CA2613125A1 - Compositions and methods for therapy and diagnosis of prostate cancer - Google Patents

Abstract

Compositions and methods for the therapy and diagnosis of cancer, such as prostate cancer, are disclosed. Compositions may comprise one or more prostate tumor proteins, immunogenic portions thereof, or polynucleotides that encode such portions. Alternatively, a therapeutic composition may comprise an antigen presenting cell that expresses a prostate tumor protein, or a T cell that is specific for cells expressing such a protein. Such compositions may be used, for example, for the prevention and treatment of diseases such as prostate cancer. Diagnostic methods based on detecting a prostate tumor protein, or mRNA encoding such a protein, in a sample are also provided.

Description

DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

CECI EST LE TOME DE _2 NOTE: Pour les tomes additionels, veiliez contacter le Bureau Canadien des Brevets.

JUMBO APPLICATIONS / PATENTS

THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.

NOTE: For additional volumes please contact the Canadian Patent Office.

COMPOSITIONS AND METHODS FOR THERAPY AND
DIAGNOSIS OF PROSTATE CANCER

TECHNICAL FIELD

The present invention relates generally to therdpy and diagnosis of cancer, such as''prostate cancer. The invention is more specifically related to polypeptides comprising at least a portion of a prostate tumor protein, and to polynucleotides encoding such polypeptides. Such 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.

BACKGROUND OF THE INVENTION

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 zv:.acnce 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.
In spite of considerable research into therapies for the disease, prostate cancer remains difficult to treat. Commonly, treatment is based on surgery and/or radiation therapy, but these methods are ineffective in a significant percentage of cases. Two previously identified prostate specific proteins - prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) - have limited therapeutic and diagnostic potential. For example, PSA
levels do not always correlate well with the presence of prostate cancer, being positive in a percentage of non-prostate cancer cases, including benign prostatic hyperplasia (BPH).
Furthermore, PSA measurements correlate with prostate volume, and do not indicate the level of metastasis.
In spite of considerable research into therapies for these and other cancers, prostate cancer remains difficult to diagnose and treat effectively.
Accordingly, there is a need in the art for improved methods for detecting and treating such cancers.
The present invention fulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and, methods for the diagnosis and therapy of cancer, such as prostate cancer. In one aspect, the present 2 invention provides polypeptides comprising at least a portion of a prostate tumor 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. Within certain embodiments, the polypeptide comprises at least an immunogenic portion of a prostate tumor protein, or a variant thereof, wherein the tumor 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:I-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 or 384-472; (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). In certain specific embodiments, such a polypeptide comprises at least a portion, or variant thereof, of a tumor 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 and 383.
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 tumor protein), expression vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.
Within other aspects, the present invention provides pharmaceutical compositions comprising a polypeptide or polynucleotide as described above and a physiologically acceptable carrier.
Within a related aspect of the present invention, vaccines are provided. Such vaccines comprise a polypeptide or polynucleotide as described above and a non-specific immune response enhancer.
The present invention further provides pharmaceutical compositions that comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to a prostate tumor protein; and (b) a physiologically acceptable carrier.
Within further aspects, 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.
Within related aspects, vaccines are provided that comprise: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) a non-specific immune response enhancer.
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.

Within related aspects, 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 a non-specific immune response enhancer.
Within further aspects, 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 fi.nther 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 tumor 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.
Within related aspects, methods are provided 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 tumor 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.
Within further aspects, 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 tumor 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.
Within further aspects, 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.
Within preferred embodiments, 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 monitoririg 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 tumor 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. Within certain embodiments, 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. Within other embodiments, 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.
In related aspects, methods are provided 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 tumor 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.
Within further aspects, 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.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS

