JP2009142284A - Composition and method for therapy and diagnosis of prostate cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide compositions and methods for the therapy and diagnosis of cancer, such as prostate cancer, and to provide diagnostic methods. <P>SOLUTION: The composition may comprise one or more prostate-specific proteins, immunogenic portions thereof, or polynucleotides that encode such portions. Alternatively, the therapeutic composition may contain antigen presenting cells that express the prostate-specific proteins, or T cells that are specific for cells expressing such proteins. Such compositions may be used, for example, for the prevention and treatment of disease such as prostate cancer. Detection of a prostate-specific protein in a sample, or mRNA encoding such a protein is also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates generally to the therapy and diagnosis of cancer, eg, prostate cancer. More particularly, the present invention relates to a polypeptide comprising at least a portion of a prostate specific protein, and a polynucleotide encoding such a polypeptide. Such polypeptides and polynucleotides can be used in vaccines and pharmaceutical compositions for the prevention and treatment of prostate cancer and the diagnosis and monitoring of such cancers.

  Prostate cancer is the most common form of cancer among men, with a 30% incidence in men over 50 years of age. Overwhelming clinical evidence shows that human prostate cancer has a tendency to metastasize to bone, and the disease inevitably progresses from androgen dependence to an androgen resistant state, increasing patient mortality. This dominant disease is the second leading cause of cancer death among men in the United States.

  Despite considerable research into the treatment of this disease, prostate cancer remains difficult to treat. In general, treatment is based on surgery and / or radiation therapy, but these methods are ineffective in a significant proportion of cases. Two previously identified prostate-specific proteins, prostate-specific antigen (PSA) and prostate acid phosphatase (PAP), have limited therapeutic and diagnostic potential. For example, PSA levels do not always correlate well with the presence of prostate cancer and are positive in a certain proportion of non-prostate cancer cases, including benign prostate hyperplasia (BPH). Furthermore, PSA measurements correlate with prostate volume and do not indicate the level of metastasis.

  Despite considerable research on therapies for these and other cancers, it is difficult to effectively diagnose and treat prostate cancer. Accordingly, there is a need in the art for improved methods for detecting and treating such cancers. The present invention satisfies these needs and provides other related advantages.

SUMMARY OF THE INVENTION Briefly stated, the present invention provides compositions and methods for the diagnosis and therapy of cancer, eg, prostate cancer. In one embodiment, the present invention provides a polypeptide comprising at least a portion of a prostate specific protein, or a variant thereof. Certain portions and other variants are immunogenic so that the ability of the variant to react with antigen-specific antisera is not substantially reduced. Within certain embodiments, the polypeptide comprises at least an immunogenic portion of a prostate-specific protein, or a variant thereof, and the oncoprotein is encoded by a polynucleotide sequence selected from the group consisting of: (A) SEQ ID NOs: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381 , 382, 384-476, 524, 526, 530, 531, 533, 535, and 536, the sequence described in any one of the above sequences under moderately stringent conditions (b) A hybridizing sequence; and (c) any complement of sequence (a) or (b). In certain specific embodiments, such a polypeptide comprises at least a portion of a protein comprising an amino acid sequence selected from the group consisting of any one of the following sequences, or a variant thereof: Consisting of: SEQ ID NOs: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527 532, 534, 537-550.

  The present invention further includes a polynucleotide encoding the aforementioned polypeptide, or a portion thereof (eg, a portion encoding at least 15 amino acid residues of prostate specific protein), an expression vector comprising such a polynucleotide, And host cells transformed or transfected with such expression vectors.

  Within another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned polypeptide or polynucleotide and a pharmaceutically acceptable carrier.

  Within the scope of the relevant aspects of the invention, a vaccine is provided. Such a vaccine comprises the aforementioned polypeptide or polynucleotide and an immunostimulant.

  The present invention further provides a pharmaceutical composition comprising (a) an antibody or antibody-binding fragment thereof that specifically binds to a prostate-specific protein, and (b) a pharmaceutically acceptable carrier. In certain embodiments, the invention includes SEQ ID NOs: 496, 504, 505, 509-517, 522, with a monoclonal antibody comprising a complementarity determining region selected from the group consisting of SEQ ID NOs: 502, 503, and 506-508. And a monoclonal antibody that specifically binds to an amino acid sequence selected from the group consisting of 541-550.

  In a further aspect, there is provided a pharmaceutical composition comprising (a) an antigen presenting cell that expresses the aforementioned polypeptide, and (b) a pharmaceutically acceptable carrier or excipient. Antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B cells.

  Within the scope of related embodiments, there is provided a vaccine comprising (a) an antigen-presenting cell that expresses the aforementioned polypeptide, and (b) an immunostimulatory agent.

  The invention further provides, in other embodiments, fusion proteins comprising at least one of the aforementioned polypeptides, as well as polynucleotides encoding such fusion proteins.

  Within the scope of related embodiments, a pharmaceutical composition is provided comprising a combination of a fusion protein, or a polynucleotide encoding the fusion protein, and a pharmaceutically acceptable carrier.

  Furthermore, within other embodiments, a vaccine comprising a combination of a fusion protein, or a polynucleotide encoding the fusion protein, and an immunostimulatory agent is provided.

  Within a further aspect, the present invention provides a method of preventing the development of cancer in a patient comprising administering to the patient a pharmaceutical composition or vaccine as described above.

  The invention further includes, within another embodiment, contacting a biological sample with a T cell that specifically reacts with a prostate specific protein, wherein the cell expressing the protein is removed from the sample. A method is provided for removing tumor cells from a biological sample, wherein the contacting step is carried out under conditions that allow for removal and for a sufficient time.

  Within the scope of related embodiments, there is provided a method of preventing the development of cancer in a patient comprising administering to the patient a biological sample treated as described above.

  Further, within other embodiments, the method comprises contacting the T cell with one or more of the following under conditions that allow for stimulation and / or expression of the T cell and for a sufficient period of time: Methods are provided for stimulating and / or expanding T cells specific for prostate tumor proteins: (i) a polypeptide as described above; (ii) a polynucleotide encoding such a polypeptide; and / or ( iii) Antigen presenting cells that express such polypeptides.

  Within a further aspect, the present invention provides a method of preventing the development of cancer in a patient, comprising administering to the patient an effective amount of the aforementioned T cell population.

  The present invention further provides a method of preventing the development of cancer in a patient comprising the steps of: (a) isolating CD4 + and / or CD8 + T cells isolated from the patient according to one or more of the following: Incubating with: (i) at least an immunogenic portion of a prostate tumor protein; (ii) a polynucleotide encoding such a polypeptide; and (iii) an antigen presenting cell expressing such a polypeptide; and (b ) Administering an effective amount of expanded T cells to the patient, thereby preventing the development of cancer in the patient. Proliferated cells can be cloned prior to administration to a patient, but this is not necessary.

  Within a further aspect, the present invention provides a method for determining the presence or absence of cancer in a patient comprising the following steps: (a) providing a biological sample obtained from the patient as described above Contacting with a binding agent that binds to the polypeptide: (b) detecting the amount of the polypeptide that binds to the binding agent in the sample; and (c) comparing the amount of said polypeptide to a predetermined cutoff value; Then the presence or absence of cancer in the patient is determined. Within preferred embodiments, the binding agent is an antibody, more preferably a monoclonal antibody. The cancer can be prostate cancer.

