased expression also being seen in colon tumor.
  • the determined DNA sequences for 10-d8, 10-hlO, l l-c8, 8-d4, 8-d9, 8-hl l, 9-fl2 and 9-f3 are provided in SEQ ID NO: 207, 208, 209, 216, 217, 220, 221 and 222, respectively.
  • the determined forward and reverse DNA sequences for 7-g6, 8-b5, 8-b6 and 8-g3 are provided in SEQ ID NO: 210 and 211; 212 and 213; 214 and 215; and 218 and 219, respectively. Comparison of these sequences with those in the gene bank revealed no significant homologies to the sequence of 9-f3.
  • the clones 10-d8, 1 l-c8 and 8-hl 1 were found to show some homology to previously isolated ESTs, while 10- hlO, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3 and 9-fl2 were found to show some homology to previously identified genes. Further characterization of 7-G6 and 8-G3 showed identity to the known genes PAP and PSA, respectively.
  • mRNA expression levels for these clones were determined using the micro-array technology described above.
  • the clones 7-G6, 8-G3, 8-B5, 8-B6, 8-D4, 8-D9, 9-F3, 9-F12, 9-H3, 10- A2, 10- A4, 11 -C9 and 11-F2 were found to be over-expressed in prostate tumor and normal prostate, with expression in other tissues tested being low or undetectable.
  • Increased expression of 8-F11 was seen in prostate tumor and normal prostate, bladder, skeletal muscle and colon.
  • Increased expression of 10-HlO was seen in prostate tumor and normal prostate, bladder, lung, colon, brain and large intestine.
  • P703PDE5 was recovered by screening of a cDNA library (#141-26) with a portion of P703P as a probe.
  • P703P6.26 was recovered from a mixture of three prostate tumor cDNAs and P703PX_23 was recovered from cDNA library (#438-48). Together, the additional sequences include all of the putative mature serine protease along with part of the putative signal sequence.
  • the putative full-length cDNA sequence for P703P is provided in SEQ ID NO: 524, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 525.
  • JP PCR subtraction
  • the determined cDNA sequences for these seven clones are provided in SEQ ID NO: 307-311, 313 and 315, respectively.
  • the remaining six clones (SEQ ID NO: 316 and 321-325) were shown to share some homology to known genes.
  • all thirteen clones showed three or more fold over-expression in prostate tissues, including prostate tumors, BPH and normal prostate as compared to normal non-prostate tissues.
  • Clones P774P, P775P and P710P showed comparatively lower expression and expression in fewer prostate tumors and BPH samples, with negative to low expression in normal prostate.
  • the full-length cDNA for P71 IP was obtained by employing the partial sequence of SEQ ID NO: 307 to screen a prostate cDNA library. Specifically, a directionally cloned prostate cDNA library was prepared using standard techniques. One million colonies of this library were plated onto LB/Amp plates.
  • Nylon membrane filters were used to lift these colonies, and the cDNAs which were picked up by these filters were denatured and cross-linked to the filters by UV light.
  • the P71 IP cDNA fragment of SEQ ID NO: 307 was radio-labeled and used to hybridize with these filters. Positive clones were selected, and cDNAs were prepared and sequenced using an automatic Perkin Elmer/ Applied Biosystems sequencer. The determined full-length sequence of P711P is provided in SEQ ID NO: 382, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 383.
  • P707P and P714P additional cDNA sequence information was derived for two clones described above, 11-C9 and 9-F3, herein after referred to as P707P and P714P, respectively (SEQ ID NO: 333 and 334).
  • P707P was found to be a splice variant of the known gene HoxB13. In contrast, no significant homologies to P714P were found.
  • Clones 8-B3, P89, P98, P130 and P201 were found to be contained within one contiguous sequence, referred to as P705P (SEQ ID NO: 335, with the predicted amino acid sequence provided in SEQ ID NO: 336), which was determined to be a splice variant of the known gene NKX 3.1. Further studies on P775P resulted in the isolation of four additional sequences (SEQ ID NO: 335, with the predicted amino acid sequence provided in SEQ ID NO: 336), which was determined to be a splice variant of the known gene NKX 3.1. Further studies on P775P resulted in the isolation of four additional sequences (SEQ ID NO: 335, with the predicted amino acid sequence provided in SEQ ID NO: 336), which was determined to be a splice variant of the known gene NKX 3.1. Further studies on P775P resulted in the isolation of four additional sequences (SEQ ID NO: 335, with the predicted amino acid sequence provided in SEQ ID NO:
  • SEQ ID NO: 473-476) which are all splice variants of the P775P gene.
  • the sequence of SEQ ID NO: 474 was found to contain two open reading frames (ORFs).
  • the predicted amino acid sequences encoded by these ORFs are provided in SEQ ID NO: 477 and 478.
  • the cDNA sequence of SEQ ID NO: 475 was found to contain an ORF which encodes the amino acid sequence of SEQ ID NO: 479.
  • the cDNA sequence of SEQ ID NO: 473 was found to contain four ORFs.
  • the predicted amino acid sequences encoded by these ORFs are provided in SEQ ID NO: 480-483.
  • Polypeptides may be synthesized on a Perkin Elmer/ Applied Biosystems 430 A peptide synthesizer using FMOC chemistry with HPTU (O-Benzotriazole-N,N,N',N'- tetramethyluronium hexafluorophosphate) activation.
  • HPTU O-Benzotriazole-N,N,N',N'- tetramethyluronium hexafluorophosphate
  • a Gly-Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method of conjugation, binding to an immobilized surface, or labeling of the peptide.
  • Cleavage of the peptides from the solid support may be carried out using the following cleavage mixture: trifluoroacetic acid:ethanedithiol:thioanisole:water:phenol (40:1 :2:2:3). After cleaving for 2 hours, the peptides may be precipitated in cold methyl-t-butyl-ether. The peptide pellets may then be dissolved in water containing 0.1%) trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC. A gradient of 0%-60% acetonitrile (containing 0.1 %> TFA) in water (containing 0.1 % TFA) may be used to elute the peptides. Following lyophilization of the pure fractions, the peptides may be characterized using electrospray or other types of mass spectrometry and by amino acid analysis.