Figure 1 illustrates the ability of T cells to kill fibroblasts expressing the representative prostate tumor 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 tumor polypeptide P502S. In each case, the number of y-interferon spots is shown for different numbers of responders. In Figure 2A, data is presented for fibroblasts pulsed with the P2S-12 peptide, as compared to fibroblasts pulsed with a control E75 peptide. In Figure 2B, data is presented for fibroblasts expressing P502S, as compared to fibroblasts expressing HER-21neu.
Figure 3 represents a peptide competition binding assay showing that the P I S# 10 peptide, derived from P501 S, binds HLA-A2. Peptide P 1 S# 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 P 1 S# 10 immunized mice to specifically lyse PIS#10-pulsed Jurkat A2Kb targets and 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 tumor polypeptide P501 S, thereby demonstrating that the PIS#10 peptide may be a naturally processed epitope of the P501 S polypeptide.
Figures 6A and 6B are graphs illustrating the specificity of a CD8+ cell line (3A-1) for a representative prostate tumor antigen (P501 S). Figure 6A shows the results of a 5'Cr release assay. The percent specific lysis is shown as a series of effector:target ratios, as indicated. Figure 6B shows the production of interferon-gamma by 3A-1 cells stimulated with autologous B-LCL transduced with P501 S, at varying effector:target rations as indicated.
SEQ ID NO: I is the determined cDNA sequence for F 1-13 SEQ ID NO: 2 is the determined 3' cDNA sequence for F1-12 SEQ ID NO: 3 is the determined 5' cDNA sequence for F I-12 SEQ ID NO: 4 is the determined 3' cDNA sequence for FI-16 SEQ ID NO: 5 is the determined 3' cDNA sequence for H 1-1 SEQ ID NO: 6 is the determined 3' eDNA sequence for H 1-9 SEQ ID NO: 7 is the determined 3' cDNA sequence for H1-4 SEQ ID NO: 8 is the determined 3' cDNA sequence for J1-17 SEQ ID NO: 9 is the determined 5' cDNA sequence for J I-17 SEQ ID NO: 10 is the determined 3' cDNA sequence for L1-12 SEQ ID NO: I 1 is the determined 5' cDNA sequence for L1-12 SEQ ID NO: 12 is the determined 3' cDNA sequence for N1-1862 SEQ ID NO: 13 is the determined 5' cDNA sequence for N1-1862 SEQ ID NO: 14 is the determined 3' cDNA sequence for J1-13 SEQ ID NO: 15 is the determined 5' cDNA sequence for J 1-13 SEQ ID NO: 16 is the determined 3' cDNA sequence for J1-19 SEQ ID NO: 17 is the determined 5' cDNA sequence for J1-19 SEQ ID NO: 18 is the determined 3' cDNA sequence for J1-25 SEQ ID NO: 19 is the determined 5' cDNA sequence for J 1-25 SEQ ID NO: 20 is the determined 5' cDNA sequence for J1.-24 SEQ ID NO: 21 is the determined 3' cDNA sequence for J 1-24 SEQ ID NO: 22 is the determined 5' cDNA sequence for K1-58 SEQ ID NO: 23 is the determined 3' cDNA sequence for K1-58 SEQ ID NO: 24 is the determined 5' cDNA sequence for K1.-63 SEQ ID NO: 25 is the determined 3' cDNA sequence for K1-63 SEQ ID NO: 26 is the determined 5' cDNA sequence for L1-4 SEQ ID NO: 27 is the determined 3' cDNA sequence for L 1-4 SEQ ID NO: 28 is the determined 5' cDNA sequence for L1-14 SEQ ID NO: 29 is the determined 3' cDNA sequence for L1-14 SEQ ID NO: 30 is the determined 3' cDNA sequence for J 1-12 SEQ ID NO: 31 is the determined 3' cDNA sequence for J1-16 SEQ ID NO: 32 is the determined 3' cDNA sequence for J1-21 SEQ ID NO: 33 is the determined 3' eDNA sequence for KI-48 SEQ ID NO: 34 is the determined 3' cDNA sequence for KI-55 SEQ ID NO: 35 is the determined 3' cDNA sequence for L1-2 SEQ ID NO: 36 is the determined 3' cDNA sequence for L1-6 SEQ ID NO: 37 is the determined 3' cDNA sequence for N1-1858 SEQ ID NO: 38 is the determined 3' cDNA sequence forNl-1860 SEQ ID NO: 39 is the determined 3' cDNA sequence for N1-1861 SEQ ID NO: 40 is the determined 3' cDNA sequence for N1-1864 SEQ ID NO: 41 is the determined cDNA sequence for P5 SEQ ID NO: 42 is the determined cDNA sequence for P8 SEQ ID NO: 43 is the determined cDNA sequence for P9 SEQ ID NO: 44 is the determined cDNA sequence for P18 SEQ ID NO: 45 is the determined eDNA sequence for P20 SEQ ID ~10: 46 is the determined cDNA sequence for P29 SEQ ID NO: 47 is the determined cDNA sequence for P30 SEQ ID NO: 48 is the determined cDNA sequence for P34 SEQ ID NO: 49 is the determined cDNA sequence for P36 SEQ ID NO: 50 is the determined cDNA sequence for P38 SEQ ID NO: 51 is the determined cDNA sequence for P39 SEQ ID NO: 52 is the determined cDNA sequence for P42 SEQ ID NO: 53 is the determined cDNA sequence for P47 SEQ ID NO: 54 is the determined cDNA sequence for P49 SEQ ID NO: 55 is the determined cDNA sequence for P50 SEQ ID NO: 56 is the determined cDNA sequence for P53 SEQ ID NO: 57 is the determined cDNA sequence for P55 SEQ ID NO: 58 is the determined cDNA sequence for P60 SEQ ID NO: 59 is the determined cDNA sequence for P64 SEQ ID NO: 60 is the determined cDNA sequence for P65 SEQ ID NO: 61 is the determined cDNA sequence for P73 SEQ ID NO: 62 is the determined eDNA sequence for P75 SEQ ID NO: 63 is the determined cDNA sequence for P76 SEQ ID NO: 64 is the determined cDNA sequence for P79 SEQ ID NO: 65 is the determined cDNA sequence for P84 SEQ ID NO: 66 is the determined cDNA sequence for P68 SEQ ID NO: 67 is the determined cDNA sequence for P80 SEQ ID NO: 68 is the determined cDNA sequence for P82 SEQ ID NO: 69 is the determined eDNA sequence for U1-3064 SEQ ID NO: 70 is the determined cDNA sequence for U1-3065 SEQ ID NO: 71 is the determined cDNA sequence for V 1-3692 SEQ ID NO: 72 is the determined cDNA sequence for 1A-3905 SEQ ID NO: 73 is the determined cDNA sequence for V1-3686 SEQ ID NO: 74 is the determined cDNA sequence for R1-2330 SEQ ID NO: 75 is the determined cDNA sequence for 1 B-3976 SEQ ID NO: 76 is the determined cDNA sequence for V 1-3679 SEQ ID NO: 77 is the determined cDNA sequence forl G-4736 SEQ ID NO: 78 is the determined cDNA sequence for 1G-4738 SEQ ID NO: 79 is the determined cDNA sequence for 1 G-4741 SEQ ID NO: 80 is the determined cDNA sequence for 1 G-4744 SEQ ID NO: 81 is the determined cDNA sequence for 1 G-4734 SEQ ID NO: 82 is the determined cDNA sequence for 1 H-4774 SEQ ID NO: 83 is the determined eDNA sequence for 1H-4781 SEQ ID NO: 84 is the determined cDNA sequence for 1 H-4785 SEQ ID NO: 85 is the determined cDNA sequence for 1 H-4787 SEQ ID NO: 86 is the determined cDNA sequence for 1H-4796 SEQ ID NO: 87 is the determined cDNA sequence for 11-4807 SEQ ID NO: 88 is the determined cDNA sequence for 1I-4810 SEQ ID NO: 89 is the determined cDNA sequence for 11-4811 SEQ ID NO: 90 is the determined cDNA sequence for 1 J-4876 SEQ ID NO: 91 is the determined cDNA sequence for 1 K-4884 SEQ ID NO: 92 is the determined cDNA sequence for 1 K-4896 SEQ ID NO: 93 is the determined cDNA sequence for 1 G-4761 SEQ ID NO: 94 is the determined cDNA sequence for I G-4762 SEQ ID NO: 95 is the determined cDNA sequence for 1 H-4766 SEQ ID NO: 96 is the determined cDNA sequence for 1H-4770 SEQ ID NO: 97 is the determined cDNA sequence for 1H-4771 SEQ ID NO: 98 is the determined cDNA sequence for IH-4772 SEQ ID NO: 99 is the determined cDNA sequence for 1 D-4297 SEQ ID NO: 100 is the determined cDNA sequence for 1D-4309 SEQ ID NO: 101 is the determined cDNA sequence for 1 D.1-4278 SEQ ID NO: 102 is the determined cDNA sequence for 1 D-4288 SEQ ID NO: 103 is the determined cDNA sequence for 1 D-4283 SEQ ID NO: 104 is the determined cDNA sequence for 1 D-4304 SEQ ID NO: 105 is the determined cDNA sequence for 1 D-4296 SEQ ID NO: 106 is the determined cDNA sequence for 1 D-4280 SEQ ID NO: 107 is the determined full length cDNA sequence for F 1-12 (also referred to as P504S) SEQ ID NO: 108 is the predicted amino acid sequence for F1-12 SEQ ID NO: 109 is the determined full length cDNA sequence for J1-17 SEQ ID NO: I 10 is the determined full length eDNA sequence for L1-12 SEQ ID NO: 111 is the determined full length cDNA sequence for N 1-1862 SEQ ID NO: 112 is the predicted amino acid sequence for J 1-17 SEQ ID NO: 113 is the predicted amino acid sequence for L1-12 SEQ ID NO: 114 is the predicted amino acid sequence for N 1-1862 SEQ ID NO: 115 is the determined cDNA sequence for P89 SEQ ID NO: 116 is the determined cDNA sequence for P90 SEQ ID NO: 117 is the determined cDNA sequence for P92 SEQ ID NO: 118 is the determined eDNA sequence for P95 SEQ ID N0: 119 is the determined cDNA sequence for P98 SEQ ID NO: 120 is the determined cDNA sequence for P102 SEQ ID NO: 121 is the determined cDNA sequence for P110 SEQ ID NO: 122 is the determined cDNA sequence for P 111 SEQ ID NO: 123 is the determined cDNA sequence for P114 SEQ ID NO: 124 is the determined cDNA sequence for Pl 15 SEQ ID NO: 125 is the determined cDNA sequence for P116 SEQ ID NO: 126 is the determined cDNA sequence for P124 SEQ ID NO: 127 is the determined cDNA sequence for P126 SEQ ID NO: 128 is the detennined cDNA sequence for P130 SEQ ID NO: 129 is the deternlined cDNA sequence for P133 SEQ ID NO: 130 is the determined cDNA sequence for P138 SEQ ID NO: 131 is the determined cDNA sequence for P143 SEQ ID NO: 132 is the determined cDNA sequence for P151 SEQ ID NO: 133 is the determined cDNA sequence for P156 SEQ ID NO: 134 is the determined cDNA sequence for P157 SEQ ID NO: 135 is the determined cDNA sequence for P166 SEQ ID NO: 136 is the determined cDN.A sequence for P176 SEQ ID NO: 137 is the determined cDNA sequence for P178 SEQ ID NO: 138 is the determined cDNA sequence for P179 SEQ ID NO: 139 is the determined cDNA sequence for P185 SEQ ID NO: 140 is the determined cDNA sequence for P192 SEQ ID NO: 141 is the determined cDNA sequence for P201 SEQ ID NO: 142 is the determined cDNA sequence for P204 SEQ ID NO: 143 is the determined cDNA sequence for P208 SEQ ID NO: 144 is the deternined cDNA sequence for P211 SEQ ID NO: 145 is the determined cDNA sequence for P213 SEQ ID NO: 146 is the determined cDNA sequence for P219 SEQ ID NO: 147 is the determined cDNA sequeiice for P237 SEQ ID NO: 148 is the determined cDNA sequence for P239 SEQ ID NO: 149 is the determined cDNA sequence for P248 SEQ ID NO: 150 is the determined cDNA sequence for P251 SEQ ID NO: 151 is the determined cDNA sequence for P255 SEQ ID NO: 152 is the determined cDNA sequence for P256 SEQ ID NO: 153 is the determined cDNA sequence for P259 SEQ ID NO: 154 is the determined cDNA sequence for P260 SEQ ID NO: 155 is the determined cDNA sequence for P263 SEQ ID 140: 156 is the determined cDNA sequence for P264 SEQ ID NO: 157 is the determined cDNA sequence for P266 SEQ ID NO: 158 is the determined cDNA sequence for P270 SEQ ID NO: 159 is the determined cDNA sequence for P272 SEQ ID NO: 160 is the determined cDNA sequence for P278 SEQ ID NO: 161 is the determined cDNA sequence for P105 SEQ ID NO: 162 is the determined cDNA sequence for P107 SEQ ID NO: 163 is the determined cDNA sequence for P137 SEQ ID NO: 164 is the determined cDNA sequence for P194 SEQ ID NO: 165 is the determined cDNA sequence for P195 SEQ ID NO: 166 is the determined cDNA sequence for P196 SEQ ID NO: 167 is the determined cDNA sequence for P220 SEQ ID NO: 168 is the determined cDNA sequence for P234 SEQ ID NO: 169 is the determined cDNA sequence for P235 SEQ ID NO: 170 is the determined cDNA sequence for P243 SEQ ID NO: 171 is the determined cDNA sequence for P703P-DEI
SEQ ID NO: 172 is the predicted amino acid sequence for P703P-DEI
SEQ ID NO: 173 is the determined cDNA sequence for P703P-DE2 SEQ ID NO: 174 is the determined cDNA sequence for P703P-DE6 SEQ ID NO: 175 is the determined cDNA sequence for P703P-DE13 SEQ ID NO: 176 is the predicted amino acid sequence for P703P-DE13 SEQ ID NO: 177 is the determined cDNA sequence for P703P-DE14 SEQ ID NO: 178 is the predicted amino acid sequence for P703P-DE14 SEQ ID NO: 179 is the determined extended cDNA sequence for 1 G-4736 SEQ ID NO: 180 is the determined extended cDNA sequence for 1G-4738 SEQ ID NO: 181 is the determined extended cDNA sequence for 1 G-4741 SEQ ID NO: 182 is the determined extended cDNA sequence for 1 G-4744 SEQ ID NO: 183 is the determined extended cDNA sequence for 1H-4774 SEQ ID NO: 184 is the determined extended cDNA sequence for I H-4781 SEQ ID NO: 185 is the determined extended cDNA sequence for I H-4785 SEQ ID NO: 186 is the determined extended cDNA sequence for I H-4787 SEQ ID NO: 187 is the determined extended cDNA sequence for 1H-4796 SEQ ID NO: 188 is the determined extended cDNA sequence for 11-4807 SEQ ID NO: 189 is the determined 3' cDNA sequence for 1I-4810 SEQ ID NO: 190 is the determined 3' cDNA sequence for 11-4811 SEQ ID NO: 191 is the determined extended cDNA sequence for 1J-4876 SEQ ID NO: 192 is the determined extended cDNA sequence for 1 K-4884 SEQ ID N,O: 193 is the determined extended cDNA sequence for 1 K-4896 SEQ ID NO: 194 is the determined extended cDNA sequence for 1 G-4761 SEQ ID NO: 195 is the determined extended cDNA sequence for I G-4762 SEQ ID NO: 196 is the determined extended cDNA sequence for 1 H-4766 SEQ ID NO: 197 is the determined 3' cDNA sequence for 1 H-4770 SEQ ID NO: 198 is the determined 3' cDNA sequence for 1 H-4771 SEQ ID NO: 199 is the determined extended cDNA sequence for 1 H-4772 SEQ ID NO: 200 is the determined extended cDNA sequence for I D-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 1 D-4288 SEQ ID NO: 203 is the determined extended cDNA sequence for I D-4283 SEQ ID NO: 204 is the determined extended cDNA sequence for 1 D-4304 SEQ ID NO: 205 is the determined extended cDNA sequence for I D-4296 SEQ ID NO: 206 is the determined extended cDNA sequence for 1 D-4280 SEQ ID NO: 207 is the determined cDNA sequence for 10-d8fwd SEQ ID NO: 208 is the determined cDNA sequence for 10-H10con 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 eDNA 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 eDNA 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 Irev SEQ ID NO: 221 is the determined cDNA sequence for g-fl 2fwd 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-A 11 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 JPTPNI3 SEQ ID 1N.O: 230 is the determined cDNA sequence for JPTPN14 SEQ ID NO: 231 is the determined eDNA sequence for JPTPN23 SEQ ID NO: 232 is the determined cDNA sequence for JPTPN24 SEQ ID NO: 233 is the determined cDNA sequence for JPTPN25 SEQ ID NO: 234 is the determined cDNA sequence for JPTPN30 SEQ ID NO: 235 is the determined cDNA sequence for JPTPN34 SEQ ID NO: 236 is the determined cDNA sequence for PTPN35 SEQ ID NO: 237 is the determined cDNA sequence for JPTPN36 SEQ ID NO: 238 is the determined cDNA sequence for JPTPN38 SEQ ID NO: 239 is the determined cDNA sequence for JPTPN39 SEQ ID NO: 240 is the determined cDNA sequence for JPTPN40 SEQ ID NO: 241 is the determined cDNA sequence for JPTPN41 SEQ ID NO: 242 is the determined cDNA sequence for JPTPN42 SEQ ID NO: 243 is the determined cDNA sequence for JPTPN45 SEQ ID NO: 244 is the determined cDNA sequence for JPTPN46 SEQ ID NO: 245 is the determined cDNA sequence for JPTPN5I
SEQ ID NO: 246 is the determined cDNA sequence for JPTPN56 SEQ ID NO: 247 is the determined eDNA sequence for PTPN64 SEQ ID NO: 248 is the determined eDNA sequence for JPTPN65 SEQ ID NO: 249 is the determined cDNA sequence for JPTPN67 SEQ ID NO: 250 is the determined cDNA sequence for JPTPN76 SEQ ID NO: 251 is the deternnined cDNA sequence for JPTPN84 SEQ ID NO: 252 is the determined cDNA sequence for JPTPN85 SEQ ID NO: 253 is the determined cDNA sequence for JPTPN86 SEQ ID NO: 254 is the determined cDNA sequence for JPTPN87 SEQ ID NO: 255 is the determined cDNA sequence for JPTPN88 SEQ ID NO: 256 is the determined cDNA sequence for JP1F1 SEQ ID NO: 257 is the determined cDNA sequence for JPIF2 SEQ ID NO: 258 is the determined cDNA sequence for JP 1 C2 SEQ ID NO: 259 is the determined cDNA sequence for JP1B1 SEQ ID NO: 260 is the determined cDNA sequence for JP1B2 SEQ ID NO: 261 is the determined cDNA sequence for JPID3 SEQ ID NO: 262 is the determined eDNA sequence for JP1A4 SEQ ID NO: 263 is the determined cDNA sequence for JP1 F5 SEQ ID NO: 264 is the determined cDNA sequence for JPIE6 SEQ ID NO: 265 is the determined cDNA sequence for JPID6 SEQ ID NO: 266 is the determined cDNA sequence for JPIB5 SEQ ID W: 267 is the determined cDNA sequence for JP1A6 SEQ ID NO: 268 is the determined cDNA sequence for JPIE8 SEQ ID NO: 269 is the determined cDNA sequence for JP 1 D7 SEQ ID NO: 270 is the determined cDNA sequence for JPI D9 SEQ ID NO: 271 is the determined cDNA sequence for JPIC10 SEQ ID NO: 272 is the determined cDNA sequence for JP1A9 SEQ ID NO: 273 is the determined cDNA sequence for JP1F12 SEQ ID NO: 274 is the determined cDNA sequence for JP1E12 SEQ ID NO: 275 is the determined cDNA sequence for JPID11 SEQ ID NO: 276 is the determined cDNA sequence for JP 1 C 11 SEQ ID NO: 277 is the determined cDNA sequence for JP1C12 SEQ ID NO: 278 is the determined cDNA sequence for JP I B 12 SEQ ID NO: 279 is the determined cDNA sequence for JP1A12 SEQ ID NO: 280 is the determined cDNA sequence for JP8G2 SEQ ID NO: 281 is the determined cDNA sequence for JP8HI
SEQ ID NO: 282 is the determined cDNA sequence for JP8H2 SEQ ID NO: 283 is the determined eDNA sequence for JP8A3 SEQ ID NO: 284 is the determined cDNA sequence for JP8A4 SEQ ID NO: 285 is the determined cDNA sequence for JP8C3 SEQ ID NO: 286 is the determined cDNA sequence for JP8G4 SEQ ID NO: 287 is the determined cDNA sequence for JP8B6 SEQ ID NO: 288 is the determined cDNA sequence for JP8D6 SEQ ID NO: 289 is the determined cDNA sequence for JP8F5 SEQ ID NO: 290 is the determined eDNA sequence for JP8A8 SEQ ID NO: 291 is the determined cDNA sequence for JP8C7 SEQ ID NO: 292 is the determined cDNA sequence for JP8D7 SEQ ID NO: 293 is the determined cDNA sequence for P8D8 SEQ ID NO: 294 is the determined cDNA sequence for JP8E7 SEQ ID NO: 295 is the determined cDNA sequence for JP8F8 SEQ ID NO: 296 is the determined cDNA sequence for JP8G8 SEQ ID NO: 297 is the determined cDNA sequence for JP8B10 SEQ ID NO: 298 is the determined cDNA sequence for JP8C10 SEQ ID NO: 299 is the determined cDNA sequence for JP8E9 SEQ ID NO: 300 is the determined cDNA sequence for JP8E10 SEQ ID NO: 301 is the determined cDNA sequence for JP8F9 SEQ ID NO: 302 is the determined eDNA sequence for JP8H9 SEQ ID NO: 303 is the determined cDNA sequence for JP8C12 SEQ ID I~D: 304 is the determined cDNA sequence for JP8E11 SEQ ID NO: 305 is the determined cDNA sequence for JP8E12 SEQ ID NO: 306 is the amino acid sequence for the peptide PS2412 SEQ ID NO: 307 is the determined cDNA sequence for P711 P
SEQ ID NO: 308 is the determined cDNA sequence for P712P
SEQ ID NO: 309 is the determined cDNA sequence for CLONE23 SEQ ID NO: 310 is the determined cDNA sequence for P774P
SEQ ID NO: 311 is the determined cDNA sequence for P775P
SEQ ID NO: 312 is the determined cDNA sequence for P715P
SEQ ID NO: 313 is the determined cDNA sequence for P710P
SEQ ID NO: 314 is the determined cDNA sequence for P767P
SEQ ID NO: 315 is the determined cDNA sequence for P768P
SEQ ID NO: 316-325 are the determined cDNA sequences of previously isolated genes SEQ ID NO: 326 is the determined cDNA sequence for P703PDE5 SEQ ID NO: 327 is the predicted amino acid sequence for P703PDE5 SEQ ID NO: 328 is the determined cDNA sequence for P703P6.26 SEQ ID NO: 329 is the predicted amino acid sequence for P703P6.26 SEQ ID NO: 330 is the determined cDNA sequence for P703PX-23 SEQ ID NO: 331 is the predicted amino acid sequence for P703PX-23 SEQ ID NO: 332 is the determined full length cDNA sequence for P509S
SEQ ID NO: 333 is the determined extended cDNA sequence for P707P (also referred to as 11-C9) SEQ ID NO: 334 is the determined eDNA 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 P1 S#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