  The present invention also provides, within other embodiments, a method of monitoring cancer progression in a patient. Such a method comprises the following steps: (a) contacting a biological sample obtained from a patient with a binding agent that binds to the aforementioned polypeptide at a first time point; (b) binding in the sample Detecting the amount of polypeptide binding to the agent; (c) repeating steps (a) and (b) using biological samples obtained from the patient at subsequent time points; and (d) step (c ) Is compared to the amount detected in step (b) and then the progression of cancer in the patient is monitored.

  The present invention also provides, within other embodiments, a method for detecting the presence or absence of cancer in a patient comprising the following steps: (a) a polynucleotide encoding a prostate specific protein Contacting an oligonucleotide that hybridizes to a biological sample obtained from a patient; (b) detecting the level of polynucleotide, preferably mRNA, that hybridizes to the oligonucleotide in the sample; ) Compare the level of polynucleotide that hybridizes to the oligonucleotide to a predetermined cutoff value, and then determine the presence or absence of cancer in the patient. Within certain embodiments, for example, polymerase chain reaction using at least one oligonucleotide primer that hybridizes to a polynucleotide encoding the aforementioned polypeptide, or to one component of such a polynucleotide. To detect the amount of mRNA. Within other embodiments, the amount of mRNA is detected by hybridization techniques using polynucleotides encoding the aforementioned polypeptides, or oligonucleotide probes that hybridize to one component of such polynucleotides. .

  In a related embodiment, there is provided a method of monitoring cancer progression in a patient comprising the following steps: (a) an oligonucleotide that hybridizes to a polynucleotide encoding a prostate specific protein; Contacting the resulting biological sample; (b) detecting the amount of polynucleotide that hybridizes to the oligonucleotide in the sample; (c) using the biological sample obtained from the patient at a subsequent time point Repeating steps (a) and (b); and (d) comparing the amount of polynucleotide detected in step (c) with the amount of polynucleotide detected in step (b) and then in the patient Monitor cancer progression.

  Within further aspects, the present invention provides antibodies, such as monoclonal antibodies, that bind to the aforementioned polypeptides, as well as diagnostic kits comprising such antibodies. Also provided are diagnostic kits comprising one or more of the aforementioned oligonucleotide probes or primers.

  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 were individually cited.

FIG. 1 shows the ability of T cells to kill fibroblasts expressing the representative prostate specific polypeptide P502S compared to control fibroblasts. As indicated, the percent lysis is shown as a series of effector: target ratios. FIGS. 2A and 2B recognize cells expressing the representative prostate specific polypeptide P502S. In each case, the number of γ-interferon spots is shown for a different number of responders. In FIG. 2A, data is presented for fibroblasts pulsed with the P2S-12 peptide compared to fibroblasts pulsed with the control E75 peptide. In FIG. 2B, data is presented for fibroblasts expressing P502S compared to fibroblasts expressing HER-2- / neu. FIG. 3 represents a peptide competition binding assay showing that the P1S # 10 peptide from P501S binds to HLA-A2. Peptide P1S # 10 inhibits HLA-A2 restricted presentation of fluM58 peptide against CTL clone D150M58 in a TNF release bioassay. D150M58 CTL is specific for the HLA-A2 binding influenza matrix peptide fluM58. Figure 4 shows the ability of T cell lines generated from P1S # 10 immunized mice to specifically lyse P1S # 10 pulsed Jurkat A2Kb targets and P501S-introduced Jurkat A2Kb targets compared to EGFP-introduced Jurkat A2Kb. Show. As indicated, the percent lysis is shown as a series of effector / target ratios. FIG. 5 shows the ability of a T cell clone to recognize and specifically lyse Jurkat A2Kb cells expressing the representative prostate specific polypeptide P501S, whereby the P1S # 10 peptide is naturally processed by the P501S polypeptide. It is proved that it can be an epitope. Figures 6A and 6B are graphs showing the specificity of the CD8 ⁺ cell line (3A-1) for a representative prostate specific antigen (P501S). FIG. 6A shows the results of the ⁵¹ Cr release assay. As indicated, the percent specific lysis is shown as a series of effector: target ratios. FIG. 6B shows interferon-gamma production by 3A-1 cells stimulated with autologous B-LCL introduced with P501S at varying effector: target ratios. FIG. 7 is a Western blot showing the expression of P501S in baculovirus. FIG. 8 shows the results of an epitope mapping test for P501S. FIG. 9 is a schematic representation of the P501S protein showing the location of the transmembrane domain and putative intracellular and extracellular domains. FIG. 10 is a genomic map showing the location of prostate genes P775P, P704P, B305D, P712P, and P774P within the Cat Eye syndrome region of chromosome 22q11.2. FIG. 11 shows the results of an ELISA assay for an antibody specific for the P501S peptide.

Sequence identification SEQ ID NO: 1 is the cDNA sequence determined for F1-13.

SEQ ID NO: 2 is the 3 ′ cDNA sequence determined for F1-12.

SEQ ID NO: 3 is the 5 ′ cDNA sequence determined for F1-12.

SEQ ID NO: 4 is the 3 ′ cDNA sequence determined for F1-16.

SEQ ID NO: 5 is the 3 ′ cDNA sequence determined for H1-1.

SEQ ID NO: 6 is the 3 ′ cDNA sequence determined for H1-9.

SEQ ID NO: 7 is the 3 ′ cDNA sequence determined for H1-4.

SEQ ID NO: 8 is the 3 ′ cDNA sequence determined for J1-17.

SEQ ID NO: 9 is the 5 ′ cDNA sequence determined for J1-17.

SEQ ID NO: 10 is the 3 ′ cDNA sequence determined for L1-12.

SEQ ID NO: 11 is the 5 ′ cDNA sequence determined for L1-12.

SEQ ID NO: 12 is the 3 ′ cDNA sequence determined for N1-1862.

SEQ ID NO: 13 is the 5 ′ cDNA sequence determined for N1-1862.

SEQ ID NO: 14 is the 3 ′ cDNA sequence determined for J1-13.

SEQ ID NO: 15 is the 5 ′ cDNA sequence determined for J1-13.

SEQ ID NO: 16 is the 3 ′ cDNA sequence determined for J1-19.

SEQ ID NO: 17 is the 5 ′ cDNA sequence determined for J1-19.

SEQ ID NO: 18 is the 3 ′ cDNA sequence determined for J1-25.

SEQ ID NO: 19 is the 5 ′ cDNA sequence determined for J1-25.

SEQ ID NO: 20 is the 5 ′ cDNA sequence determined for J1-24.

SEQ ID NO: 21 is the 3 ′ cDNA sequence determined for J1-24.

SEQ ID NO: 22 is the 5 ′ cDNA sequence determined for K1-58.

SEQ ID NO: 23 is the 3 ′ cDNA sequence determined for K1-58.

SEQ ID NO: 24 is the 5 ′ cDNA sequence determined for K1-63.

SEQ ID NO: 25 is the 3 ′ cDNA sequence determined for K1-63.