  • TFA trifluoroacetic acid
  • a cDNA library generated from prostate primary tumor mRNA as described above was subtracted with cDNA from normal prostate.
  • the subtraction was performed using a PCR- based protocol (Clontech), which was modified to generate larger fragments.
  • tester and driver double stranded cDNA were separately digested with five restriction enzymes that recognize six-nucleotide restriction sites (Mlul, Mscl, PvuII, Sail and Stul). This digestion resulted in an average cDNA size of 600 bp, rather than the average size of 300 bp that results from digestion with Rsal according to the Clontech protocol. This modification did not affect the subtraction efficiency.
  • Two tester populations were then created with different adapters, and the driver library remained without adapters.
  • the tester and driver libraries were then hybridized using excess driver cDNA.
  • driver was separately hybridized with each of the two tester cDNA populations. This resulted in populations of (a) unhybridized tester cDNAs, (b) tester cDNAs hybridized to other tester cDNAs, (c) tester cDNAs hybridized to driver cDNAs and (d) unhybridized driver cDNAs.
  • the two separate hybridization reactions were then combined, and rehybridized in the presence of additional denatured driver cDNA.
  • a fifth population (e) was generated in which tester cDNA with one adapter hybridized to tester cDNA with the second adapter. Accordingly, the second hybridization step resulted in enrichment of differentially expressed sequences which could be used as templates for PCR amplification with adaptor-specific primers.
  • This PCR-based subtraction technique normalizes differentially expressed cDNAs so that rare transcripts that are overexpressed in prostate tumor tissue may be recoverable. Such transcripts would be difficult to recover by traditional subtraction methods.
  • seventy-seven further clones were identified. Sequences of these partial cDNAs are provided in SEQ ID NO: 29 to 305. Most of these clones had no significant homology to database sequences.
  • JPTPN23 (SEQ ID NO: 231; similarity to pig valosin-containing protein), JPTPN30 (SEQ ID NO: 234; similarity to rat mRNA for proteasome subunit), JPTPN45 (SEQ ID NO: 243; similarity to rat norvegicus cytosolic NADP-dependent isocitrate dehydrogenase), JPTPN46 (SEQ ID NO: 244; similarity to human subclone H8 4 d4 DNA sequence), JP1D6 (SEQ ID NO: 265; similarity to G.
  • JP8D6 SEQ ID NO: 288; similarity to human BAC clone RG016J04
  • JP8F5 SEQ ID NO: 289; similarity to human subclone H8 3 b5 DNA sequence
  • JP8E9 SEQ ID NO: 299; similarity to human Alu sequence
  • P790P Further studies on the clone of SEQ ID NO: 352 (referred to as P790P) led to the isolation of the full-length cDNA sequence of SEQ ID NO: 526. The corresponding predicted amino acid is provided in SEQ ID NO: 527. Data from two quantitative PCR experiments indicated that P790P is over-expressed in 11/15 tested prostate tumor samples and is expressed at low levels in spinal cord, with no expression being seen in all other normal samples tested. Data from further PCR experiments and microarray experiments showed over-expression in normal prostate and prostate tumor with little or no expression in other tissues tested. P790P was subsequently found to show significant homology to a previously identified G-protein coupled prostate tissue receptor. EXAMPLE 6 PEPTIDE PRIMING OF MICE AND PROPAGATION OF CTL LINES
  • This Example illustrates the preparation of a CTL cell line specific for cells expressing the P502S gene.
  • mice expressing the transgene for human HLA A2Kb were immunized with P2S#12 peptide (VLGWVAEL; SEQ ID NO: 306), which is derived from the P502S gene (also referred to herein as Jl-17, SEQ ID NO: 8), as described by Theobald et al., Proc. Natl. Acad. Sci. USA 92:11993-11997, 1995 with the following modifications.
  • mice were immunized with lOO ⁇ g of P2S#12 and 120 ⁇ g of an I-A b binding peptide derived from hepatitis B Vims protein emulsified in incomplete Freund's adjuvant. Three weeks later these mice were sacrificed and using a nylon mesh single cell suspensions prepared.
  • P2S#12 line was cloned by limiting dilution analysis with peptide pulsed EL4 A2Kb tumor cells (1 x 10 4 cells/ well) as stimulators and A2 transgenic spleen cells as feeders ( 5 x 10 5 cells/ well) grown in the presence of 30U/ml IL-2. On day 14, cells were restimulated as before. On day 21, clones that were growing were isolated and maintained in culture. Several of these clones demonstrated significantly higher reactivity (lysis) against human fibroblasts (HLA A2Kb expressing) transduced with P502S than against control fibroblasts. An example is presented in Figure 1.
  • P2S #12 represents a naturally processed epitope of the P502S protein that is expressed in the context of the human HLA A2Kb molecule.
  • This Example illustrates the preparation of murine CTL lines and CTL clones specific for cells expressing the P501S gene.
  • mice were immunized with the P1S#10 peptide (SEQ ID NO: 337), which is derived from the P501S gene (also referred to herein as Ll-12, SEQ ID NO: 110).
  • the P1S#10 peptide was derived by analysis of the predicted polypeptide sequence for P501S for potential HLA-A2 binding sequences as defined by published HLA-A2 binding motifs (Parker, KC, et al, J. Immunol, 152:163, 1994). P1S#10 peptide was synthesized as described in Example 4, and empirically tested for HLA-A2 binding using a T cell based competition assay.
  • Predicted A2 binding peptides were tested for their ability to compete HLA-A2 specific peptide presentation to an HLA-A2 restricted CTL clone (D150M58), which is specific for the HLA-A2 binding influenza matrix peptide fluM58.
  • D150M58 CTL secretes TNF in response to self-presentation of peptide fluM58.