15 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 deten-nined 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 P781 P
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: 366.
SEQ ID NO: 377 is the predicted amino acid sequence encoded by the sequence of SEQ ID
NO: 372.
SEQ ID NO: 378 is the predicted amino acid sequence encoded by the sequence of SEQ ID
NO: 373.

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: 375.
SEQ ID NO: 381 is the determined cDNA sequence for B716P.
SEQ ID NO: 382 is the determined full-length cDNA sequence for P711P.
SEQ ID N'O: 383 is the predicted amino acid sequence for P711P.
SEQ ID NO: 384 is the cDNA sequence for P1000C.
SEQ ID NO: 385 is the cDNA sequence for CGI-82.
SEQ ID NO:386 is the eDNA 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 22548.
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:411 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 N0:417 is the cDNA sequence for 22573.
SEQ ID N0:418 is the cDNA sequence for 22575.
SEQ ID N0:419 is the cDNA sequence for 22580.
SEQ ID 1V0:420 is the cDNA sequence for 22581.
SEQ ID N0:421 is the cDNA sequence for 22582.
SEQ ID N0:422 is the cDNA sequence for 22583.
SEQ ID N0:423 is the cDNA sequence for 22584.
SEQ ID N0:424 is the cDNA sequence for 22585.
SEQ ID N0:425 is the cDNA sequence for 22586.
SEQ ID N0:426 is the cDNA sequence for 22587.
SEQ ID N0:427 is the cDNA sequence for 22588.
SEQ ID N0:428 is the cDNA sequence for 22589.
SEQ ID N0:429 is the cDNA sequence for 22590.
SEQ ID N0:430 is the cDNA sequence for 22591.
SEQ ID N0:431 is the cDNA sequence for 22592.
SEQ ID N0:432 is the cDNA sequence for 22593.
SEQ ID N0:433 is the cDNA sequence for 22594.
SEQ ID N0:434 is the cDNA sequence for 22595.
SEQ ID N0:435 is the eDNA sequence for 22596.
SEQ ID N0:436 is the cDNA sequence for 22847.
SEQ ID N0:437 is the cDNA sequence for 22848.
SEQ ID N0:438 is the cDNA sequence for 22849.
SEQ ID N0:439 is the cDNA sequence for 22851.
SEQ ID N0:440 is the cDNA sequence for 22852.
SEQ ID N0:441 is the cDNA sequence for 22853.
SEQ ID N0:442 is the cDNA sequence for 22854.
SEQ ID N0:443 is the cDNA sequence for 22855.
SEQ ID N0:444 is the cDNA sequence for 22856.
SEQ ID N0:445 is the cDNA sequence for 22857.
SEQ ID N0:446 is the cDNA sequence for 23601.
SEQ ID N0:447 is the cDNA sequence for 23602.
SEQ ID N0:448 is the eDNA sequence for 23605.
SEQ ID N0:449 is the cDNA sequence for 23606.
SEQ ID N0:450 is the cDNA sequence for 23612.

WO 00/04149 PCTlUS99/15838 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 I~O: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 NOs:462-467 are cDNA sequences for known genes.
SEQ ID NOs:468-471 are cDNA sequences for P7lOP.
SEQ ID NO:472 is a cDNA sequence for P1001C.
DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to compositions and methods for the therapy and diagnosis of cancer, such as prostate cancer.
The compositions described herein may include prostate tumor 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 tumor protein or a variant thereof. A "prostate tumor protein" is a protein that is expressed in 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 normal tissue, as determined using a representative assay provided herein. Certain prostate tumor proteins are tumor 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.

WO 00/04149 PCT/(JS99/15838 The present invention is based on the discovery of human prostate tumor proteins. Sequences of polynucleotides encoding certain tumor proteins, or portions thereof, are provided in SEQ ID NOs:1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 or 384-472. Sequences of polypeptides comprising at least a portion of a tumor protein are provided in SEQ ID NOs:112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380 and 383.

PROSTATE TUMOR PROTEIN POLYNUCLEOTIDES
Any polynucleotide that encodes a prostate tumor 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 tumor protein. More preferably, a polynucleotide encodes an immunogenic portion of a prostate tumor 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 tumor 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 tumor 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 tumor protein or a portion thereof.
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 protein- - - 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. 183, Academic Press, Inc., San Diego, CA;
Higgins, D.G. and Sharp, P.M. (1989) CABIOS 5:151-153; Myers, E.W. and Muller W.
(1988) CABIOS 4:11-17; Robinson, E.D. (1971) Comb. Theor 11:105; Santou, N.
Nes, M.
(1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J. (1983) Proc. Natl. Acad., Sci.
USA 80:726-730.
Preferably, 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 tumor 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.
It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many 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 tumor than in nonnal 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 93:10614-10619, 1996 and Heller et al., Proc. Natl.
Acad. Sci. USA 94:2150-2155, 1997). Alternatively, polypeptides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as prostate tumor cells. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, 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 tumor cDNA library) using well known techniques.
Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, 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.
For hybridization techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32P) 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.
Alternatively, there are numerous amplification techniques for obtaining a full length coding sequence from a partial cDNA sequence. Within such techniques, amplific'ation 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 lulown 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. 16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another such technique is known as "rapid amplification of cDNA ends" or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR
(Lagerstrom et al., PCR Methods Applic. 1:111-19, 1991) and walking PCR
(Parker et al., Nucl. Acids. Res. 19:3055-60, 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. 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.
Certain nucleic acid sequences of cDNA molecules encoding at least a portion of a prostate tumor protein are provided in SEQ ID NOs:1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 or 384-472. Isolation of these polynucleotides is described below. Each of these prostate tumor proteins was overexpressed in prostate tumor 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 Adelmari'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 tumor protein, or portion thereof, provided that the DNA is incorporated 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. In addition, or alternatively, 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 tumor polypeptide, and administering the transfected cells to the patient).
A portion of a sequence complementary to a coding sequence (i.e., an antisense polynucleotide) may also be used as a probe or to modulate gene expression.
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 tumor 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)).
Alternatively, 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' 0-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. For example, 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 expressiori'-vectors, replication vectors, probe generation vectors and sequencing vectors. In general, 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.
Within certain embodiments, 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 purposes, as described below. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus).
Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. A
retroviral vector may additionally transfer or incorporate 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.
Other formulations for therapeutic purposes include 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.