SEQ ID NO: 26 is the 5 ′ cDNA sequence determined for L1-4.

SEQ ID NO: 27 is the 3 ′ cDNA sequence determined for L1-4.

SEQ ID NO: 28 is the 5 ′ cDNA sequence determined for L1-14.

SEQ ID NO: 29 is the 3 ′ cDNA sequence determined for L1-14.

SEQ ID NO: 30 is the 3 ′ cDNA sequence determined for J1-12.

SEQ ID NO: 31 is the 3 ′ cDNA sequence determined for J1-16.

SEQ ID NO: 32 is the 3 ′ cDNA sequence determined for J1-21.

SEQ ID NO: 33 is the 3 ′ cDNA sequence determined for K1-48.

SEQ ID NO: 34 is the 3 ′ cDNA sequence determined for K1-55.

SEQ ID NO: 35 is the 3 ′ cDNA sequence determined for L1-2.

SEQ ID NO: 36 is the 3 ′ cDNA sequence determined for L1-6.

SEQ ID NO: 37 is the 3 ′ cDNA sequence determined for N1-1858.

SEQ ID NO: 38 is the 3 ′ cDNA sequence determined for N1-1860.

SEQ ID NO: 39 is the 3 ′ cDNA sequence determined for N1-1861.

SEQ ID NO: 40 is the 3 ′ cDNA sequence determined for J1-1864.

SEQ ID NO: 41 is the cDNA sequence determined for P5.

SEQ ID NO: 42 is the cDNA sequence determined for P8.

SEQ ID NO: 43 is the cDNA sequence determined for P9.

SEQ ID NO: 44 is the cDNA sequence determined for P18.

SEQ ID NO: 45 is the cDNA sequence determined for P20.

SEQ ID NO: 46 is the cDNA sequence determined for P29.

SEQ ID NO: 47 is the cDNA sequence determined for P30.

SEQ ID NO: 48 is the cDNA sequence determined for P34.

SEQ ID NO: 49 is the cDNA sequence determined for P36.

SEQ ID NO: 50 is the cDNA sequence determined for P38.

SEQ ID NO: 51 is the cDNA sequence determined for P39.

SEQ ID NO: 52 is the cDNA sequence determined for P42.

SEQ ID NO: 53 is the cDNA sequence determined for P47.

SEQ ID NO: 54 is the cDNA sequence determined for P49.

SEQ ID NO: 55 is the cDNA sequence determined for P50.

SEQ ID NO: 56 is the cDNA sequence determined for P53.

SEQ ID NO: 57 is the cDNA sequence determined for P55.

SEQ ID NO: 58 is the cDNA sequence determined for P60.

SEQ ID NO: 59 is the cDNA sequence determined for P64.

SEQ ID NO: 60 is the cDNA sequence determined for P65.

SEQ ID NO: 61 is the cDNA sequence determined for P73.

SEQ ID NO: 62 is the cDNA sequence determined for P75.

SEQ ID NO: 63 is the cDNA sequence determined for P76.

SEQ ID NO: 64 is the cDNA sequence determined for P79.

SEQ ID NO: 65 is the cDNA sequence determined for P84.

SEQ ID NO: 66 is the cDNA sequence determined for P68.

SEQ ID NO: 67 is the cDNA sequence determined for P80.

SEQ ID NO: 68 is the cDNA sequence determined for P82.

SEQ ID NO: 69 is the cDNA sequence determined for U1-3064.

SEQ ID NO: 70 is the cDNA sequence determined for U1-3065.

SEQ ID NO: 71 is the cDNA sequence determined for V1-3692.

SEQ ID NO: 72 is the cDNA sequence determined for 1A-3905.

SEQ ID NO: 73 is the cDNA sequence determined for V1-3686.

SEQ ID NO: 74 is the cDNA sequence determined for R1-2330.

SEQ ID NO: 75 is the cDNA sequence determined for 1B-3976.

SEQ ID NO: 76 is the cDNA sequence determined for V1-3679.

SEQ ID NO: 77 is the cDNA sequence determined for 1G-4736.

SEQ ID NO: 78 is the cDNA sequence determined for 1G-4739.

SEQ ID NO: 79 is the cDNA sequence determined for 1G-4741.

SEQ ID NO: 80 is the cDNA sequence determined for 1G-4744.

SEQ ID NO: 81 is the cDNA sequence determined for 1G-4734.

SEQ ID NO: 82 is the cDNA sequence determined for 1H-4774.

SEQ ID NO: 83 is the cDNA sequence determined for 1H-4781.

SEQ ID NO: 84 is the cDNA sequence determined for 1H-4785.

SEQ ID NO: 85 is the cDNA sequence determined for 1H-4787.

SEQ ID NO: 86 is the cDNA sequence determined for 1H-4796.

SEQ ID NO: 87 is the cDNA sequence determined for 1I-4807.

SEQ ID NO: 88 is the cDNA sequence determined for 1I-4810.

SEQ ID NO: 89 is the cDNA sequence determined for 1I-4811.

SEQ ID NO: 90 is the cDNA sequence determined for 1J-4876.

SEQ ID NO: 91 is the cDNA sequence determined for 1K-4884.

SEQ ID NO: 92 is the cDNA sequence determined for 1K-4896.

SEQ ID NO: 93 is the cDNA sequence determined for 1G-4761.

SEQ ID NO: 94 is the cDNA sequence determined for 1G-4762.

SEQ ID NO: 95 is the cDNA sequence determined for 1H-4766.

SEQ ID NO: 96 is the cDNA sequence determined for 1H-4770.

SEQ ID NO: 97 is the cDNA sequence determined for 1H-4771.

SEQ ID NO: 98 is the cDNA sequence determined for 1H-4772.

SEQ ID NO: 99 is the cDNA sequence determined for 1D-4297.

SEQ ID NO: 100 is the cDNA sequence determined for 1D-4309.

SEQ ID NO: 101 is the cDNA sequence determined for 1D.1-4278.

SEQ ID NO: 102 is the cDNA sequence determined for 1D-4288.

SEQ ID NO: 103 is the cDNA sequence determined for 1D-4283.

SEQ ID NO: 104 is the cDNA sequence determined for 1D-4304.

SEQ ID NO: 105 is the cDNA sequence determined for 1D-4296.

SEQ ID NO: 106 is the cDNA sequence determined for 1D-4280.

SEQ ID NO: 107 is the full length cDNA sequence determined for F1-12 (also referred to as P504S).

SEQ ID NO: 108 is the amino acid sequence predicted for F1-12.

SEQ ID NO: 109 is the full-length cDNA sequence determined for J1-17.

SEQ ID NO: 110 is the full-length cDNA sequence determined for L1-12 (also referred to as P501S).

SEQ ID NO: 111 is the full-length cDNA sequence determined for N1-1862 (also referred to as P503S).

SEQ ID NO: 112 is the predicted amino acid sequence for J1-17.

SEQ ID NO: 113 is the amino acid sequence predicted for L1-12 (also referred to as P501S).

SEQ ID NO: 114 is the amino acid sequence predicted for N1-1862 (also referred to as P503S).