  • test peptides at 100-200 ⁇ g/ml were added to cultures of D150M58 CTL in order to bind HLA-A2 on the CTL. After thirty minutes, CTL cultured with test peptides, or control peptides, were tested for their antigen dose response to the fluM58 peptide in a standard TNF bioassay.
  • peptide P1S#10 competes HLA-A2 restricted presentation of fluM58, demonstrating that peptide P1 S#10 binds HLA- A2.
  • mice expressing the transgene for human HLA A2Kb were immunized as described by Theobald et al. (Proc. Natl. Acad. Sci. USA 92: 11993-11997, 1995) with the following modifications. Mice were immunized with 62.5 ⁇ g of PIS #10 and 120 ⁇ g of an I-A b binding peptide derived from Hepatitis B Vims protein emulsified in incomplete Freund's adjuvant. Three weeks later these mice were sacrificed and single cell suspensions prepared using a nylon mesh.
  • a PlS#10-specific CTL line was cloned by limiting dilution analysis with peptide pulsed EL4 A2Kb tumor cells (1 x 10 4 cells/ well) as stimulators and A2 transgenic spleen cells as feeders ( 5 x 10 5 cells/ well) grown in the presence of 30U/ml IL-2. On day 14, cells were restimulated as before. On day 21, viable clones were isolated and maintained in culture. As shown in Figure 5, five of these clones demonstrated specific cytolytic reactivity against P501S-transduced Jurkat A2Kb targets. This data indicates that P1S#10 represents a naturally processed epitope of the P501S protein that is expressed in the context of the human HLA-A2.1 molecule.
  • the prostate-specific antigen Ll-12 as described above, is also referred to as P501S.
  • HLA A2Kb Tg mice (provided by Dr L. Sherman, The Scripps Research Institute, La Jolla, CA) were immunized with 100 ⁇ g P501S in the vector VR1012 either intramuscularly or intradermally. The mice were immunized three times, with a two week interval between immunizations. Two weeks after the last immunization, immune spleen cells were cultured with Jurkat A2Kb-P501S transduced stimulator cells. CTL lines were stimulated weekly. After two weeks of in vitro
  • This Example illustrates the ability of T cells specific for a prostate tumor polypeptide to recognize human tumor.
  • Human CD8 + T cells were primed in vitro to the P2S-12 peptide (SEQ ID NO: 306) derived from P502S (also referred to as Jl-17) using dendritic cells according to the protocol of Van Tsai et al. (Critical Reviews in Immunology 75:65-75, 1998).
  • the resulting CD8 + T cell microcultures were tested for their ability to recognize the P2S-12 peptide presented by autologous fibroblasts or fibroblasts which were transduced to express the P502S gene in a ⁇ -interferon ELISPOT assay (see Lalvani et al., J. Exp. Med. 756:859-865, 1997).
  • T cells were assayed in duplicate on 10 4 fibroblasts in the presence of 3 ⁇ g/ml human ⁇ 2 - microglobulin and 1 ⁇ g/ml P2S-12 peptide or control E75 peptide.
  • T cells were simultaneously assayed on autologous fibroblasts transduced with the P502S gene or as a control, fibroblasts transduced with HER-2/ «ew.
  • the fibroblasts were treated with 10 ng/ml ⁇ -interferon for 48 hours to upregulate class I MHC expression.
  • FIG. 2A demonstrates that there was a strong increase in the number of ⁇ -interferon spots with increasing numbers of T cells on fibroblasts pulsed with the P2S-12 peptide (solid bars) but not with the control E75 peptide (open bars). This shows the ability of these T cells to specifically recognize the P2S-12 peptide.
  • FIG. 2B this microculture also demonstrated an increase in the number of ⁇ -interferon spots with increasing numbers of T cells on fibroblasts transduced to express the P502S gene but not the HER-2/ «ew gene.
  • This Example illustrates the ability of a prostate-specific antigen to elicit a CTL response in blood of normal humans.
  • DC dendritic cells
  • monocyte cultures derived from PBMC of normal donors by growth for five days in RPMI medium containing 10% human serum, 50 ng/ml GMCSF and 30 ng/ml IL-4.
  • DC were infected overnight with recombinant P501S-expressing vaccinia vims at an M.O.I, of 5 and matured for 8 hours by the addition of 2 micrograms/ml CD40 ligand.
  • Vims was inactivated by UV irradiation, CD8 + cells were isolated by positive selection using magnetic beads, and priming cultures were initiated in 24- well plates.
  • CD8+ lines were identified that specifically produced interferon- gamma when stimulated with autologous P501S-transduced fibroblasts.
  • the P501S-specific activity of cell line 3A-1 could be maintained following additional stimulation cycles on autologous B-LCL transduced with P501S.
  • Line 3A-1 was shown to specifically recognize autologous B-LCL transduced to express P501S, but not EGFP-transduced autologous B-LCL, as measured by cytotoxicity assays ( 51 Cr release) and interferon-gamma production (Interferon-gamma Elispot; see above and Lalvani et al., J. Exp. Med. 756:859-865, 1997). The results of these assays are presented in Figures 6 A and 6B.
  • the 9-mer peptide p5 (SEQ ID NO: 338) was derived from the P703P antigen (also referred to as P20).
  • the p5 peptide is immunogenic in human HLA-A2 donors and is a naturally processed epitope.
  • Antigen specific human CD8+ T cells can be primed following repeated in vitro stimulations with monocytes pulsed with p5 peptide.
  • These CTL specifically recognize p5-pulsed and P703P-transduced target cells in both ELISPOT (as described above) and chromium release assays.
  • immunization of HLA-A2Kb transgenic mice with p5 leads to the generation of CTL lines which recognize a variety of HLA-A2Kb or HLA-A2 transduced target cells expressing P703P.
  • HLA-A2Kb transgenic mice were immunized subcutaneously in the footpad with 100 ⁇ g of p5 peptide together with 140 ⁇ g of hepatitis B vims core peptide (a Th peptide) in Freund's incomplete adjuvant.