PROSTATE TUMOR POLYPEPTIDES
Within the context of the present invention, polypeptides may comprise at least an immunogenic portion of a prostate tumor protein or a variant thereof, as described herein. As noted above, a "prostate tumor protein" is a protein that is expressed by prostate tumor cells. Proteins that are prostate tumor 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 (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. 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 tumor protein or a varialnt 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., (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. As used herein, 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). Such antisera and antibodies may be prepared as described herein, and using well known techniques. An immunogenic portion of a native prostate tumor 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. For example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the invnobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example,'ZSI-labeled Protein A.
As noted above, a composition may comprise a variant of a native prostate tumor protein. A polypeptide "variant," as used herein, is a polypeptide that differs from a native prostate tumor protein in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the polypeptide is not substantially diminished.
In other words, 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.
Preferably, 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. For example, 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.
Other groups of amino acids that may represent conservative changes include:
(1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, 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.
As noted above, 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. For example, 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 and higher eukaryotic cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO. Supernatants from suitable hostlvector 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.
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. For example, 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. 85:2149-2146, 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.
Within certain specific embodiments, 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 tumor protein. A
fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility 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. Preferably, 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. Briefly, 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 incorporated 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. Other near neutral amino acids, such as thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S.
Patent No. 4,935,233 and U.S. Patent No. 4,751,180. The linker sequence may generally be from I
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.
Similarly, 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. Preferably 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., 336:86-91, 1997).
Within preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B
(WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D
derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D
fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenzae virus, NS 1(hemaglutinin). Typically, the N-tenninal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as WO 00/04149 PCT/[3S99/15838 amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain 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 Biotechriology 10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA
may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.
In general, 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. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system.
Preferably, such 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.
BINDING AGENTS
The present invention further provides agents, such as antibodies and antigen-binding fragments thereof, that specifically bind to a prostate tumor protein.
As used herein, an antibody, or antigen-binding fragment thereof, is said to "specifically bind" to a prostate tumor protein if it reacts at a detectable level (within, for example, an ELISA) with a prostate tumor protein, and does not react detectably with unrelated proteins under similar conditions.
As used herein, "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. In general, two compounds are said to "bind," in the context of the present invention, when the binding constant for complex formation exceeds about 103 L/mol. The 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 tumor 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. To determine whether a binding agent satisfies this requirement, biological samples (e.g., blood, sera, urine and/or tumor biopsies) from patients with and without a cancer (as determined using standard clinical tests) may be assayed as described herein for the presence of polypeptides that bind to the binding agent. It will be apparent that 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. For example, a binding agent may be a ribosome, with or without a peptide component, an RNA
molecule or a polypeptide. In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, 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. In one technique, 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.
Alternatively, particularly for relatively short polypeptides, 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 incorporating 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. 6:511 -519, 1976, and improvements thereto. Briefly, 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. For example, 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 I 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. In addition, 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.
Within certain embodiments, the use of 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 iLlanual, 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 9DY,1z3I, 125I, 131I, '86Re, 'ggRe, 21 '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. For example, 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.
Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. 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.
It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, IL), may be employed as the linker group.
Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or' oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Patent No. 4,671,958, to Rodwell et al.
Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group which is cleavable during or upon internalization 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.), by serum complement-mediated hydrolysis (e.g., U.S.
Patent No. 4,671,958, to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Patent No. 4,569,789, to Blattler et al.).
It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody.
Regardless of the particular embodiment, 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. For example, 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. For example, 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 tumor protein. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, 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 CEPRATET"" system, available from Ce11Pro Inc., Bothell WA (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). Alternatively, 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 tumor polypeptide, polynucleotide encoding a prostate tumor polypeptide and/or an antigen presenting cell (APC) that expresses such a polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide.
Preferably, a prostate tumor 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 tumor polypeptide if the T
cells 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.
For example, 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 incorporated into DNA). Contact with a prostate tumor polypeptide (100 ng/ml - 100 g/ml, preferably 200 ng/ml - 25 g/ml) for 3 - 7 days should result in at least a two fold increase in proliferation of the T cells. Contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN-y) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells that have been activated in response to a prostate tumor polypeptide, polynucleotide or polypeptide-expressing APC may be CD4' and/or CD8'. Prostate tumor protein-specific T cells may be expanded using standard techniques. Within preferred embodiments, 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.
For therapeutic purposes, CD4+ or CD8+ T cells that proliferate in response to a prostattY tumor 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. For example, the T cells can be re-exposed to a prostate tumor 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 tumor polypeptide. Alternatively, one or more T cells that proliferate in the presence of a prostate tumor protein can be expanded in number by cloning.
Methods for cloning cells are well known in the art, and include limiting dilution.

PHARMACEUTICAL COMPOSITIONS AND VACCINES
Within certain aspects, polypeptides, polynucleotides, T cells and/or binding agents disclosed herein may be incorporated 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 a non-specific immune response enhancer. A non-specific immune response enhancer may be any substance that enhances an immune response to an exogenous antigen. Examples of non-specific immune response enhancers include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see e.g., Fullerton, U.S. Patent No. 4,235,877).
Vaccine preparation is generally described in, for example, M.F. Powell and M.J.
Newman, eds., "Vaccine Design (the subunit and adjuvant approach)," Plenum Press (NY, 1995).
Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be present, either incorporated 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.
As noted above, 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 15:143-198, 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-Guerrin) that expresses an inununogenic portion of the polypeptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus; or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N. Y.
Acad. Sci.
569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Patent Nos.
4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Patent No. 4,777,127; GB
2,200,651;
EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Aead. Sci. USA 91:215-219, 1994;
Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993.
Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993.
The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. 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.
For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, 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 (e.g., polylactate polyglycolate) 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. Alternatively, compositions of the present invention may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.
Any of a variety of non-specific immune response enhancers may be employed in the vaccines of this invention. For example, 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.
Within the vaccines provided herein, the adjuvant composition is preferably designed to induce an immune response predominantly of the Thl type. High levels of Th 1-type cytokines (e.g., IFN-y, IL-2 and IL- 12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6, IL-10 and TNF-(3) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Thl- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Th1-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. (Hamilton, MT; see US Patent Nos.
4,436,727; 4,877,611; 4,866,034 and 4,912,094). 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. For example, 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.
The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration). Such 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. Camers for use within such formulations are biocompatible, and may also be biodegradable;
preferably the formulation provides a relatively constant level of active component release.
The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
Any of a variety of delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets tumor cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. 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.
Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 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. 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro) and based on the lack of differentiation markers of B cells (CD19 and CD20), T cells (CD3), monocytes (CD14) and natural killer cells (CD56), as determined using standard assays. Dendritic cells may, of course, be engineered to express specific cell-WO 00/04149 PCT/[7S99/15838 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.
As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 1998).
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. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL- 13 and/or TNFa to cultures of monocytes harvested from peripheral blood.
Alternatively, 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, TNFa, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce 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 construed 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 Fcy receptor, mannose receptor and DEC-205 marker. 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 CD11) and costimulatory molecules (e.g., CD40, CD80 and CD86).
APCs may generally be transfected with a polynucleotide encoding a prostate tumor protein (or portion or other variant thereof) such that the prostate tumor 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 purposes, 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, for example, 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 tumor polypeptide, DNA
(naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.

CANCER THERAPY
In further aspects of the present invention, the compositions described herein may be u'sed for immunotherapy of cancer, such as prostate cancer. Within such methods, pharmaceutical compositions and vaccines are typically administered to a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A
patient may or may not be afflicted with cancer. Accordingly, 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.
Within certain embodiments, 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).
Within other embodiments, 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. Examples of 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. As noted above, 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. In particular, antigen-presenting cells, such as dendritic, macrophage, monocyte, fibroblast or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or dther expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al., Immunological Reviews 157:177, 1997).
Alternatively, 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.
Routes and frequency of administration of the therapeutic compositions disclosed herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally.
Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 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. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 100 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.
In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic andlor prophylactic benefit. Such 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 tumor 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.'-In general, a cancer may be detected in a patient based on the presence of one or more prostate tumor proteins and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, urine and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a cancer such as prostate cancer. In addition, such 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. In general, a prostate tumor sequence should be present at a level that is at least three fold higher in tumor tissue than in normal tissue There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, 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.
In a preferred embodiment, 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.
Such 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. Alternatively, 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 WO 00/04149 PCTlUS99/15838 agent. Suitable polypeptides for use within such assays include full length prostate tumor 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 tumor protein may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
Alternatively, 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. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, 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). Inunobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption 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. In general, 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.
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. For example, 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).
In certain embodiments, 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, conunonly 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.

More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked.
Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20TM (Sigma Chemical Co., St. Louis, MO). 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 incub~mion. In general, an appropriate contact time (i.e., incubation 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. Preferably, 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. Those of ordinary skill in the art will recognize that 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 20T"'. 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.
To determine the presence or absence of a cancer, such as prostate cancer, 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. In one preferred embodiment, 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. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the cancer. In an altemate preferred embodiment, 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. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true 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 encldses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, 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.
In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer.
In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, 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. In 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. Typically, the 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. In general, 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. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about I 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.
Of course, numerous other assay protocols exist that are suitable for use with the tumor proteins or binding agents of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use prostate tumor polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such prostate tumor protein specific antibodies may correlate with the presence of a cancer.
A cancer may also, or alternatively, be detected based on the presence of T
cells that specifically react with a prostate tumor protein in a biological sample. Within certain methods, a biological sample comprising CD4+ and/or CD8' T cells isolated from a patient is incubated with a prostate tumor polypeptide, a polynucleotide encoding such a polypeptiYde 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.
For example, 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 tumor 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 tumor polypeptide to serve as a control. For CD4+ T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8+ 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.
As noted above, a cancer may also, or alternatively, be detected based on the level of mRNA encoding a prostate tumor protein in a biological sample. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a prostate tumor 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 tumor protein. The amplified eDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding a prostate tumor protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the tumor protein in a biological sample.
To permit hybridization under assay conditions, 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 tumor protein that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, 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. In a preferred embodiment, 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 and 381.
Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR
Technology, Stockton Press, NY, 1989).
One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, 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.
In another embodiment, the disclosed compositions may be used as markers for the progression of cancer. In this embodiment, 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. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a cancer is progressing in those patients in whom the level of polypeptide or polynucleotide detected increases over time. In contrast, 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 perfonned 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. Such binding agents may also be used in histological applications. AlternativeIy, polynucleotide probes may be used within such applications.
As noted above, to improve sensitivity, multiple prostate tumor 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 tumor protein markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for tumor proteins provided herein may be combined with assays for other known tumor antigens.