SEQ ID NO: 115 is the cDNA sequence determined for P89.

SEQ ID NO: 116 is the cDNA sequence determined for P90.

SEQ ID NO: 117 is the cDNA sequence determined for P92.

SEQ ID NO: 118 is the cDNA sequence determined for P95.

SEQ ID NO: 119 is the cDNA sequence determined for P98.

SEQ ID NO: 120 is the cDNA sequence determined for P102.

SEQ ID NO: 121 is the cDNA sequence determined for P110.

SEQ ID NO: 122 is the cDNA sequence determined for P111.

SEQ ID NO: 123 is the cDNA sequence determined for P114.

SEQ ID NO: 124 is the cDNA sequence determined for P115.

SEQ ID NO: 125 is the cDNA sequence determined for P116.

SEQ ID NO: 126 is the cDNA sequence determined for P124.

SEQ ID NO: 127 is the cDNA sequence determined for P126.

SEQ ID NO: 128 is the cDNA sequence determined for P130.

SEQ ID NO: 129 is the cDNA sequence determined for P133.

SEQ ID NO: 130 is the cDNA sequence determined for P138.

SEQ ID NO: 131 is the cDNA sequence determined for P143.

SEQ ID NO: 132 is the cDNA sequence determined for P151.

SEQ ID NO: 133 is the cDNA sequence determined for P156.

SEQ ID NO: 134 is the cDNA sequence determined for P157.

SEQ ID NO: 135 is the cDNA sequence determined for P166.

SEQ ID NO: 136 is the cDNA sequence determined for P176.

SEQ ID NO: 137 is the cDNA sequence determined for P178.

SEQ ID NO: 138 is the cDNA sequence determined for P179.

SEQ ID NO: 139 is the cDNA sequence determined for P185.

SEQ ID NO: 140 is the cDNA sequence determined for P192.

SEQ ID NO: 141 is the cDNA sequence determined for P201.

SEQ ID NO: 142 is the cDNA sequence determined for P204.

SEQ ID NO: 143 is the cDNA sequence determined for P208.

SEQ ID NO: 144 is the cDNA sequence determined for P211.

SEQ ID NO: 145 is the cDNA sequence determined for P213.

SEQ ID NO: 146 is the cDNA sequence determined for P219.

SEQ ID NO: 147 is the cDNA sequence determined for P237.

SEQ ID NO: 148 is the cDNA sequence determined for P239.

SEQ ID NO: 149 is the cDNA sequence determined for P248.

SEQ ID NO: 150 is the cDNA sequence determined for P251.

SEQ ID NO: 151 is the cDNA sequence determined for P255.

SEQ ID NO: 152 is the cDNA sequence determined for P256.

SEQ ID NO: 153 is the cDNA sequence determined for P259.

SEQ ID NO: 154 is the cDNA sequence determined for P260.

SEQ ID NO: 155 is the cDNA sequence determined for P263.

SEQ ID NO: 156 is the cDNA sequence determined for P264.

SEQ ID NO: 157 is the cDNA sequence determined for P266.

SEQ ID NO: 158 is the cDNA sequence determined for P270.

SEQ ID NO: 159 is the cDNA sequence determined for P272.

SEQ ID NO: 160 is the cDNA sequence determined for P278.

SEQ ID NO: 161 is the cDNA sequence determined for P105.

SEQ ID NO: 162 is the cDNA sequence determined for P107.

SEQ ID NO: 163 is the cDNA sequence determined for P137.

SEQ ID NO: 164 is the cDNA sequence determined for P194.

SEQ ID NO: 165 is the cDNA sequence determined for P195.

SEQ ID NO: 166 is the cDNA sequence determined for P196.

SEQ ID NO: 167 is the cDNA sequence determined for P220.

SEQ ID NO: 168 is the cDNA sequence determined for P234.

SEQ ID NO: 169 is the cDNA sequence determined for P235.

SEQ ID NO: 170 is the cDNA sequence determined for P243.

SEQ ID NO: 171 is the cDNA sequence determined for P703P-DE1.

SEQ ID NO: 172 is the amino acid sequence predicted for P703P-DE1.

SEQ ID NO: 173 is the cDNA sequence determined for P703P-DE2.

SEQ ID NO: 174 is the cDNA sequence determined for P703P-DE6.

SEQ ID NO: 175 is the cDNA sequence determined for P703P-DE13.

SEQ ID NO: 176 is the amino acid sequence predicted for P703P-DE13.

SEQ ID NO: 177 is the cDNA sequence determined for P703P-DE14.

SEQ ID NO: 178 is the predicted amino acid sequence for P703P-DE14.

SEQ ID NO: 179 is the extended cDNA sequence determined for 1G-4736.

SEQ ID NO: 180 is the extended cDNA sequence determined for 1G-4738.

SEQ ID NO: 181 is the extended cDNA sequence determined for 1G-4741.

SEQ ID NO: 182 is the extended cDNA sequence determined for 1G-4744.

SEQ ID NO: 183 is the extended cDNA sequence determined for 1H-4774.

SEQ ID NO: 184 is the extended cDNA sequence determined for 1H-4781.

SEQ ID NO: 185 is the extended cDNA sequence determined for 1H-4785.

SEQ ID NO: 186 is the extended cDNA sequence determined for 1H-4787.

SEQ ID NO: 187 is the extended cDNA sequence determined for 1H-4796.

SEQ ID NO: 188 is the extended cDNA sequence determined for 1I-4807.

SEQ ID NO: 189 is the 3 ′ cDNA sequence determined for 1I-4810.

SEQ ID NO: 190 is the 3 ′ cDNA sequence determined for 1I-4811.

SEQ ID NO: 191 is the extended cDNA sequence determined for 1J-4876.

SEQ ID NO: 192 is the extended cDNA sequence determined for 1K-4884.

SEQ ID NO: 193 is the extended cDNA sequence determined for 1K-4896.

SEQ ID NO: 194 is the extended cDNA sequence determined for 1G-4761.

SEQ ID NO: 195 is the extended cDNA sequence determined for 1G-4762.

SEQ ID NO: 196 is the extended cDNA sequence determined for 1H-4766.

SEQ ID NO: 197 is the 3 ′ cDNA sequence determined for 1H-4770.

SEQ ID NO: 198 is the 3 ′ cDNA sequence determined for 1H-4771.

SEQ ID NO: 199 is the extended cDNA sequence determined for 1H-4772.

SEQ ID NO: 200 is the extended cDNA sequence determined for 1D-4309.

SEQ ID NO: 201 is the extended cDNA sequence determined for 1D.1-4278.

SEQ ID NO: 202 is the extended cDNA sequence determined for 1D-4288.

SEQ ID NO: 203 is the extended cDNA sequence determined for 1D-4283.

SEQ ID NO: 204 is the extended cDNA sequence determined for 1D-4304.

SEQ ID NO: 205 is the extended cDNA sequence determined for 1D-4296.

SEQ ID NO: 206 is the extended cDNA sequence determined for 1D-4280.

SEQ ID NO: 207 is the cDNA sequence determined for 10-d8fwd.

SEQ ID NO: 208 is the cDNA sequence determined for 10-H10con.

SEQ ID NO: 209 is the cDNA sequence determined for 11-C8rev.