  • spleen cells from immunized mice were stimulated in vitro with peptide-pulsed LPS blasts.
  • CTL activity was assessed by chromium release assay five days after primary in vitro stimulation.
  • Retrovirally transduced cells expressing the control antigen P703P and HLA-A2Kb were used as targets. CTL lines that specifically recognized both p5-pulsed targets as well as P703P-expressing targets were identified.
  • DC Dendritic cells
  • monocyte cultures derived from PBMC of normal human donors by culturing for five days in RPMI medium containing 10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml human IL-4. Following culture, the DC were pulsed with 1 ug/ml p5 peptide and cultured with CD8+ T cell enriched PBMC.
  • CTL lines were restimulated on a weekly basis with p5-pulsed monocytes. Five to six weeks after initiation of the CTL cultures, CTL recognition of p5-pulsed target cells was demonstrated. CTL were additionally shown to recognize human cells transduced to express P703P, demonstrating that p5 is a naturally processed epitope.
  • the determined DNA sequence of this ORF is provided in SEQ ID NO: 531.
  • This frameshift generates a protein sequence (provided in SEQ ID NO: 532) of 293 amino acids that contains the C-terminal domain common to the other isoforms of B305D but that differs in the N-terminal region.
  • B305D The expression levels of B305D in a variety of tumor and normal tissues were examined by real time PCR and by Northern analysis. The results indicated that B305D is highly expressed in breast tumor, prostate tumor, normal prostate and normal testes, with expression being low or undetectable in all other tissues examined (colon tumor, lung tumor, ovary tumor, and normal bone marrow, colon, kidney, liver, lung, ovary, skin, small intestine, stomach).
  • DC dendritic cells
  • monocyte cultures derived from PBMC of normal human donors by growing for five days in RPMI medium containing 10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml human IL-4.
  • DC were infected overnight with recombinant P501S vaccinia vims at a multiplicity of infection (M.O.I) of five, and matured overnight by the addition of 3 ⁇ g/ml CD40 ligand. Vims was inactivated by UV irradiation.
  • CD8+ T cells were isolated using a magnetic bead system, and priming cultures were initiated using standard culture techniques.
  • cDNA encoding P501S was fragmented by various restriction digests, and sub-cloned into the retroviral expression vector pBIB-KS. Retroviral supernatants were generated by transfection of the helper packaging line Phoenix- Ampho. Supernatants were then used to transduce Jurkat/ A2Kb cells for CTL screening. CTL were screened in IFN-gamma ELISPOT assays against these A2Kb targets transduced with the "library" of P501S fragments.
  • Initial positive fragments P501S/H3 and P501S/F2 were sequenced and found to encode amino acids 106-553 and amino acids 136-547, respectively, of SEQ ID NO: 113.
  • a truncation of H3 was made to encode amino acid residues 106-351 of SEQ ID NO: 113, which was unable to stimulate the CTL, thus localizing the epitope to amino acid residues 351-547. Additional fragments encoding amino acids 1-472 (Fragment A) and amino acids 1-351 (Fragment B) were also constmcted. Fragment A but not Fragment B stimulated the CTL thus localizing the epitope to amino acid residues 351-472. Overlapping 20-mer and 18-mer peptides representing this region were tested by pulsing Jurkat/A2Kb cells versus CTL in an IFN-gamma assay.
  • Primary B cells were derived from PBMC of a homozygous HLA-A2 donor by culture in CD40 ligand and IL- 4, transduced at high frequency with recombinant P501S in the vector pBIB, and selected with blastocidin-S.
  • purified CD8+ T cells were cultured with autologous CD40 ligand + IL-4 derived, P501S-transduced B cells in a 96- well microculture format. These CTL microcultures were re-stimulated with P501S-transduced B cells and then assayed for specificity.
  • This Example describes the isolation of certain prostate-specific polypeptides from a prostate tumor cDNA library.
  • a human prostate tumor cDNA expression library as described above was screened using microarray analysis to identify clones that display at least a three fold over-expression in prostate tumor and/or normal prostate tissue, as compared to non-prostate normal tissues (not including testis). 372 clones were identified, and 319 were successfully sequenced. Table I presents a summary of these clones, which are shown in SEQ ID NOs:385-400. Of these sequences SEQ ID NOs:386, 389, 390 and 392 correspond to novel genes, and SEQ ID NOs: 393 and 396 correspond to previously identified sequences. The others (SEQ ID NOs:385, 387, 388, 391, 394, 395 and 397-400) correspond to known sequences, as shown in Table I.
  • CGI-82 showed 4.06 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 43% of prostate tumors, 25% normal prostate, not detected in other normal tissues tested.
  • L-iditol-2 dehydrogenase showed 4.94 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 90%) of prostate tumors, 100%) of normal prostate, and not detected in other normal tissues tested.
  • Ets transcription factor PDEF showed 5.55 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 47% prostate tumors, 25% normal prostate and not detected in other normal tissues tested.
  • hTGRl showed 9.11 fold over-expression in prostate tissues as compared to other normal tissues tested.
  • prostate tumors It was over-expressed in 63% of prostate tumors and is not detected in normal tissues tested including normal prostate.
  • KIAA0295 showed 5.59 fold over- expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 47%) of prostate tumors, low to undetectable in normal tissues tested including normal prostate tissues.
  • Prostatic acid phosphatase showed 9.14 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 67% of prostate tumors, 50% of normal prostate, and not detected in other normal tissues tested.
  • Transglutaminase showed 14.84 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over- expressed in 30% of prostate tumors, 50% of normal prostate, and is not detected in other normal tissues tested.
  • High density lipoprotein binding protein showed 28.06 fold over- expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 97% of prostate tumors, 75% of normal prostate, and is undetectable in all other normal tissues tested.