DIAGNOSTIC KITS
The present invention further provides 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. For example, one container within a kit may contain a monoclonal antibody or fragmenf thereof that specifically binds to a prostate tumor 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.
Alternatively, a kit may be designed to detect the level of mRNA encoding a prostate tumor protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a prostate tumor 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 tumor protein.
The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES
EXAMPLE I
ISOLATION AND CHARACTERIZATION OF PROSTATE TUMOR POLYPEPTIDES
This Example describes the isolation of certain prostate tumor 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-dT 18 primer. Double-stranded cDNA was synthesized. ligated with EcoRl/BAXI adaptors (Invitrogen, San Diego, CA) and digested with NotI.
Following size fractionation with Chroma Spin-1000 columns (Clontech, Palo Alto, CA), the cDNA was ligated into the EcoRI/NotI site of pCDNA3.l (Invitrogen) and transformed into ElectroMax E. coli DHIOB cells (BRL Life Technologies) by electroporation.
Using the same procedure, 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' independent colonies, with 70% of clones having an insert and the average insert size being 1745 base pairs. The normal pancreas eDNA
library contained 3.3 x 106 independent colonies, with 69% of clones having inserts and the average insert size being 1120 base pairs. For both libraries, 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, 84:189-199, 1994) with some modifications. Specifically, a prostate tumor-specific subtracted cDNA
library was generated as follows. Normal pancreas cDNA library (70 jig) was digested with EcoRl, Notl, and Sful, followed by a filling-in reaction with DNA polymerase Kienow fragment. After phenol-chloroform extraction and ethanol precipitation, the DNA was dissolved in 100 l of H2O, heat-denatured and mixed with 100 1 (100 g) of Photoprobe biotin (Vector Laboratories, Burlingame, CA). As recommended by the manufacturer, the resulting mixture was irradiated with a 270 W sunlamp on ice for 20 minutes. Additional Photoprobe biotin (50 gl) was added and the biotinylation reaction was repeated. After extraction with butanol five times, the DNA was ethanol-precipitated and dissolved in 23 pl H,O to form the driver DNA.
To form the tracer DNA, 10 g prostate tumor cDNA library was digested with BamHI and XhoI, phenol chloroform extracted and passed through Chroma spin-400 columns (Clontech). Following ethanol precipitation, the tracer DNA was dissolved in 5 111 H20. Tracer DNA was mixed with 15 l driver DNA and 20 i of 2 x hvbridization buffer (1.5 M NaCI/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]).
The reaction mixture was then subjected to a streptavidin treatment followed by phenol/chloroform extraction. This process was repeated three more times. Subtracted DNA was precipitated, dissolved in 12 l H,O, mixed with 8 l driver DNA and 20 l of 2 x hybridization buffer, and subjected to a hybridization at 68 C for 2 hours (short hybridization [SH]). After removal of biotinylated double-stranded DNA, subtracted cDNA was ligated into BamHI/XhoI site of chloramphenicol resistant pBCSK' (Stratagene, La Jolla, CA
92037) and transformed into ElectroMax E. coli DH l OB cells by electroporation to generate a prostate tumor specific subtracted cDNA library (referred to as "prostate subtraction 1").
To analyze the subtracted cDNA library, 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 F1-13, F1-12, F1-16, Hl-1, H1-9 and H1-4, were shown to be abundant in the subtracted prostate-specific cDNA library. The determined 3' and 5' cDNA sequences for F 1-12 are provided in SEQ ID
NO: 2 and 3, respectively, with determined 3' cDNA sequences for F 1-13, F 1-16, H 1-1, H 1-9 and H1-4 being provided in SEQ ID NO: 1 and 4-7, respectively.
The eDNA 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, F 1-13, F 1-16, H 1-1, and H 1-4, 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 11. H1-9 was found to be identical to a previously identified human autonomously replicating sequence. No significant homologies to the cDNA
sequence for F1-12 were found.
Subsequent studies led to the isolation of a full-length cDNA sequence for F 1-12. This sequence is provided in SEQ ID NO: 107, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 108.
To clone less abundant prostate tumor specific genes, 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 11.
Specifically, 1 g each of human glandular kallikrein, PSA and mitochondria cytochrome C
oxidase subunit II cDNAs in pCDNA3.l 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".
Twenty-two 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 J1-17, L1-12, NI-1862, J1-13, J1-19, JI-25. J1-24, KI-58, KI-63, I.l-4 and LI-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 J1-12, JI-16, JI-21, K1-48, KI-55, LI-2, L1-6, N1-1858, N1-1860, N1-1861, NI-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, (J1-17, LI-12 and N1-1862; SEQ ID NOS: 8-9, 10-11 and 12-13, respectively). Of the remaining two most abundant species, one (J 1-12;
SEQ ID NO:30) was found to be identical to the previously identified human pulmonary surfactant-associated protein, and the other (KI-48; SEQ ID NO:33) was determined to have some homology to R.
norvegicus mRNA for 2-arylpropionyl-CoA epimerase. Of the 17 less abundant cDNA
clones isolated from the subtracted prostate tumor specific cDNA library with spike, four (J 1-16, K1-55, L1-6 and N1-1864; SEQ ID NOS:31, 34, 36 and 40, respectively) were found to be identical to previously identified sequences, two (J1-21 and N1-1860; SEQ
ID NOS: 32 and 38, respectively) were found to show some homology to non-human sequences, and two (Ll-2 and N1-1861; SEQ ID NOS: 35 and 39, respectively) were found to show some homology to known human sequences. No significant homologies were found to the polypeptides J1-13, JI-19, J1-24, J1-25, K1-58, K1-63, LI-4, L1-14 (SEQ ID
NOS: 14-15, 16-17, 20-21, 18-19, 22-23, 24-25, 26-27, 28-29, respectively).
Subsequent studies led to the isolation of full length cDNA sequences for J 1-17, LI-12 and N1-1862 (SEQ ID NOS: 109-111, respectively). The corresponding predicted amino acid sequences are provided in SEQ ID NOS: 112-114. L1-12 is also referred to as P501 S.
In a further experiment, four additional clones were identified by subtracting a prostate tumor cDNA library with normal prostate cDNA prepared from a pool of three normal prostate poly A+ RNA (referred to as "prostate subtraction 2"). The determined cDNA se ; quences for these clones, hereinafter referred to as U I-3064, U l-3065, V 1-3692 and 1A-3905,'are provided in SEQ ID NO: 69-72, respectively. Comparison of the determined sequences with those in the gene bank revealed no significant homologies to U
1-3065.
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, J1-17, pulmonary surfactant-associated protein, mitochondrial DNA, cytochrome c oxidase subunit II, N 1-1862, autonomously replicating sequence, L 1-12 and tumor expression enhanced gene. Four additional clones, hereinafter referred to as V 1-3686, R 1-23 30, IB-3976 and V 1-3679, were isolated. The determined cDNA sequences for these clones are provided in SEQ ID NO:73-76, respectively. Comparison of these sequences with those in the gene bank revealed no significant honiologies to VI-3686 and RI-2330.
Further analysis of the three prostate subtractions described above (prostate subtraction 2, subtracted prostate tumor specific cDNA library with spike, and prostate subtraction spike 2) resulted in the identification of sixteen additional clones, referred to as 1 G-4736, IG-4738, IG-4741, IG-4744, 1 G-4734, IH-4774, 1 H-4781, 1 H-4785, 1 H-4787, 1 H-4796, 11-4810, 11-4811, 1 J-4876, 1 K-4884 and 1 K-4896. 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 IG-4741, 1G-4734, 11-4807, 1J-4876 and IK-4896 (SEQ ID NOS: 79, 81, 87, 90 and 92, respectively). Further analysis of the isolated clones led to the determination of extended cDNA sequences for I G-4736, 1 G-473 8, 1 G-4741, I G-4744, 1 H-4774, I H-4781, IH-4785, 1 H-4787, 1 H-4796, 11-4807, IJ-4876, IK-4884 and IK-4896, provided in SEQ ID
NOS: 179-188 and 191-193, respectively, and to the determination of additional partial cDNA sequences for 11-4810 and 11-4811, provided in SEQ ID NOS: 189 and 190, respectively.
Additional studies with prostate subtraction spike 2 resulted in the isolation of three more clones. Their sequences were determined as described above and compared to the most recent GenBank. All three clones were found to have homology to known genes, which are Cysteine-rich protein, KIAA0242, and KIAA0280 (SEQ ID NO: 317, 319, and 320, respectively). Further analysis of these clones by Synteni microarray (Synteni, Palo Alto, CA) demonstrated that all three clones were over-expressed in most prostate tumors and prostate BPH, as well as in the majority of normal prostate tissues tested, but low expression in all other normal tissues.
An additional subtraction was performed by subtracting a normal prostate cDNA library witli normal pancreas cDNA (referred to as "prostate subtraction 3"). This led to the identification of six additional clones referred to as I G-4761, 1 G-4762, 1 H-4766, I H-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 I G-4761 and I H-4771 (SEQ ID
NOS: 93 and 97, respectively). Further analysis of the isolated clones led to the determination of extended cDNA sequences for 1 G-476 1, 1 G-4762, 1 H-4766 and 1 H-4772 provided in SEQ
ID NOS: 194-196 and 199, respectively, and to the determination of additional partial cDNA
sequences for 1 H-4770 and IH-4771, provided in SEQ ID NOS: 197 and 198, respectively.
Subtraction of a prostate tumor cDNA library, prepared from a pool of polyA+
RNA from three prostate cancer patients, with a normal pancreas eDNA library (prostate subtraction 4) led to the identification of eight clones, referred to as 1 D-4297, 1 D-4309, 1 D.1-4278, ID-4288, 1 D-4283, 1 D-4304, ID-4296 and ID-4280 (SEQ ID NOS: 99-107).
'rhese sequences were compared to those in the gene bank as described above. No significant homologies were found to I D-4283 and ID-4304 (SEQ ID NOS: 103 and 104, respectively).
Further analysis of the isolated clones led to the determination of extended eDNA sequences for ID-4309, 1 D.1-4278, ID-4288, 1 D-4283, 1 D-4304, 1 D-4296 and 1 D-4280, provided in SEQ ID NOS: 200-206, respectively.
cDNA clones isolated in prostate subtraction I and prostate subtraction 2, described above, 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 eDNA 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.

WO 00/04149 PCTlUS99/15838 Additional, studies led to the isolation of the full-length cDNA sequence for P509S. This sequence is provided in SEQ ID NO: 332, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 339.

DETERMINATION OF TISSUE SPECIFICITY OF PROSTATE TUMOR
POLYPEPTIDES
Using gene specific primers, mRNA expression levels for the representative prostate tumor polypeptides F 1-15, H 1-1, J 1-17 (also referred to as P502S), L 1-12 (also referred to -as P501S), F1-12 (also referred to as P504S) and N1-1862 (also referred to as P503S) were examined in a variety of normal and tumor tissues using RT-PCR.
Briefly, total RNA was extracted from a variety of normal and tumor tissues using Trizol reagent as described above. First strand synthesis was carried out using 1-2 g of total RNA with SuperScript II reverse transcriptase (BRL Life Technologies) at 42 C for one hour. The cDNA was then amplified by PCR with gene-specific primers. To ensure the semi-quantitative nature of the RT-PCR, P-actin was used as an internal control for each of the tissues examined. First, serial dilutions of the first strand cDNAs were prepared and RT-PCR assays were performed using (3-actin specific primers. A dilution was then chosen that enabled the linear range amplification of the (3-actin template and which was sensitive enough to reflect the differences in the initial copy numbers. Using these conditions, the P-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. F 1-16 was found to be expressed at high levels in prostate tumor tissue, colon tumor and normal prostate, and at lower levels in normai liver, skin and testes, with expression being undetectable in the other tissues examined. H1-1 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. J1-17 (P502S) and L1-12 (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. N1-1862 (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 F1-16, H1-1, J1-17 (P502S), N1-1862 (P503S) and L1-12 (P5O1S) are either prostate specific or are expressed at significantly elevated levels in prostate.
Further RT-PCR studies showed that F1-12 (P504S) is over-expressed in 60%
of prostate tumors, detectable in normal kidney but not detectable in all other tissues tested.
Similarly, RI-2330 was shown to be over-expressed in 40% of prostate tumors, detectable in normal kidney and liver, but not detectable in all other tissues tested. U1-3064 was found to be over-expressed in 60% of prostate tumors, and also expressed in breast and colon tumors, but was not detectable in normal tissues.
RT-PCR characterization of R 1-2330, Ul-3064 and 1 D-4279 showed that these three antigens are over-expressed in prostate and/or prostate tumors.
Northern analysis with four prostate tumors, two normal prostate samples, two BPH prostates, and normal colon, kidney, liver, lung, pancrease, skeletal muscle, brain, stomach, testes, small intestine and bone marrow, showed that L1-12 (P501S) is over-expressed in prostate tumors and normal prostate, while being undetectable in other normal tissues tested. J1-17 (P502S) was detected in two prostate tumors and not in the other tissues tested. N1-1862 (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. F1-12 (P504S) was found to be highly expressed in two prostate tumors and to be undetectable in all other tissues tested.
The microarray technology described above was used to determine the expression levels of representative antigens described herein in prostate tumor, breast tumor and the following normal tissues: prostate, liver, pancreas, skin, bone marrow, brain, breast, adrenal gland, bladder, testes, salivary gland, large intestine, kidney, ovary, lung, spinal cord, skeletal muscle and colon. L1-12 (P501S) was found to be over-expressed in normal prostate and prostate tumor, with some expression being detected in normal skeletal muscle. Both J 1-12 and F1-12 (P504S) were found to be over-expressed in prostate tumor, with expression being lower or undetectable in all other tissues tested. N 1-1862 (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. R1-2330 was found to be over-expressed in prostate tumor and normal prostate, and to be expressed at lower levels in all other tissues tested. ID-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.
Further microarray analysis to specifically address the extent to which P501 S
(SEQ ID NO: 110) was expressed in breast tumor revealed moderate over-expression not only in breast tumor, but also in nietastatic breast tumor (2/31), with negligible to low expression in normal tissues. This data suggests that P501 S may be over-expressed in various breast tumors as well as in prostate tumors.
The expression levels of 32 ESTs (expressed sequence tags) described by Vasmatzis et al. (Proc. Natl. Acad Sci. USA 95:300-304, 1998) in a variety of tumor and normal tissues were examined by microarray technology as described above. Two of these clones ~referred to as P1000C and PIOOIC) 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 P 1000C and P 1001 C are provided in SEQ ID
NO: 384 and 472, respectively. 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 P1000C.
The expression of the polypeptide encoded by the full length cDNA sequence for F1-12 (also referred to as P504S; SEQ ID NO: 108) was investigated by immunohistochemical analysis. Rabbit-anti-P504S polyclonal antibodies were generated against the full length P504S protein by standard techniques. Subsequent isolation and characterization of the polyclonal antibodies were also performed by techniques well known in the art. Immunohistochemical analysis showed that the P504S polypeptide was expressed in 100% of prostate carcinoma samples tested (n=5).
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.
This data indicates that the P504S polypeptide is present in prostate cancer tissues, and that there are qualitative and quantitative differences in the staining between benign prostatic hyperplasia tissues and prostate cancer tissues, suggesting that this polypeptide may be detected selectively in prostate tumors and therefore be useful in the diagnosis of prostate cancer.