SEQ ID NO: 210 is the cDNA sequence determined for 7.g6fwd.

SEQ ID NO: 211 is the cDNA sequence determined for 7.g6rev.

SEQ ID NO: 212 is the cDNA sequence determined for 8-b5fwd.

SEQ ID NO: 213 is the cDNA sequence determined for 8-b5rev.

SEQ ID NO: 214 is the cDNA sequence determined for 8-b6fwd.

SEQ ID NO: 215 is the cDNA sequence determined for 8-b6rev.

SEQ ID NO: 216 is the cDNA sequence determined for 8-d4fwd.

SEQ ID NO: 217 is the cDNA sequence determined for 8-d9rev.

SEQ ID NO: 218 is the cDNA sequence determined for 8-g3fwd.

SEQ ID NO: 219 is the cDNA sequence determined for 8-g3rev.

SEQ ID NO: 220 is the cDNA sequence determined for 8-h11rev.

SEQ ID NO: 221 is the cDNA sequence determined for g-f12fwd.

SEQ ID NO: 222 is the cDNA sequence determined for g-f3rev.

SEQ ID NO: 223 is the cDNA sequence determined for P509S.

SEQ ID NO: 224 is the cDNA sequence determined for P510S.

SEQ ID NO: 225 is the cDNA sequence determined for P703DE5.

SEQ ID NO: 226 is the cDNA sequence determined for 9-A11.

SEQ ID NO: 227 is the cDNA sequence determined for 8-C6.

SEQ ID NO: 228 is the cDNA sequence determined for 8-H7.

SEQ ID NO: 229 is the cDNA sequence determined for JPTPN13.

SEQ ID NO: 230 is the cDNA sequence determined for JPTPN14.

SEQ ID NO: 231 is the cDNA sequence determined for JPTPN23.

SEQ ID NO: 232 is the cDNA sequence determined for JPTPN24.

SEQ ID NO: 233 is the cDNA sequence determined for JPTPN25.

SEQ ID NO: 234 is the cDNA sequence determined for JPTPN30.

SEQ ID NO: 235 is the cDNA sequence determined for JPTPN34.

SEQ ID NO: 236 is the cDNA sequence determined for PTPN35.

SEQ ID NO: 237 is the cDNA sequence determined for JPTPN36.

SEQ ID NO: 238 is the cDNA sequence determined for JPTPN38.

SEQ ID NO: 239 is the cDNA sequence determined for JPTPN39.

SEQ ID NO: 240 is the cDNA sequence determined for JPTPN40.

SEQ ID NO: 241 is the cDNA sequence determined for JPTPN41.

SEQ ID NO: 242 is the cDNA sequence determined for JPTPN42.

SEQ ID NO: 243 is the cDNA sequence determined for JPTPN45.

SEQ ID NO: 244 is the cDNA sequence determined for JPTPN46.

SEQ ID NO: 245 is the cDNA sequence determined for JPTPN51.

SEQ ID NO: 246 is the cDNA sequence determined for JPTPN56.

SEQ ID NO: 247 is the cDNA sequence determined for PTPN64.

SEQ ID NO: 248 is the cDNA sequence determined for JPTPN65.

SEQ ID NO: 249 is the cDNA sequence determined for JPTPN67.

SEQ ID NO: 250 is the cDNA sequence determined for JPTPN76.

SEQ ID NO: 251 is the cDNA sequence determined for JPTPN84.

SEQ ID NO: 252 is the cDNA sequence determined for JPTPN85.

SEQ ID NO: 253 is the cDNA sequence determined for JPTPN86.

SEQ ID NO: 254 is the cDNA sequence determined for JPTPN87.

SEQ ID NO: 255 is the cDNA sequence determined for JPTPN88.

SEQ ID NO: 256 is the cDNA sequence determined for JP1F1.

SEQ ID NO: 257 is the cDNA sequence determined for JP1F2.

SEQ ID NO: 258 is the cDNA sequence determined for JP1C2.

SEQ ID NO: 259 is the cDNA sequence determined for JP1B1.

SEQ ID NO: 260 is the cDNA sequence determined for JP1B2.

SEQ ID NO: 261 is the cDNA sequence determined for JP1D3.

SEQ ID NO: 262 is the cDNA sequence determined for JP1A4.

SEQ ID NO: 263 is the cDNA sequence determined for JP1F5.

SEQ ID NO: 264 is the cDNA sequence determined for JP1E6.

SEQ ID NO: 265 is the cDNA sequence determined for JP1D6.

SEQ ID NO: 266 is the cDNA sequence determined for JP1B5.

SEQ ID NO: 267 is the cDNA sequence determined for JP1A6.

SEQ ID NO: 268 is the cDNA sequence determined for JP1E8.

SEQ ID NO: 269 is the cDNA sequence determined for JP1D7.

SEQ ID NO: 270 is the cDNA sequence determined for JP1D9.

SEQ ID NO: 271 is the cDNA sequence determined for JP1C10.

SEQ ID NO: 272 is the cDNA sequence determined for JP1A9.

SEQ ID NO: 273 is the cDNA sequence determined for JP1F12.

SEQ ID NO: 274 is the cDNA sequence determined for JP1E12.

SEQ ID NO: 275 is the cDNA sequence determined for JP1D11.

SEQ ID NO: 276 is the cDNA sequence determined for JP1C11.

SEQ ID NO: 277 is the cDNA sequence determined for JP1C12.

SEQ ID NO: 278 is the cDNA sequence determined for JP1B12.

SEQ ID NO: 279 is the cDNA sequence determined for JP1A12.

SEQ ID NO: 280 is the cDNA sequence determined for JP8G2.

SEQ ID NO: 281 is the cDNA sequence determined for JP8H1.

SEQ ID NO: 282 is the cDNA sequence determined for JP8H2.

SEQ ID NO: 283 is the cDNA sequence determined for JP8A3.

SEQ ID NO: 284 is the cDNA sequence determined for JP8A4.

SEQ ID NO: 285 is the cDNA sequence determined for JP8C3.

SEQ ID NO: 286 is the cDNA sequence determined for JP8G4.

SEQ ID NO: 287 is the cDNA sequence determined for JP8B6.

SEQ ID NO: 288 is the cDNA sequence determined for JP8D6.

SEQ ID NO: 289 is the cDNA sequence determined for JP8F5.

SEQ ID NO: 290 is the cDNA sequence determined for JP8A8.

SEQ ID NO: 291 is the cDNA sequence determined for JP8C7.

SEQ ID NO: 292 is the cDNA sequence determined for JP8D7.

SEQ ID NO: 293 is the cDNA sequence determined for P8D8.

SEQ ID NO: 294 is the cDNA sequence determined for JP8E7.

SEQ ID NO: 295 is the cDNA sequence determined for JP8F8.

SEQ ID NO: 296 is the cDNA sequence determined for JP8G8.

SEQ ID NO: 297 is the cDNA sequence determined for JP8B10.

SEQ ID NO: 298 is the cDNA sequence determined for JP8C10.

SEQ ID NO: 299 is the cDNA sequence determined for JP8E9.

SEQ ID NO: 300 is the cDNA sequence determined for JP8E10.