  • CGI-69 showed 3.56 fold over-expression in prostate tissues as compared to other normal tissues tested. It is a low abundant gene, detected in more than 90% of prostate tumors, and in 75% normal prostate tissues. The expression of this gene in normal tissues was very low.
  • KIAA0122 showed 4.24 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 57% of prostate tumors, it was undetectable in all normal tissues tested including normal prostate tissues.
  • Novel clone 23399 showed 4.09 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 27% of prostate tumors and was undetectable in all normal tissues tested including normal prostate tissues. Novel clone 23320 showed 3.15 fold over- expression in prostate tissues as compared to other normal tissues tested. It was detectable in all prostate tumors and 50%> of normal prostate tissues. It was also expressed in normal colon and trachea. Other normal tissues do not express this gene at high level.
  • This Example describes the use of an electronic subtraction technique to identify prostate-specific antigens.
  • prostate-specific genes present in the GenBank human EST database were identified by electronic subtraction (similar to that described by Vasmatizis et al., Proc. Natl. Acad. Sci. USA 95:300-304, 1998).
  • the sequences of EST clones (43,482) derived from various prostate libraries were obtained from the GenBank public human EST database.
  • Each prostate EST sequence was used as a query sequence in a BLASTN (National Center for Biotechnology Information) search against the human EST database. All matches considered identical (length of matching sequence >100 base pairs, density of identical matches over this region > 70%>) were grouped (aligned) together in a cluster.
  • Clusters containing more than 200 ESTs were discarded since they probably represented repetitive elements or highly expressed genes such as those for ribosomal proteins. If two or more clusters shared common ESTs, those clusters were grouped together into a "supercluster,” resulting in 4,345 prostate superclusters.
  • Each supercluster was analyzed in terms of the ESTs within the supercluster.
  • the tissue source of each EST clone was noted and used to classify the superclusters into four groups: Type 1- EST clones found in the Plus group libraries only; no expression detected in Minus or Other group libraries; Type 2- EST clones derived from the Plus and Other group libraries only; no expression detected in the Minus group; Type 3- EST clones derived from the Plus, Minus and Other group libraries, but the number of ESTs derived from the Plus group is higher than in either the Minus or Other groups; and Type 4- EST clones derived from Plus, Minus and Other group libraries, but the number derived from the Plus group is higher than the number derived from the Minus group.
  • This analysis identified 4,345 breast clusters (see Table III). From these clusters, 3,172 EST clones were ordered from Research Genetics, Inc., and were received as frozen glycerol stocks in 96-well plates.
  • the EST clone inserts were PCR-amplified using amino-linked PCR primers for Synteni microarray analysis. When more than one PCR product was obtained for a particular clone, that PCR product was not used for expression analysis.
  • 2,528 clones from the electronic subtraction method were analyzed by microarray analysis to identify electronic subtraction breast clones that had high levels of tumor vs. normal tissue mRNA. Such screens were performed using a Synteni (Palo Alto, CA) microarray, according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl Acad. Sci. USA 95:10614-10619, 1996 and Heller et al., Proc. Natl.
  • This Example describes the isolation of additional prostate-specific polypeptides from a prostate tumor cDNA library.
  • a human prostate tumor cDNA expression library as described above was screened using microarray analysis to identify clones that display at least a three fold over-expression in prostate tumor and/or normal prostate tissue, as compared to non-prostate normal tissues (not including testis).
  • 142 clones were identified and sequenced. Certain of these clones are shown in SEQ ID NO: 454-467. Of these sequences, SEQ ID NO: 459-461 represent novel genes. The others (SEQ ID NO: 454-458 and 461-467) correspond to known sequences.
  • This Example describes the full length cloning of P710P.
  • the prostate cDNA library described above was screened with the P710P fragment described above.
  • One million colonies were plated on LB/Ampicillin plates. Nylon membrane filters were used to lift these colonies, and the cDNAs picked up by these filters were then denatured and cross-linked to the filters by UV light.
  • the P710P fragment was radiolabeled and used to hybridize with the filters. Positive cDNA clones were selected and their cDNAs recovered and sequenced by an automatic Perkin Elmer/Applied Biosystems Division Sequencer. Four sequences were obtained, and are presented in SEQ ID NO: 468-471 These sequences appear to represent different splice variants of the P710P gene.
  • This example describes the expression and purification of the prostate-specific antigen P501S in E. coli, baculovirus and mammalian cells.
  • DNA amplification was performed using 5 ⁇ l 10X Pfu buffer, 1 ⁇ l 20 mM dNTPs, 1 ⁇ l each of the PCR primers at 10 ⁇ M concentration, 40 ⁇ l water, 1 ⁇ l Pfu DNA polymerase (Stratagene, La Jolla, CA) and 1 ⁇ l DNA at 100 ng/ ⁇ l. Denaturation at 95°C was performed for 30 sec, followed by 10 cycles of 95°C for 30 sec, 60°C for 1 min and by 72°C for 3 min. 20 cycles of 95°C for 30 sec, 65°C for 1 min and by 72°C for 3 min, and lastly by 1 cycle of 72°C for 10 min.
  • the PCR product was cloned to Ral2m/pET17b using Hindlll and EcoRI. The sequence of the resulting fusion construct (referred to as Ral2-P501S-F) was confirmed by DNA sequencing.
  • the fusion construct was transformed into BL21(DE3)pLysE, pLysS and CodonPlus E. coli (Stratagene) and grown overnight in LB broth with kanamycin. The resulting culture was induced with IPTG. Protein was transferred to PVDF membrane and blocked with 5% non-fat milk (in PBS-Tween buffer), washed three times and incubated with mouse anti-His tag antibody (Clontech) for 1 hour. The membrane was washed 3 times and probed with HRP-Protein A (Zymed) for 30 min. Finally, the membrane was washed 3 times and developed with ECL (Amersham). No expression was detected by Western blot.