ISOLATION AND CHARACTERIZATION OF PROSTATE TUMOR POLYPEPTIDES
BY PCR-BASED SUBTRACTION

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 sub-cloned into the vector pT7 Blue T-vector (Novagen, Madison, WI) and transformed into XL-I Blue MRF' E. coli (Stratagene). DNA was isolated from independent clones and sequenced using a Perkin Elmer/Applied Biosystems Division Automated Sequencer Model 373A.
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.
Further studies using the PCR-based methodology described above resulted in the isolation of more than 180 additional clones, of which 23 clones were found to show no significant homologies to known sequences. The determined cDNA sequences for these clones are provided in SEQ ID NO: 115-123, 127, 131, 137, 145, 147-151, 153, 156-158 and 160. Twenty-three clones (SEQ ID NO: 124-126, 128-130, 132-136, 138-144. 146, 152, 154, 155 and 159) were found to show some homology to previously identified ESTs. An additional ten clones (SEQ ID NO: 161-170) were found to have some degree of homology to known genes. Larger 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 DE1, DE13 and DE14 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.
mRNA Expression levels for representative clones in tumor tissues (prostate (n=5), breast (n=2), colon and lung) normal tissues (prostate (n=5), colon, kidney, liver, lung (n=2), ovary (n=2), skeletal muscle, skin, stomach, small intestine and brain), and activated and non-activated PBMC was determined by RT-PCR as described above. Expression was examined in one sample of each tissue type unless otherwise indicated.
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. P20, a portion of the P703P gene, was found to be highly expressed in normal prostate and prostate tumor, compared to all twelve normal tissues tested. A
modest increase in expre~sion of P20 in breast tumor (n=2), colon tumor and lung tumor was seen compared to all normal tissues except lung (l of 2). Increased expression of P18 was found in normal prostate, prostate tumor and breast tumor conlpared to other normal tissues except lung and stomach. A modest increase in expression of P5 was observed in normal prostate compared to most other normal tissues. However, some elevated expression was seen in normal lung and PBMC. Elevated expression of P5 was also observed in prostate tumors (2 of 5), breast tumor and one lung tumor sample. For 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.
Further studies resulted in the isolation of twelve additional clones, hereinafter referred to as 10-d8, 10-h10, 11-c8, 7-g6, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3, 8-h11, 9-fl 2 and 9-0.
The determined DNA sequences for 10-d8, 10-h10, 11-c8, 8-d4, 8-d9, 8-h11, 9-f12 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, 11-c8 and 8-hi I were found to show some homology to previously isolated ESTs, while 10-h10, 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-F 12, 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 undetectabie.

Increased expression of 8-F11 was seen in prostate tumor and normal prostate, bladder, skeletal muscle and colon. Increased expression of 10-H10 was seen in prostate tumor and normal prostate, bladder, lung, colon, brain and large intestine. Increased expression of 9-B 1 was seen in prostate tumor, breast tumor, and normal prostate, salivary gland, large intestine and skin, with increased expression of 11-C8 being seen in prostate tumor, and normal prostate and large intestine.
An additional cDNA fragment derived from the PCR-based normal prostate subtraction, described above, was found to be prostate specific by both micro-array technology and RT-PCR. The determined cDNA sequence of this clone (referred to as 9-A11) is provided in SEQ ID NO: 226. Comparison of this sequence with those in the public databases revealed 99% identity to the known gene HOXB 13.
Further studies led to the isolation of the clones 8-C6 and 8-H7. The determined cDNA sequences for these clones are provided in SEQ ID NO: 227 and 228, respectively. These sequences were found to show some homology to previously isolated ESTs.
PCR and hybridization-based methodologies were employed to obtain longer cDNA sequences for clone P20 (also referred to as P703P), yielding three additional cDNA
fragments that progressively extend the 5' end of the gene. These fragments, referred to as P703PDE5, P703P6.26, and P703PX-23 (SEQ ID NO: 326, 328 and 330, with the predicted corresponding amino acid sequences being provided in SEQ ID NO: 327, 329 and 331, respectively) contain additional 5' sequence. 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. Further studies using a PCR-based subtraction library of a prostate tumor pool subtracted against a pool of normal tissues (referred to as JP: PCR subtraction) resulted in the isolation of thirteen additional clones, seven of which did not share any significant homology to known GenBank sequences. The determined cDNA sequences for these seven clones (P71 IP, P712P, novel 23, P774P, P775P, P710P and P768P) 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. By microarray analysis, 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 P711P, P712P, novel 23 and P768P showed over-expression in most prostate tumors and BPH tissues tested (n=29), and in the majority of normal prostate tissues (n=4), but background to low expression levels in all normal 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 P711 P 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 1P 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.
Using PCR and hybridization-based methodologies, 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).
After comparison with the most recent GenBank, P707P was found to be a splice variant of the known gene HoxB 13. In contrast, no significant homologies to P714P were found.
Clones 8-B3, P89, P98, P130 and P201 (as disclosed in U.S. Patent Application No. 09/020,956, filed February 9, 1998) 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.

SYNTHESIS OF POLYPEPTIDES

Polypeptides may be synthesized on a Perkin Elmer/Applied Biosystems 430A
peptide synthesizer using FMOC chemistry with HPTU (O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) activation. 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.

FURTHER ISOLATION AND CHARACTERIZATION OF PROSTATE TUMOR
POLYPEPTIDES BY PCR-BASED SUBTRACTION

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.
Within this protocol, tester and driver double stranded cDNA were separately digested with five restriction enzymes that recognize six-nucleotide restriction sites (MIuI, MscI, Pvull, Sall 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.
In the first hybridization step, 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. Following this second hybridization, in addition to populations (a) through (d), 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.
The ends were then filled in, and PCR amplification was performed using adaptor-specific primers. Only population (e), which contained tester cDNA
that did not hybridize to driver cDNA, was amplified exponentially. A second PCR
amplification step was then performed, to reduce background and further enrich differentially expressed sequences.
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.

In addition to genes known to be overexpressed in prostate tumor, 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.
Exceptions were JPTPN23 (SEQ ID NO: 231; similarity to pig valosin-containing protein), JPTPN30 (SEQ ID NO: 234; similarity to rat mRNA for proteasome subunit), (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. gallus dynein light chain-A), 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), and JP8E9 (SEQ ID NO: 299;
similarity to human Alu sequence).
Additional studies using the PCR-based subtraction library consisting of a prostate tumor pool subtracted against a normal prostate pool (referred to as PT-PN PCR
subtraction) yielded three additional clones. Comparison of the cDNA sequences of these clones with the most recent release of GenBank revealed no significant homologies to the two clones referred to as P715P and P767P (SEQ ID NO: 312 and 314). The remaining clone was found to show some homology to the known gene KIAA0056 (SEQ ID NO: 318). Using microarray analysis to measure mRNA expression levels in various tissues, all three clones were found to be over-expressed in prostate tumors and BPH tissues.
Specifically, clone P715P was over-expressed in most prostate tumors and BPH tissues by a factor of three or greater, with elevated expression seen in the majority of nonnal prostate samples and in fetal tissue, but negative to low expression in all other normal tissues. Clone P767P was over-expressed in several prostate tumors and BPH tissues, with moderate expression levels in half of the normal prostate samples, and background to low expression in all other normal tissues tested.

Further analysis, by microarray as described above, of the PT-PN PCR
subtraction library and of a DNA subtraction library containing cDNA from prostate tumor subtracted with a pool of normal tissue cDNAs, led to the isolation of 27 additional clones (SEQ ID NO: 340-365 and 381) which were determined to be over-expressed in prostate tumor. The clones of SEQ ID NO: 341, 342, 345, 347, 348, 349, 351, 355-359, 361, 362 and 364 were also found to be expressed in normal prostate. Expression of all 26 clones in a variety of normal tissues was found to be low or undetectable, with the exception of P544S
(SEQ ID NO: 356) which was found to be. expressed in small intestine. Of the 26 clones, 10 (SEQ ID NO: 340-349) were found to show some homology to previously identified sequences. No significant homologies were found to the clones of SEQ ID NO:
350-365.

PEPTIDE PRIMING OF MICE AND PROPAGATION OF CTL LINES

6.1. This Example illustrates the preparation of a CTL cell line specific for cells expressing the P502S gene.
Mice expressing the transgene for human HLA A2.1 (provided by Dr L.
Sherman, The Scripps Research Institute, La Jolla, CA) were immunized with P2S#12 peptide (VLGWVAEL; SEQ ID NO: 306), which is derived from the P502S gene (also referred to herein as J1-17, 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 100 g of P2S#12 and 120 g of an I-Ab binding peptide derived from hepatitis B Virus protein emulsified in incomplete Freund's adjuvant. Three weeks later these mice were sacrificed and using a nylon mesh single cell suspensions prepared. Cells were then resuspended at 6 x 10b cells/ml in complete media (RPMI-1640; Gibco BRL, Gaithersburg, MD) containing 10% FCS, 2mM Glutamine (Gibco BRL), sodium pyruvate (Gibco BRL), non-essential amino acids (Gibco BRL), 2 x 10"5 M 2-mercaptoethanol, 50U/ml penicillin and streptomycin, and cultured in the presence of inadiated (3000 rads) P2S# 12-pulsed (5mg/mi P2S#12 and 10mg/mi (32-microglobulin) LPS blasts (A2 transgenic spleens cells cultured in the presence of 7 g/ml dextran sulfate and 25 g/ml LPS for 3 days). Six days later, cells (5 x I0S/ml) were restimulated with 2.5 x 106/ml peptide pulsed irradiated (20,000 rads) EL4A2Kb cells (Sherman et al, Science 258:815-818, 1992) and 3 x 106/ml A2 transgenic spleen feeder cells. Cells were cultured in the presence of 20U/ml IL-2. Cells continued to be restimulated on a weekly basis as described, in preparation for cloning the line.
P2S#12 line was cloned by limiting dilution analysis with peptide pulsed EL4 A2Kb tumor cells (1 x 10' cells/ well) as stimulators and A2 transgenic spleen cells as feeders ( 5 x 105 cells/ well) grown in the presence of 30U/ml IL-2. On day 14, cells were WO 00/04149 PCT/[JS99/15838 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 A2.1 expressing) transduced with P502S than against control fibroblasts. An example is presented in Figure 1.
This data indicates that P2S #12 represents a naturally processed epitope of the P502S protein that is expressed in the context of the human HLA A2.1 molecule.