SEQ ID NO: 301 is the cDNA sequence determined for JP8F9.

SEQ ID NO: 302 is the cDNA sequence determined for JP8H9.

SEQ ID NO: 303 is the cDNA sequence determined for JP8C12.

SEQ ID NO: 304 is the cDNA sequence determined for JP8E11.

SEQ ID NO: 305 is the cDNA sequence determined for JP8E12.

SEQ ID NO: 306 is the amino acid sequence for peptide PS2 # 12.

SEQ ID NO: 307 is the cDNA sequence determined for P711P.

SEQ ID NO: 308 is the cDNA sequence determined for P712P.

SEQ ID NO: 309 is the cDNA sequence determined for CLONE23.

SEQ ID NO: 310 is the cDNA sequence determined for P774P.

SEQ ID NO: 311 is the cDNA sequence determined for P775P.

SEQ ID NO: 312 is the cDNA sequence determined for P715P.

SEQ ID NO: 313 is the cDNA sequence determined for P710P.

SEQ ID NO: 314 is the cDNA sequence determined for P767P.

SEQ ID NO: 315 is the cDNA sequence determined for P768P.

SEQ ID NOs: 316-325 are determined cDNA sequences of previously isolated genes.

SEQ ID NO: 326 is the cDNA sequence determined for P703PDE5.

SEQ ID NO: 327 is the amino acid sequence predicted for P703PDE5.

SEQ ID NO: 328 is the cDNA sequence determined for P703P6.26.

SEQ ID NO: 329 is the amino acid sequence predicted for P703P6.26.

SEQ ID NO: 330 is the cDNA sequence determined for P703PX-23.

SEQ ID NO: 331 is the predicted amino acid sequence for P703PX-23.

SEQ ID NO: 332 is the full length cDNA sequence determined for P509S.

SEQ ID NO: 333 is the extended cDNA sequence determined for P707P (also referred to as 11-C9).

SEQ ID NO: 334 is the cDNA sequence determined for P714P.

SEQ ID NO: 335 is the cDNA sequence determined for P705P (also referred to as 9-F3).

SEQ ID NO: 336 is the amino acid sequence predicted for 705P.

SEQ ID NO: 337 is the amino acid sequence of peptide P1S # 10.

SEQ ID NO: 338 is the amino acid sequence of peptide p5.

SEQ ID NO: 339 is the predicted amino acid sequence for peptide P509S.

SEQ ID NO: 340 is the cDNA sequence determined for P778P.

SEQ ID NO: 341 is the cDNA sequence determined for P786P.

SEQ ID NO: 342 is the cDNA sequence determined for P789P.

SEQ ID NO: 343 is the cDNA sequence determined for a clone showing homology to human MM46 mRNA.

SEQ ID NO: 344 is the cDNA sequence determined for a clone exhibiting homology to human TNF-alpha stimulated ABC protein (ABC50) mRNA.

SEQ ID NO: 345 is the cDNA sequence determined for a clone showing homology to human E-cadherin mRNA.

SEQ ID NO: 346 is the cDNA sequence determined for a clone showing homology to human nucleus-encoded mitochondrial serine hydroxymethyltransferase (SHMT).

SEQ ID NO: 347 is the cDNA sequence determined for a clone showing homology to human natural resistance-related macrophage protein 2 (NRAMP2).

SEQ ID NO: 348 is the cDNA sequence determined for a clone showing homology to human phosphoglucomutase-related protein protein (PGMRP).

SEQ ID NO: 349 is the cDNA sequence determined for a clone showing homology to the human mRNA of the proteosome subunit p40.

SEQ ID NO: 350 is the cDNA sequence determined for P777P.

SEQ ID NO: 351 is the cDNA sequence determined for P779P.

SEQ ID NO: 352 is the cDNA sequence determined for P790P.

SEQ ID NO: 353 is the cDNA sequence determined for P784P.

SEQ ID NO: 354 is the cDNA sequence determined for P776P.

SEQ ID NO: 355 is the cDNA sequence determined for P780P.

SEQ ID NO: 356 is the cDNA sequence determined for P544S.

SEQ ID NO: 357 is the cDNA sequence determined for P745S.

SEQ ID NO: 358 is the cDNA sequence determined for P782P.

SEQ ID NO: 359 is the cDNA sequence determined for P783P.

SEQ ID NO: 360 is the cDNA sequence determined for the unknown 17984.

SEQ ID NO: 361 is the cDNA sequence determined for P787P.

SEQ ID NO: 362 is the cDNA sequence determined for P788P.

SEQ ID NO: 363 is the cDNA sequence determined for the unknown 17994.

SEQ ID NO: 364 is the cDNA sequence determined for P781P.

SEQ ID NO: 365 is the cDNA sequence determined for P785P.

SEQ ID NOs: 366-375 are the cDNA sequences determined for the B305D splice variant.

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 cDNA sequence determined for B716P.

SEQ ID NO: 382 is the full length cDNA sequence determined for P711P.

SEQ ID NO: 383 is the amino acid sequence predicted for P711P.

SEQ ID NO: 384 is the cDNA sequence of P1000C.

SEQ ID NO: 385 is the cDNA sequence of CGI-82.

SEQ ID NO: 386 is the cDNA sequence of 23320.

SEQ ID NO: 387 is the cDNA sequence of CGI-69.

SEQ ID NO: 388 is the cDNA sequence of L-iditol-2-dehydrogenase.

SEQ ID NO: 389 is the cDNA sequence of 23379.

SEQ ID NO: 390 is the cDNA sequence of 23381.

SEQ ID NO: 391 is the cDNA sequence of KIAA0122.

SEQ ID NO: 392 is the cDNA sequence of 23399.

SEQ ID NO: 393 is the cDNA sequence of a previously identified gene.

SEQ ID NO: 394 is the cDNA sequence of HCLBP.

SEQ ID NO: 395 is the cDNA sequence of transglutaminase.

SEQ ID NO: 396 is the cDNA sequence of a previously identified gene.

SEQ ID NO: 397 is the cDNA sequence of PAP.

SEQ ID NO: 398 is the cDNA sequence of Ets transcription factor PDEF.

SEQ ID NO: 399 is the cDNA sequence of hTGR.

SEQ ID NO: 400 is the cDNA sequence of KIAA0295.

SEQ ID NO: 401 is the 22545 cDNA sequence.

SEQ ID NO: 402 is the 22547 cDNA sequence.

SEQ ID NO: 403 is the 22548 cDNA sequence.

SEQ ID NO: 404 is the 22550 cDNA sequence.

SEQ ID NO: 405 is the 22551 cDNA sequence.

SEQ ID NO: 406 is the 22552 cDNA sequence.

SEQ ID NO: 407 is the 22553 cDNA sequence.

SEQ ID NO: 408 is the cDNA sequence of 22558.

SEQ ID NO: 409 is the 22562 cDNA sequence.

SEQ ID NO: 410 is the 22565 cDNA sequence.

SEQ ID NO: 411 is the cDNA sequence of 22567.

SEQ ID NO: 412 is the 22568 cDNA sequence.

SEQ ID NO: 413 is the cDNA sequence of 22570.