  • the Ral2-P501S-N fusion construct was used for expression in BL21(DE3)pLysE, pLysS and CodonPlus, essentially as described above. Using Western blot analysis, protein bands were observed at the expected molecular weight of 36 kDa. Some high molecular weight bands were also observed, probably due to aggregation of the recombinant protein. No expression was detected by Western blot when the Ral2-P501S-F fusion was used for expression in BL21 CodonPlus by CE6 phage.
  • a fusion constmct comprising a C-terminal portion of P501S (amino acids 257-553 of SEQ ID NO: 113) located down-stream of the first 30 amino acids of the M. tuberculosis antigen Ral2 (SEQ ID NO: 484) was prepared as follows.
  • P501S DNA was used to perform PCR using the primers AW026 (SEQ ID NO: 488) and AW003 (SEQ ID NO: 486).
  • AW026 is a sense cloning primer that contains a Hindlll site. DNA amplification was performed essentially as described above. The resulting PCR product was cloned to Ral2 in pET17b at the Hindlll and EcoRI sites.
  • Ral2-P501S-C The sequence for the fusion constmct (referred to as Ral2-P501S-C) was confirmed.
  • the Ral2-P501S-C fusion construct was used for expression in BL21(DE3)pLysE, pLysS and CodonPlus, as described above.
  • a small amount of protein was detected by Western blot, with some molecular weight aggregates also being observed.
  • Expression was also detected by Western blot when the Ral2-P501S-C fusion was used for expression in BL21CodonPlus induced by CE6 phage.
  • the Bac-to-Bac baculovirus expression system (BRL Life Technologies, Inc.) was used to express P501S protein in insect cells.
  • Full-length P501S (SEQ ID NO: 113) was amplified by PCR and cloned into the Xbal site of the donor plasmid pFastBacI.
  • the recombinant bacmid and baculovirus were prepared according to the manufacturer's isntructions.
  • the recombinant baculovims was amplified in Sf9 cells and the high titer viral stocks were utilized to infect High Five cells (Invitrogen) to make the recombinant protein.
  • the localization of recombinant P501S in the insect cells was investigated as follows.
  • the insect cells overexpressing P501S were fractionated into fractions of nucleus, mitochondria, membrane and cytosol. Equal amounts of protein from each fraction were analyzed by Western blot with a monoclonal antibody against P501S. Due to the scheme of fractionation, both nucleus and mitochondria fractions contain some plasma membrane components. However, the membrane fraction is basically free from mitochondria and nucleus. P501S was found to be present in all fractions that contain the membrane component, suggesting that P501S may be associated with plasma membrane of the insect cells expressing the recombinant protein.
  • P501S Full-length P501S (553AA) was cloned into various mammalian expression vectors, including pCEP4 (Invitrogen), pVR1012 (Vical, San Diego, CA) and a modified form of the retroviral vector pBMN, referred to as pBIB.
  • Transfection of p501S/pCEP4 into CHO-K cells was carried out using GenePorter transfection reagent (Gene Therapy Systems, San Diego, CA). Briefly, 15 ⁇ l of GenePorter was diluted in 500 ⁇ l of semm-free media and incubated at room temperature for 10 min. The GenePorter/media mixture was added to 2 ⁇ g of plasmid DNA that was diluted in 500 ⁇ l of serum-free media, mixed briefly and incubated for 30 min at room temperature. CHO-K cells were rinsed in PBS to remove semm proteins, and the GenePorter/DNA mix was added and incubated for 5 hours. The transfected cells were then fed an equal volume of 2x media and incubated for 24-48 hours.
  • FACS analysis of P501S transiently infected CHO-K cells demonstrated surface expression of P501S. Expression was detected using rabbit polyclonal antisera raised against a P501S peptide, as described below. Flow cytometric analysis was performed using a FaCScan (Becton Dickinson), and the data were analyzed using the Cell Quest program.
  • a murine monoclonal antibody directed against the carboxy-terminus of the prostate-specific antigen P501S was prepared as follows.
  • a t ncated fragment of P501S (amino acids 355-526 of SEQ ID NO: 113) was generated and cloned into the pET28b vector (Novagen) and expressed in E. coli as a thioredoxin fusion protein with a histidine tag.
  • the trx-P501S fusion protein was purified by nickel chromatography, digested with thrombin to remove the trx fragment and further purified by an acid precipitation procedure followed by reverse phase HPLC.
  • mice were immunized with tmncated P501S protein. Serum bleeds from mice that potentially contained anti-P501S polyclonal sera were tested for P501S-specific reactivity using ELISA assays with purified P501S and trx-P501S proteins. Serum bleeds that appeared to react specifically with P501S were then screened for P501S reactivity by Western analysis. Mice that contained a P501S-specific antibody component were sacrificed and spleen cells were used to generate anti-P501S antibody producing hybridomas using standard techniques. Hybridoma supernatants were tested for P501S-specific reactivity initially by ELISA, and subsequently by FACS analysis of reactivity with P501S transduced cells.
  • a monoclonal hybridoma referred to as 10E3 was chosen for further subcloning.
  • a number of subclones were generated, tested for specific reactivity to P501S using ELISA and typed for IgG isotype. The results of this analysis are shown below in Table V.
  • the monoclonal antibody 10E3-G4-D3 was selected for further study.
  • 10E3-G4-D3 for P501S was examined by FACS analysis. Specifically, cells were fixed (2% formaldehyde, 10 minutes), permeabilized (0.1 %> saponin, 10 minutes) and stained with 10E3-G4-D3 at 0.5 - 1 ⁇ g/ml, followed by incubation with a secondary, FITC-conjugated goat anti-mouse lg antibody (Pharmingen, San Diego, CA). Cells were then analyzed for FITC fluorescence using an Excalibur fluorescence activated cell sorter. For FACS analysis of transduced cells, B-LCL were retrovirally transduced with P501S.
  • B-LCL were infected with a vaccinia vector that expresses P501S.
  • B-LCL transduced with a different antigen (P703P) and uninfected B- LCL vectors were utilized.