6.2. This Example illustrates the preparation of murine CTL lines and CTL
clones specific for cells expressing the P501 S gene.
This series of experiments were performed similarly to that described above.
Mice were immunized with the PIS#10 peptide (SEQ ID NO: 337), which is derived from the P501 S gene (also referred to herein as L 1-12, SEQ ID NO: 110). The P 1 S# 10 peptide was derived by analysis of the predicted polypeptide sequence for P501 S for potential HLA-A2 binding sequences as defined by published HL,A-A2 binding motifs (Parker, KC, et al, J.
Immunol., 152:163, 1994). P 1 S# 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. In the competition assay, test peptides at 100-200 gg/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. As shown in Figure 3, peptide PIS#10 competes HLA-A2 restricted presentation of fluM58, demonstrating that peptide P 1 S# 10 binds HLA-A2.
Mice expressing the transgene for human HLA A2.1 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 P 1 S# 10 and 120 g of an I-Ab binding peptide derived from Hepatitis B Virus protein emulsified in incomplete Freund's adjuvant. Three weeks later these mice were sacrificed and single cell suspensions prepared using a nylon mesh. Cells were then resuspended at 6 x 106 cells/ml in complete media (as described above) and cultured in the presence of irradiated (3000 rads) P I S#
10-pulsed (2 g/ml PIS#10 and 10mg/ml [32-microglobulin) LPS blasts (A2 transgenic spleens cells cultured in the presence of 7 g/ml dextran sulfate and 25 g/ml LPS for 3 days). Six days later cells (5 x 195/ml) were restimulated with 2.5 x 106/mi peptide-pulsed irradiated (20,000 rads) EL4A2Kb cells, as described above, and 3 x 106/ml A2 transgenic spleen feeder cells.
Cells were cultured in the presence of 20 U/ml IL-2. Cells were restimulated on a weekly basis in preparation for cloning. After three rounds of in vitro stimulations, one line was generated that recognized P 1 S# 10-pulsed Jurkat A2Kb targets and P501 S-transduced Jurkat targets as shown in Figure 4.
A P 1 S# 10-specific CTL line was cloned by limiting dilution analysis with peptide pulsed EL4 A2Kb tumor cells (1 x 104 cells/ well) as stimulators and A2 transgenic spleen cglls as feeders ( 5 x 105 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 P501 S-transduced Jurkat A2Kb targets. This data indicates that P I S#
10 represents a naturally processed epitope of the P501 S protein that is expressed in the context of the human HLA-A2.1 molecule.

ABILITY OF HUMAN T CELLS TO RECOGNIZE PROSTATE TUMOR
POLYPEPTIDES
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 J1-17) using dendritic cells according to the protocol of Van Tsai et al. (Critical Reviews in Immunology 18:65-75, 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 y-interferon ELISPOT assay (see. Lalvani et al., J. Exp. Med.
186:859-865, 1997). Briefly, titrating numbers of T cells were assayed in duplicate on 10' fibroblasts in the presence of 3 g/ml human p,-microglobulin and 1 ug/ml P2S-12 peptide or control E75 peptide. In addition, T cells were simultaneously assayed on autologous fibroblasts transduced with the P502S gene or as a control, fibroblasts transduced with HER-2/neu. Prior to the assay, the fibroblasts were treated with 10 ng/ml y-interferon for 48 hours to upregulate class I MHC expression. One of the microcultures (#5) demonstrated strong recognition of both peptide pulsed fibroblasts as well as transduced fibroblasts in a y-interferon ELISPOT
assay. Figure 2A demonstrates that there was a strong increase in the number of y-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. As shown in Figure 2B, this microculture also demonstrated an increase in the number of y-interferon spots with increasing numbers of T

cells on fibroblasts transduced to express the P502S gene but not the HER-2/neu gene. These results provide additional confirmatory evidence that the P2S-12 peptide is a naturally processed epitope of the P502S protein. Furthermore, this also demonstrates that there exists in the human T cell repertoire, high affinity T cells which are capable of recognizing this epitope. These T cells should also be capable of recognizing human tumors which express the P502S gene.

PRIMING OF CTL IN VIVO USING NAKED DNA IMMUNIZATION WITH A
PROSTATE ANTIGEN

The prostate tumor antigen Li-12, as described above, is also referred to as P501 S. HLA A2Kb Tg mice (provided by Dr L. Sherman, The Scripps Research Institute, La Jolla, CA) were immunized with 100 g VR10132-P501 S 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-P501 S transduced stimulator cells. CTL lines were stimulated weekly.
After two weeks of in vitro stimulation, CTL activity was assessed against P501 S transduced targets. Two out of 8 mice developed strong anti-P501 S CTL responses. These results demonstrate that P501 S contains at least one naturally processed A2-restricted CTL epitope.

GENERATION OF HUMAN CTL IN VITRO USING WHOLE GENE PRIMING AND
STIMULATION TECHNIQUES WITH PROSTATE TUMOR ANTIGEN

Using in vitro whole-gene priming with P501 S-retrovirally transduced autologous fibroblasts (see, for example, Yee et al, The Journal of Immunology, 157(9):4079-86, 1996), human CTL lines were derived that specifically recognize autologous fibroblasts transduced with P501 S(also known as L 1-12), as determined by interferon-y ELISPOT
analysis as described above. Using a panel of HLA-mismatched fibroblast lines transduced with P501 S, these CTL lines were shown to be restricted HLA-A2 class I
allele. Specifically, dendritic cells (DC) were differentiated from 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. Following culture, DC
were infected overnight with recombinant P501 S vaccinia virus at a multiplicity of infection (M.O.I) of five, and matured overnight by the addition of 3 g/ml CD40 ligand.
Virus was inactivated by UV irradiation. CD8+ T cells were isolated using a magnetic bead system, and priming cultures were initiated using standard culture techniques. Cultures were restimulated every 7-10 days using autologous primary fibroblasts retrovirally transduced with P501S.
Following four stimulation cycles, CD8+ T cell lines were identified that specifically produced interferon-y when stimulated with P501 S-transduced autologous fibroblasts. The P501 S-specific activity could be sustained by the continued stimulation of the cultures with P501 S-transduced fibroblasts in the presence of IL-15. A panel of HLA-mismatched fibroblast lines transduced with P501 S were generated to define the restriction allele of the response. By measuring interferon-y in an ELISPOT assay, the P501 S specific response was shown to be restricted by HLA-A2. These results demonstrate that a CD8+ CTL
response to P501 S can be elicited.

IDENTIFICATION OF A NATURALLY PROCESSED CTL EPITOPE CONTAINED
WITHIN A PROSTATE TUMOR ANTIGEN

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 CD8+ T cells can be primed following repeated in vitro stimulations with monocytes pulsed with p5 peptide. These CTL
specifically recognize p5-pulsed target cells in both ELISPOT (as described above) and chromium release assays. Additionally, immunization of HLA-A2 transgenic mice with p5 leads to the generation of CTL lines which recognize a variety of P703P transduced target cells expressing either HLA-A2Kb or HLA-A2. Specifically, HLA-A2 transgenic mice were immunized subcutaneously in the footpad with 100 g of p5 peptide together with 140 g of hepatitis B virus core peptide (a Th peptide) in Freund's incomplete adjuvant.
Three weeks post immunization, 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.
Human in vitro priming experiments demonstrated that the p5 peptide is immunogenic in humans. Dendritic cells (DC) were differentiated from 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/mi human IL-4.
Following culture, the DC were pulsed with p5 peptide and cultured with GM-CSF
and IL-4 together 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.

EXPRESSION OF A BREAST TUMOR-DERIVED ANTIGEN
IN PROSTATE
Isolation of the antigen B305D from breast tumor by differential display is described in US Patent Application No. 08/700,014, filed August 20, 1996.
Several different splice forms of this antigen were isolated. The determined eDNA sequences for these splice forms are provided in SEQ ID NO: 366-375, with the predicted amino acid sequences corresponding to the sequences of SEQ ID NO: 292, 298 and 301-303 being provided in SEQ
ID NO: 299-306, respectively.
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 tumor 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).

ELICITATION OF PROSTATE TUMOR ANTIGEN-SPECIFIC CTL RESPONSES IN
HUMAN BLOOD
I
This Example illustrates the ability of a prostate tumor antigen to elicit a CTL
response in blood of normal humans.
Autologous dendritic cells (DC) were differentiated from 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. Following culture, DC were infected ovemight with recombinant P501 S-expressing vaccinia virus at an M.O.I. of 5 and matured for 8 hours by the addition of 2 micrograms/mi CD40 ligand. Virus was inactivated by UV irradiation, CD8+ cells were isolated by positive selection using magnetic beads, and priming cultures were initiated in 24-well plates. Following five stimulation cycles, CD8+
lines were identified that specifically produced interferon-gamma when stimulated with autologous P501 S-transduced fibroblasts. The P501 S-specific activity of cell line 3A-1 could be maintained following additional stimulation cycles on autologous B-LCL
transduced with P501S. Line 3A-I was shown to specifically recognize autologous B-LCL
transduced to express P501 S, but not EGFP-transduced autologous B-LCL, as measured by cytotoxity assays (S'Cr release) and interferon-gamma production (Interferon-gamma Elispot; see above and Lalvani et al., J Exp. Med. 186:859-865, 1997). The results of these assays are presented in Figures 6A and 6B.

IDENTIFICATION OF PROSTATE TUMOR ANTIGENS
BY MICROARRAY ANALYSIS

This Example describes the isolation of certain prostate tumor 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.

Table I
Sununary of Prostate Tumor Antigens Known Genes Previously identified Genes Novel Genes T-cell gamma chain P504S 23379 (SEQ

ID NO:389) Kallikrein P1000C 23399 (SEQ
ID NO:392) Vector P501 S 23320 (SEQ
ID NO:386) CGI-82 protein mRNA (23319; SEQ ID P503S 23381 (SEQ
NO:385) ID NO:390) Ald. 6 Dehyd. P784P
L-iditol-2 dehydrogenase (23376; SEQ ID P502S
NO:3 88 ) Ets transcription factor PDEF (22672; SEQ P706P
ID NO:398) hTGR (22678; SEQ ID NO:399) 19142.2, bangur.seq (22621; SEQ
ID NO:396) KIAA0295(22685; SEQ ID NO:400) 5566.1 Wang(23404; SEQ ID
NO:393) Prostatic Acid Phosphatase(22655; SEQ ID P712P
NO:397) SUBSTITUTE SHEET (RULE 26) transglutaminase (22611; SEQ ID NO:395) P778P
HDLBP (23508; SEQ ID NO:394) CGI-69 Protein(23367; SEQ ID NO:387) KIAA0122(23383; SEQ ID NO:391) TEEG

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. hTGR1 showed 9.11 fold over-expression in prostate tissues as compared to other normal tissues tested. 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 (HDLBP) 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.
19142.2 bangur showed 23.25 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 97% of prostate tumors and 100% of normal prostate. It was undetectable in other normal tissues tested. 5566.1 Wang showed 3.31 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 97% of prostate tumors, 75% normal prostate and was also over-expressed in normal bone marrow, pancreas, and activated PBMC. Novel clone 23379 showed 4.86 fold over-expression in prostate tissues as compared to other normal tissues tested. It was detectable in 97% of prostate tumors and 75% normal prostate and is undetectable in all other normal tissues tested. 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.

IDENTIFICATION OF PROSTATE TUMOR ANTIGENS
BY ELECTRONIC SUBTRACTION

This Example describes the use of an electronic subtraction technique to identify prostate tumor antigens.
Potential prostate-specific genes present in the GenBank human EST database were identified by electronic subtraction (similar to that described by Vasmatizis et al., Proc.
1Vat1. 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.