SEQ ID NO: 414 is the 22571 cDNA sequence.

SEQ ID NO: 415 is the 22572 cDNA sequence.

SEQ ID NO: 416 is the cDNA sequence of 22573.

SEQ ID NO: 417 is the cDNA sequence of 22573.

SEQ ID NO: 418 is the 22575 cDNA sequence.

SEQ ID NO: 419 is the 22580 cDNA sequence.

SEQ ID NO: 420 is the 22581 cDNA sequence.

SEQ ID NO: 421 is the cDNA sequence of 22582.

SEQ ID NO: 422 is the cDNA sequence of 22583.

SEQ ID NO: 423 is the cDNA sequence of 22584.

SEQ ID NO: 424 is the 22585 cDNA sequence.

SEQ ID NO: 425 is the 22586 cDNA sequence.

SEQ ID NO: 426 is the cDNA sequence of 22587.

SEQ ID NO: 427 is the 22588 cDNA sequence.

SEQ ID NO: 428 is the 22589 cDNA sequence.

SEQ ID NO: 429 is the 22590 cDNA sequence.

SEQ ID NO: 430 is the cDNA sequence of 22591.

SEQ ID NO: 431 is the cDNA sequence of 22592.

SEQ ID NO: 432 is the cDNA sequence of 22593.

SEQ ID NO: 433 is the cDNA sequence of 22594.

SEQ ID NO: 434 is the 22595 cDNA sequence.

SEQ ID NO: 435 is the 22596 cDNA sequence.

SEQ ID NO: 436 is the 22847 cDNA sequence.

SEQ ID NO: 437 is the 22848 cDNA sequence.

SEQ ID NO: 438 is the cDNA sequence of 22849.

SEQ ID NO: 439 is the cDNA sequence for 22851.

SEQ ID NO: 440 is the cDNA sequence of 22852.

SEQ ID NO: 441 is the cDNA sequence of 22853.

SEQ ID NO: 442 is the 22854 cDNA sequence.

SEQ ID NO: 443 is the cDNA sequence of 22855.

SEQ ID NO: 444 is the 22856 cDNA sequence.

SEQ ID NO: 445 is the cDNA sequence of 22857.

SEQ ID NO: 446 is the cDNA sequence of 23601.

SEQ ID NO: 447 is the cDNA sequence of 23602.

SEQ ID NO: 448 is the cDNA sequence of 23603.

SEQ ID NO: 449 is the cDNA sequence of 23606.

SEQ ID NO: 450 is the cDNA sequence of 23612.

SEQ ID NO: 451 is the cDNA sequence of 23614.

SEQ ID NO: 452 is the cDNA sequence of 23618.

SEQ ID NO: 453 is the cDNA sequence of 23622.

SEQ ID NO: 454 is the cDNA sequence of folate hydrolase.

SEQ ID NO: 455 is the cDNA sequence of the LIM protein.

SEQ ID NO: 456 is the cDNA sequence of a known gene.

SEQ ID NO: 457 is the cDNA sequence of a known gene.

SEQ ID NO: 458 is the cDNA sequence of a previously identified gene.

SEQ ID NO: 459 is the cDNA sequence of 23045.

SEQ ID NO: 460 is the cDNA sequence of 23032.

SEQ ID NO: 461 is the cDNA sequence of 23054.

SEQ ID NOs: 462 to 467 are cDNA sequences of known genes.

SEQ ID NOs: 468-471 are cDNA sequences of P710P.

SEQ ID NO: 472 is the cDNA sequence of P1001C.

SEQ ID NO: 473 is the determined cDNA sequence of the first splice variant of P775P (also referred to as 27505).

SEQ ID NO: 474 is the determined cDNA sequence of the second splice variant of P775P (also referred to as 19947).

SEQ ID NO: 475 is the determined cDNA sequence of the third splice variant of P775P (also called 19941).

SEQ ID NO: 476 is the determined cDNA sequence of the fourth splice variant of P775P (also referred to as 19937).

SEQ ID NO: 477 is the first deduced amino acid sequence encoded by SEQ ID NO: 474.

SEQ ID NO: 478 is the second deduced amino acid sequence encoded by the sequence of SEQ ID NO: 474.

SEQ ID NO: 479 is the deduced amino acid sequence encoded by the sequence of SEQ ID NO: 475.

SEQ ID NO: 480 is the first deduced amino acid sequence encoded by the sequence of SEQ ID NO: 473.

SEQ ID NO: 481 is the second deduced amino acid sequence encoded by the sequence of SEQ ID NO: 473.

SEQ ID NO: 482 is the third deduced amino acid sequence encoded by the sequence of SEQ ID NO: 473.

SEQ ID NO: 483 is the fourth deduced amino acid sequence encoded by the sequence of SEQ ID NO: 473.

SEQ ID NO: 484 is the first 30 amino acids of M. tuberculosis antigen Ra12.

SEQ ID NO: 485 is the PCR primer AW025.

SEQ ID NO: 486 is PCR primer AW003.

SEQ ID NO: 487 is the PCR primer AW027.

SEQ ID NO: 488 is the PCR primer AW026.

SEQ ID NOs: 489-501 are peptides used in the epitope mapping test.

SEQ ID NO: 502 is the determined cDNA sequence of the complementarity determining region for anti-P503S monoclonal antibody 20D4.

SEQ ID NO: 503 is the determined cDNA sequence of the complementarity determining region of anti-P503S monoclonal antibody JA1.

SEQ ID NOs: 504 and 505 are peptides used in epitope mapping.

SEQ ID NO: 506 is the determined cDNA sequence of the complementarity determining region of anti-P703P monoclonal antibody 8H2.

SEQ ID NO: 507 is the determined cDNA sequence of the complementarity determining region of anti-P703P monoclonal antibody 7H8.

SEQ ID NO: 508 is the determined cDNA sequence of the complementarity determining region of anti-P703P monoclonal antibody 2D4.

SEQ ID NOs: 509-522 are peptides used in the epitope mapping test.

SEQ ID NO: 523 is the mature form of P703P used to generate antibodies to P703P.

SEQ ID NO: 524 is the predicted full-length cDNA sequence of P703P.

SEQ ID NO: 525 is the deduced amino acid sequence encoded by SEQ ID NO: 524.

SEQ ID NO: 526 is the full-length cDNA sequence of P790P.

SEQ ID NO: 527 is the deduced amino acid sequence of P790P.

SEQ ID NOs: 528 and 529 are PCR primers.

SEQ ID NO: 530 is the cDNA sequence of the splice variant of SEQ ID NO: 336.

SEQ ID NO: 531 is the cDNA sequence of the open reading frame of SEQ ID NO: 530.

SEQ ID NO: 532 is the deduced amino acid sequence encoded by the sequence of SEQ ID NO: 531.

SEQ ID NO: 533 is the DNA sequence of the predicted ORF of P775P.

SEQ ID NO: 534 is the deduced amino acid sequence encoded by SEQ ID NO: 533.

SEQ ID NO: 535 is the first full-length cDNA sequence of P510S.

SEQ ID NO: 536 is the second full length cDNA sequence of P510S.

SEQ ID NO: 537 is the deduced amino acid sequence encoded by SEQ ID NO: 535.