  • 10E3-G4-D3 was shown to bind with P501S-transduced B-LCL and also with P501S-infected B-LCL, but not with either uninfected cells or P703P-transduced cells.
  • B-LCL were transduced with P501S or HLA-B8 as a control antigen and either fixed and permeabilized as described above or directly stained with
  • 10E3-G4-D3 for P501S was also demonstrated by Western blot analysis. Lysates from the prostate tumor cell lines Lncap, DU-145 and PC-3, from P501S- transiently transfected HEK293 cells, and from non-transfected HEK293 cells were generated. Western blot analysis of these lysates with 10E3-G4-D3 revealed a 46 kDa immunoreactive band in Lncap, PC-3 and P501 S-transfected HEK cells, but not in DU-145 cells or non-transfected HEK293 cells.
  • P501S mRNA expression is consistent with these results since semi-quantitative PCR analysis revealed that P501S mRNA is expressed in Lncap, to a lesser but detectable level in PC-3 and not at all in DU-145 cells.
  • Bacterially expressed and purified recombinant P501S (referred to as P501SStr2) was recognized by 10E3-G4-D3 (24 kDa), as was full-length P501S that was transiently expressed in HEK293 cells using either the expression vector VR1012 or pCEP4. Although the predicted molecular weight of P501S is 60.5 kDa, both transfected and "native" P501S run at a slightly lower mobility due to its hydrophobic nature.
  • Immunohistochemical analysis was performed on prostate tumor and a panel of normal tissue sections (prostate, adrenal, breast, cervix, colon, duodenum, gall bladder, ileum, kidney, ovary, pancreas, parotid gland, skeletal muscle, spleen and testis). Tissue samples were fixed in formalin solution for 24 hours and embedded in paraffin before being sliced into 10 micron sections. Tissue sections were permeabilized and incubated with 10E3-G4-D3 antibody for 1 hr. HRP-labeled anti-mouse followed by incubation with DAB chromogen was used to visualize P501S immunoreactivity. P501S was found to be highly expressed in both normal prostate and prostate tumor tissue but was not detected in any of the other tissues tested.
  • Purified antibody 10E3-G4-D3 was added at 2 fold dilutions (1000 ng - 16 ng) in PBST and incubated for 30 minutes at room temperature. This was followed by washing 6 times with PBST and subsequently incubating with HRP-conjugated donkey anti-mouse IgG (H+L)Affinipure F(ab') fragment (Jackson Immunoresearch, West Grove, PA) at 1 :20000 for 30 minutes. Plates were then washed and incubated for 15 minutes in tetramethyl benzidine. Reactions were stopped by the addition of IN sulfuric acid and plates were read at 450 nm using an ELISA plate reader. As shown in Fig.
  • a fragment of P503S (amino acids 113-241 of SEQ ID NO: 114) was expressed and purified from bacteria essentially as described above for P501S and used to immunize both rabbits and mice.
  • Mouse monoclonal antibodies were isolated using standard hybridoma technology as described above.
  • Rabbit monoclonal antibodies were isolated using Selected Lymphocyte Antibody Method (SLAM) technology at Immgenics Pharmaceuticals (Vancouver, BC, Canada). Table VI, below, lists the monoclonal antibodies that were developed against P503S.
  • SLAM Selected Lymphocyte Antibody Method
  • CDRs complementarity determining regions
  • PBST PBS containing 0.1% Tween 20
  • Purified rabbit monoclonal antibodies diluted in PBST were added to the wells and incubated for 30 min at room temperature. This was followed by washing 6 times with PBST and incubation with Protein-A HRP conjugate at a 1 :2000 dilution for a further 30 min. Plates were washed six times in PBST and incubated with tetramethylbenzidine (TMB) substrate for a further 15 min. The reaction was stopped by the addition of IN sulfuric acid and plates were read at 450 nm using at ELISA plate reader. ELISA with the mouse monoclonal antibodies was performed with supernatants from tissue culture run neat in the assay.
  • TMB tetramethylbenzidine
  • the antibodies were tested by FACS analysis on a cell line that stably expressed P503S to confirm that the antibodies bind to cell surface epitopes.
  • Cells stably transfected with a control plasmid were employed as a negative control.
  • Cells were stained live with no fixative.
  • 0.5 ug of anti-P503S monoclonal antibody was added and cells were incubated on ice for 30 min before being washed twice and incubated with a FITC-labelled goat anti-rabbit or mouse secondary antibody for 20 min. After being washed twice, cells were analyzed with an Excalibur fluorescent activated cell sorter.
  • the monoclonal antibodies 1C3, 1D12, 9C12, 20D4 and JAl, but not 8D3, were found to bind to a cell surface epitope of P503S.
  • P503S was found to be highly expressed in prostate tissue, with lower levels of expression being observed in cervix, colon, ileum and kidney, and no expression being observed in adrenal, breast, duodenum, gall bladder, ovary, pancreas, parotid gland, skeletal muscle, spleen and testis.
  • the monoclonal antibody 20D4 detected the appropriate molecular weight 14 kDa recombinant P503S (amino acids 113-241) and the 23.5 kDa species in the HEK293 cell lysates transfected with full length P503S.
  • Other anti-P503S monoclonal antibodies displayed similar specificity by Western blot.
  • Rabbits were immunized with either a tmncated (P703Ptrl; SEQ ID NO: 172) or full-length mature form (P703Pfl; SEQ ID NO: 523) of recombinant P703P protein was expressed in and purified from bacteria as described above. Affinity purified polyclonal antibody was generated using immunogen P703Pfl or P703Ptrl attached to a solid support. Rabbit monoclonal antibodies were isolated using SLAM technology at Immgenics Pharmaceuticals. Table VII below lists both the polyclonal and monoclonal antibodies that were generated against P703P.
  • CDRs complementarity determining regions
  • the polyclonal antibody 2594 was found to bind to the peptides of SEQ ID NO: 511-514, with the polyclonal antibody 9427 binding to the peptides of SEQ ID NO: 515-517.