WO 00/04149 PCTlUS99115838 Records for the 479 human cDNA libraries represented in the GenBank release were downloaded to create a database of these cDNA library records. These 479 cDNA
libraries were grouped into three groups, Plus (normal prostate and prostate tumor libraries, and breast cell lines, in which expression was desired), Minus (libraries from other normal adult tissues, in which expression was not desirable), and Other (fetal tissue, infant tissue, tissue found only in women, non-prostate tumors and cell lines other than prostate cell lines, in which expression was considered to be irrelevant). A summary of these library groups is presented in Table II.

Table II
Prostate cDNA Libraries and ESTs Library # of Libraries # of ESTs Plus 25 43,482 Normal 11 18,875 Tumor 11 21,769 Cell lines 3 2,838 Minus 166 Other 287 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 found in the Plus and Other group libraries only; no expression detected in the Minus group; Type 3- EST clones found in the Plus, Minus and Other group libraries, but the expression in the Plus group is higher than in either the Minus or Other groups; and Type 4- EST clones found in Plus, Minus and Other group libraries, but the expression in the Plus group is higher than the expression in 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.

Table III
Prostate Cluster Summarv # of # of ESTs Type Superclusters Ordered Total 4345 3172 The 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. In total, 2,528 clones from the electronic subtraction method were analyzed by microarray analysis to identify electronic subtraction breast clones that had high 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 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997). Within these analyses, the clones were arrayed on the chip, which was then probed with fluorescent probes generated from normal and tumor prostate cDNA, as well as various other normal tissues. The slides were scanned and the fluorescence intensity was measured.
Clones with an expression ratio greater than 3 (i.e., the level in prostate tumor cDNA was at least three times the level in normal prostate cDNA) were identified as prostate tumor-specific sequences (Table IV). The sequences of these clones are provided in SEQ ID
NOs:401-453, with certain novel sequences shown in SEQ ID NOs:407, 413, 416-419, 422, 426, 427 and 450.

Table IV
Prostate-tumor Specific Clones SEQ ID NO. Sequence Comments Designation 401 22545 previously identified P 1000C
402 22547 previouslv identified P704P

SUBSTITUTE SHEET (RULE 26) 403 22548 known 404 22550 known 406 22552 prostate secretory protein 94 407 22553 novel 408 22558 previously identified P509S
409 22562 glandular kallikrein 410 22565 previously identified P1000C

412 22568 B 1006C (breast tumor antigen) 413 22570 novel 415 22572 previously identified P706P
416 22573 novel 417 22574 novel 418 22575 novel 419 22580 novel 421 22582 prostatic secretory protein 94 422 22583 novel 423 22584 prostatic secretory protein 94 424 22585 prostatic secretory protein 94 425 22586 known 426 22587 novel 427 22588 novel 429 22590 known 431 22592 known 432 22593 Previously identified P777P
433 22594 T cell receptor gamma chain 434 22595 Previously identified P705P
435 22596 Previously identified P707P

43 7 22848 known 438 22849 prostatic secretory protein 57 SUBSTITUTE SHEET (RULE 26) WO 00104149 PCT/[IS99/15838 442 22854 previously identified P509S
443 22855 previously identified P705P
444 22856 previously identified P774P

446 23601 previously identified P777P

450 23612 novel 452 23618 previously identified P1000C
453 23622 previously identified P705P

FURTHER IDENTIFICATION OF PROSTATE TUMOR ANTIGENS
BY MICROARRAY ANALYSIS

This Example describes the isolation of additional prostate tumor 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 NOs:454-467. Of these sequences SEQ ID NOs:459-461 correspond to novel genes. The others (SEQ ID NOs:454-458 and 461-467) correspond to known sequences.

This Example describes the full length cloning of P71 OP.

SUBSTITUTE SHEET (RULE 26) 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 ciDNAs recovered and sequenced by an automatic ABI Sequencer. Four sequences were obtained, and are presented in SEQ ID NOs:468-471.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.
Accordingly, the present invention is not limited except as by the appended claims.

DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

CECI EST LE TOME DE _2 NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.

JUMBO APPLICATIONS / PATENTS

THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.

NOTE: For additional volumes please contact the Canadian Patent Office.

Claims (52)

1. ~An isolated polypeptide comprising an amino acid sequence that is encoded by a polynucleotide sequence recited in any one of SEQ ID NOs: 225, 326, 328 or 330, or a complementary sequence thereto.

2. ~An isolated polypeptide comprising a sequence recited in any one of SEQ ID

NOs: 327, 329, 331 or 338.

3. ~An isolated polynucleotide encoding a prostate tumor protein, or a variant thereof, wherein the tumor protein comprises an amino acid sequence that is encoded by a polynucleotide comprising a sequence recited in any one of SEQ ID NOs: 225, 326, 328 or 330, or a complement of any of the foregoing sequences.

4. ~An isolated polynucleotide comprising a sequence recited in any one of SEQ
ID
NOs: 225, 326, 328 or 330.

5. ~An isolated polynucleotide complementary to a polynucleotide according to claim 3.

6. ~An expression vector comprising a polynucleotide according to claim 3.

7. ~A host cell transformed or transfected with an expression vector according to claim 6.

8. ~An expression vector comprising a polynucleotide according to claim 5.

9. ~A host cell transformed or transfected with an expression vector according to claim 6.

10. ~A pharmaceutical composition comprising a polypeptide according to claim 1, in combination with a physiologically acceptable carrier.

11. ~A vaccine comprising a polypeptide according to claim 1, in combination with a non-specific immune response enhancer.

12. ~A vaccine according to claim 11, wherein the non-specific immune response enhancer is an adjuvant.

13. ~A vaccine according to claim 11, wherein the non-specific immune response enhancer induces a predominantly Type I response.

14. ~A pharmaceutical composition comprising a polynucleotide according to claim 4, in combination with a physiologically acceptable carrier.

15. ~A vaccine comprising a polynucleotide according to claim 3, in combination with a non-specific immune response enhancer.

16. ~A vaccine according to claim 15, wherein the non-specific immune response enhancer is an adjuvant.

17. ~A vaccine according to claim 15, wherein the non-specific immune response enhancer induces a predominantly Type I response.

18. ~A pharmaceutical composition comprising an antigen-presenting cell that expresses a polypeptide according to claim 1, in combination with a pharmaceutically acceptable carrier of excipient.

19. ~A pharmaceutical composition-according to claim 18, wherein the antigen presenting cell is a dentritic cell or a macrophage.

20. ~A vaccine comprising an antigen-presenting cell that expresses a polypeptide according to claim 1, in combination with a non-specific immune response enhancer.

21. ~A vaccine according to claim 20, wherein the non-specific immune response enhancer is an adjuvant.

22. ~A vaccine according to claim 20, wherein the non-specific immune response enhancer incudes a predominantly Type I response.

23. ~A vaccine according to claim 20, wherein the antigen-presenting cell is a dendritic cell.

24. ~A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a polypeptide according to claim 1, and thereby inhibiting the development of a cancer in the patient.

25. ~A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a polynucleotide according to claim 3, and thereby inhibiting the development of a cancer in the patient.

26. ~A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of an antigen-presenting cell that expresses a polypeptide according to claim 1, and thereby inhibiting the development of a cancer in the patient.

27. ~A method according to claim 26, wherein the antigen-presenting cell is a dendritic cell.

28. ~A method according to any one of claim 24-26, wherein the cancer is prostate cancer.

29. ~A fusion protein comprising at least one polypeptide according to claim 1.

30. ~A fusion protein according to claim 29, wherein the fusion protein comprises an expression enhancer that increases expression of the fusion protein in a host cell transfected with a polynucleotide encoding he fusion protein.

31. ~A fusion protein according to claim 29, wherein the fusion protein comprises a T
helper epitope that is not present within the polypeptide of claim 1.

32. ~A fusion protein according to claim 29, wherein the fusion protein comprises an affinity tag.

33. ~An isolated polynucleotide encoding a fusion protein according to any one of claims 29 to 32.

34. ~A pharmaceutical composition comprising a fusion protein according to claim 29, in combination with a physiologically acceptable carrier.

35. A vaccine comprises a fusion protein according to claim 29, in combination with a non-specific immune response enhancer.

36. A vaccine according to claim 35, wherein the non-specific immune response enhancer is an adjuvant.

37. A vaccine according to claim 35, wherein the non-specific immune response enhancer induces a predominantly Type I response.

38. A pharmaceutical composition comprising a polynucleotide according to claim 33, in combination with a physiologically acceptable carrier.

39. A vaccine comprising a polynucleotide according to claim 33, in combination with a non-specific immune response enhancer.

40. A vaccine according to claim 39, wherein the non-specific immune response enhancer is an adjuvant.

41. A vaccine according to claim 39, wherein the non-specific immune response enhancer induces a predominantly Type I response.

42. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a pharmaceutical composition according to claim 34 or claim 38.

43. A method of inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a vaccine according to claim 35 or claim 39.

44. A method for removing tumor cells from a biological sample, comprising contacting a biological sample with T cells that specifically react with a prostate tumor protein, wherein the tumor protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group cnsisting of (i) polynucleotide recited in any one of SEQ ID NOs: 225, 326, 328 or 330; and (ii) complements of the foregoing polynucleotides;

wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of cells expressing the prostate tumor protein from the sample.

45. A method according to claim 44, wherein the biological sample is blood or a fraction thereof.

46. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient a biological sample treated according to the method of claim 44.

47. A method for stimulating and/or expanding T cells specific for a prostate tumor protein, comprising contacting T cells with one or more of:
(i) a polypeptide according to claim 1;
(ii) a polypeptide encoded by a polynucleotide comprising a sequence provided in any one of SEQ ID NOs: 225, 326, 328 or 330;
(iii) a polynucleotide encoding a polypeptide of (i) or (ii); and/or (iv) an antigen presenting cell that expresses a polypeptide of (i) or (ii);
under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells.

48. An isolated T cell population, comprising T cells prepared according to the method of claim 47.

49. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a T cell population according to claim 48.

50. A method 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 at least one component selected from the group consisting of:
(i) a polypeptide according to claim 1;
(ii) a polypeptide encoded by a polynucleotide comprising a sequence of any one of SEQ ID NOs: 225, 326, 328 or 330;
(iii) a polynucleotide encoding a polypeptide of (i) of (ii); or (iv) an antigen-presenting cell that expresses a polypeptide of (i) or (ii);
such that T cell proliferate; and (b) administering to the patient an effective amount of proliferated T cells, and thereby inhibiting the development of a cancer in the patient.

51. A method 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 at least one component selected from the group consisting of:
(i) a polypeptide according to claim 1;
(ii) a polypeptide encoded by a polynucleotide comprising a sequence of any one of SEQ ID NOs: 225, 326, 328 or 330;
(iii) a polynucleotide encoding a polypeptide of (i) or (ii); or (iv) an antigen-presenting cell that expresses a polypeptide of (i) or (ii);
such that T cells proliferate;
(b) cloning at least one proliferated cell; and (c) administering to the patient an effective amount of the cloned T cells, and thereby inhibiting the development of a cancer in the patient.

52. An isolated polypeptide according to any one of claims 1 to 5 or 33, an expression vector according to claim 6 or 8, a host cell according to claim 7 or 9, a pharmaceutical composition according to claim 10, 14, 18 to 19, 34 or 38, a vaccine according to any one of claims 11 to 13, 15 to 17, 20, to 23, 35 to 37 or 39 to 41, a method according to any one of claims 24 to 28, 42 to 47 or 49 to 51, a fusion protein according to any one of claims 29 to 32 or an isolated T cell according to claim 48 substantially as hereinbefore described with reference to the Figures and/or Examples.