  • the specificity of the anti-P703P antibodies was determined by Western blot analysis as follows. SDS-PAGE was performed on (1) bacterially expressed recombinant antigen;
  • lysates and supernatants from cells transfected with full length P703P a 30 kDa band corresponding to P703P was observed.
  • lysates from HEK293 cells stably transfected with a control plasmid were also tested and were negative for P703P expression.
  • Other anti-P703P antibodies showed similar results. Immunohistochemical studies were performed as described above, using anti-P703P monoclonal antibody. P703P was found to be expressed at high levels in normal prostate and prostate tumor tissue but was not detectable in all other tissues tested (breast tumor, lung tumor and normal kidney).
  • This example describes studies demonstrating that the prostate-specific antigen P501S is expressed on the surface of cells, together with studies to determine the probable chromosomal location of P501S.
  • the protein P501S (SEQ ID NO: 113) is predicted to have 11 transmembrane domains. Based on the discovery that the epitope recognized by the anti-P501S monoclonal antibody 10E3-G4-D3 (described above in Example 17) is intracellular, it was predicted that following transmembrane determinants would allow the prediction of extracellular domains of P501S.
  • Fig. 9 is a schematic representation of the P501S protein showing the predicted location of the transmembrane domains and the intracellular epitope described in Example 17. Underlined sequence represents the predicted transmembrane domains, bold sequence represents the predicted extracellular domains, and italized sequence represents the predicted intracellular domains.
  • Sequence that is both bold and underlined represents sequence employed to generate polyclonal rabbit serum.
  • the location of the transmembrane domains was predicted using HHMTOP as described by Tusnady and Simon (Principles Governing Amino Acid Composition of Integral Membrane Proteins: Applications to Topology Prediction, J. Mol. Biol 255:489-506, 1998).
  • the P501S domain flanked by the transmembrane domains corresponding to amino acids 274-295 and 323-342 is predicted to be extracellular.
  • the peptide of SEQ ID NO: 518 corresponds to amino acids 306-320 of P501S and lies in the predicted extracellular domain.
  • the peptide of SEQ ID NO: 519 which is identical to the peptide of SEQ ID NO: 518 with the exception of the substitution of the histidine with an asparginine, was synthesized as described above. A Cys-Gly was added to the C-terminus of the peptide to facilitate conjugation to the carrier protein.
  • Cleavage of the peptide from the solid support was carried out using the following cleavage mixture: trifluoroacetic acid:ethanediol:thioanisol:water:phenol (40:1 :2:2:3). After cleaving for two hours, the peptide was precipitated in cold ether. The peptide pellet was then dissolved in 10% v/v acetic acid and lyophilized prior to purification by C18 reverse phase hplc. A gradient of 5-60% acetonitrile (containing 0.05% TFA) in water (containing 0.05% TFA) was used to elute the peptide. The purity of the peptide was verified by hplc and mass spectrometry, and was determined to be >95%. The purified peptide was used to generate rabbit polyclonal antisera as described above.
  • P501S surface expression of P501S was examined by FACS analysis.
  • Cells were stained with the polyclonal anti-P501S peptide serum at 10 ⁇ g/ml, washed, incubated with a secondary FITC-conjugated goat anti-rabbit lg antibody (ICN), washed and analyzed for FITC fluorescence using an Excalibur fluorescence activated cell sorter.
  • ICN secondary FITC-conjugated goat anti-rabbit lg antibody
  • B- LCL were retrovirally transduced with P501S.
  • B-LCL transduced with an irrelevant antigen (P703P) or nontransduced were stained in parallel.
  • Lncap, PC-3 and DU-145 were utilized.
  • Prostate tumor cell lines were dissociated from tissue culture plates using cell dissociation medium and stained as above. All samples were treated with propidium iodide (PI) prior to FACS analysis, and data was obtained from Pi-excluding (i.e. intact and non-permeabilized) cells.
  • the rabbit polyclonal semm generated against the peptide of SEQ ID NO: 519 was shown to specifically recognize the surface of cells transduced to express P501S, demonstrating that the epitope recognized by the polyclonal serum is extracellular.
  • peripheral membranes from Lncap cells were isolated and subjected to Western blot analysis.
  • Lncap cells were lysed using a dounce homogenizer in 5 ml of homogenization buffer (250 mM sucrose, 10 mM HEPES, lmM EDTA, pH 8.0, 1 complete protease inhibitor tablet (Boehringer Mannheim)). Lysate samples were spun at 1000 g for 5 min at 4 °C. The supernatant was then spun at 8000g for 10 min at 4 °C. Supernatant from the 8000g spin was recovered and subjected to a 100,000g spin for 30 min at 4 °C to recover peripheral membrane.
  • homogenization buffer 250 mM sucrose, 10 mM HEPES, lmM EDTA, pH 8.0, 1 complete protease inhibitor tablet (Boehringer Mannheim)
  • FACS analysis was employed, utilizing either B-LCL transduced with P501S or the irrelevant antigen P703P, of B-LCL infected with vaccinia vims- expressing P501S.
  • For surface expression dead and non-intact cells were excluded from the analysis as described above.
  • For intracellular staining cells were fixed and permeabilized as described above. Rabbit polyclonal serum generated against the peptide of SEQ ID NO: 548, which corresponds to amino acids 181-198 of P501S, was found to recognize a surface epitope of P501S.
  • the chromosomal location of P501S was determined using the GeneBridge 4 Radiation Hybrid panel (Research Genetics).
  • the PCR primers of SEQ ID NO: 528 and 529 were employed in PCR with DNA pools from the hybrid panel according to the manufacturer's directions. After 38 cycles of amplification, the reaction products were separated on a 1.2% agarose gel, and the results were analyzed through the Whitehead Institute/MIT Center for Genome Research web server (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) to determine the probable chromosomal location.
  • P501S was mapped to the long arm of chromosome 1 at WI-9641 between q32 and q42.

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