SEQ ID NO: 538 is the deduced amino acid sequence encoded by SEQ ID NO: 536.

SEQ ID NO: 539 is the peptide P501S-370.

SEQ ID NO: 540 is the peptide P501S-376.

SEQ ID NOs: 541-550 are P501S epitopes.

SEQ ID NO: 551 corresponds to amino acids 543-553 of P501S.

Detailed Description of the Invention As noted above, the present invention generally relates to compositions and methods for treating and diagnosing cancer, eg, prostate cancer. The compositions described herein include prostate specific polypeptides, polynucleotides encoding such polypeptides, binding agents such as antibodies, antigen presenting cells (APC) and / or immune system cells (eg, T Cell). The polypeptides of the present invention generally comprise at least a portion of a prostate specific protein (eg, an immunogenic portion) or a variant thereof. A “prostate-specific protein” is measured in prostate tumor cells at a level that is at least 2-fold, preferably at least 5-fold greater than the level of expression in normal tissue, as measured using the representative assays provided herein. Is a protein expressed in Certain prostate-specific proteins are oncoproteins that react detectably (by immunoassay, eg, ELISA or Western blot) with the antisera of patients suffering from prostate cancer. The polynucleotides of the invention generally comprise DNA or RNA sequences that encode all or part of such polypeptides, or DNA or RNA sequences that are complementary to such sequences. The antibody is generally an immune system protein, or antigen-binding fragment thereof, that can bind to the aforementioned polypeptide. Antigen presenting cells include dendritic cells, macrophages, fibroblasts and B cells that express the aforementioned polypeptides. T cells that can be used in such compositions are generally T cells that are specific for the aforementioned polypeptides.

  The present invention is based on the discovery of human prostate specific proteins. The polynucleotide sequence encoding a particular prostate specific protein, or portion thereof, is SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332- 335, 340-375, 381, 382, 384-476, 524, 526, 530, 531, 533, 535, and 536. Polypeptide sequences comprising at least a portion of a prostate specific protein are SEQ ID NOs: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, and 537-550.

Prostate Specific Protein Polynucleotides Any polynucleotide encoding a prostate specific protein described herein or a portion or variant thereof is encompassed by the present invention. Preferred polynucleotides comprise at least 15 contiguous nucleotides, preferably 30 contiguous nucleotides, more preferably at least 45 contiguous nucleotides encoding a portion of a prostate specific protein. More preferably, the polynucleotide encodes an immunogenic portion of a prostate specific protein. Polynucleotides that are complementary to such sequences are also encompassed by the present invention. A polynucleotide can be single-stranded (coding or antisense) or double-stranded and can be a DNA (genomic, cDNA or synthetic) or RNA molecule. RNA molecules include HnRNAs that contain introns and correspond to DNA molecules in a one-to-one manner, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences can be present in the polynucleotides of the invention, but this is not necessary and the polynucleotide can bind to other molecules and / or support materials, Is not necessarily required.

  A polynucleotide can comprise a native sequence (ie, an endogenous sequence encoding a prostate-specific protein or portion thereof) or can comprise a variant of such a sequence. A polynucleotide variant can contain one or more substitutions, additions, deletions and / or insertions such that the immunogenicity of the encoded polypeptide is not reduced with respect to the native protein. The effect on the immunogenicity of the encoded polypeptide can generally be assessed as described herein. The variant preferably has at least about 70% identity, more preferably at least about 80% identity, most preferably at least about 90% to a polynucleotide sequence encoding a native prostate specific protein or portion thereof. Shows the identity. The term “mutant” (mutant) also encompasses homologous genes of heterologous origin.

  Two polynucleotides or polypeptide sequences are said to be "identical" when the nucleotide or amino acid sequences in the two sequences are identical when aligned with the greatest correspondence, as described below. Typically, a comparison between two sequences is performed by comparing the sequences over a comparison window that identifies and compares local regions of sequence similarity. A “comparison window” as used herein can compare a sequence with a reference sequence at the same number of flanking positions after the two sequences are optimally aligned, at least about 20, usually 30 to about 75, meaning 40 to about 50 adjacent segments.

  Optimal sequence alignment for comparison can be performed according to the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) Using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, MO ) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D. C. Vol. 5, Suppl. 3, 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, California; Higgins, DG and Sharp, PM (1989) CABIOS 5: 151-153; Myers, EW and Muller W. (1988) CABIOS 4: 11-17; Robinson, ED (1971) Comb. Theor. 11: 105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-the Principles and Prcactice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman DJ (1983) Proc. Natl. Acad. Sci. USA 80: 726-730.

  Preferably, the “percent sequence identity” is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the reference sequence for optimal alignment of the two sequences (this is Part of the sequence of the polynucleotide or polypeptide in the comparison window is 20% or greater, usually 5-15% or 10-12% additions or deletions (compared to no additions or deletions) That is, it can comprise a gap). The number of matching positions is calculated by determining the number of positions where the same nucleobase or amino acid residue is present in both sequences, and the number of matching positions is calculated as the total number of positions in the reference sequence (i.e. The percentage is calculated by dividing by (size) and multiplying the result by 100 to get the percentage of sequence identity.

  A variant may also be substantially homologous to a native gene, or a portion or complement thereof, separately. Such variants can hybridize under moderately stringent conditions to naturally occurring DNA sequences (or complementary sequences) encoding native prostate specific proteins. Suitable moderate stringent conditions include the following conditions: 5 × SSC, 0.5% SDS, pre-wash in a solution of 1.0 mM EDTA (pH 8.0); 50 ° C. to 65 ° C., 5 × SSC Overnight hybridization in; then wash for 20 minutes at 65 ° C. using 2 ×, 0.5 × and 0.2 × SSC each containing 0.1% SDS.

  As one skilled in the art will appreciate, as a result of the degeneracy of the genetic code, there are numerous nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that change due to different codon usage are specifically included within the scope of the present invention. In addition, alleles (alleles) of genes comprising the polynucleotide sequences provided herein are within the scope of the invention. Aryl is an endogenous gene that is altered as a result of one or more mutations in the nucleotide, such as deletions, additions and / or substitutions. The resulting mRNA and protein can have altered structure or function, but they are not necessary. Aryls can be identified by standard techniques (eg, hybridization, amplification and / or database sequence comparison).

  Polynucleotides can be produced by various techniques. For example, the polynucleotide may have at least prostate-specific expression as measured using tumor-associated expression (i.e., using a representative assay provided herein, as described in greater detail below). Can be identified by screening a cDNA microarray for 5 times greater expression). Such screening is performed using Synteni microarrays (Palo Alto, Calif.) According to the manufacturer's instructions (and 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, the polypeptide can be amplified from cDNA that is prepared from cells that express the proteins described herein, eg, prostate specific cells. Under stringent conditions, the polynucleotide can be amplified by polymerase chain reaction (PCR). For this approach, sequence-specific primers can be designed based on the sequences provided herein and can be purchased or synthesized based on the sequences provided herein.

  The full length gene can be isolated from a suitable library (eg, a prostate specific cDNA library) using the portion amplified according to well-known techniques. In such techniques, a library (cDNA or genome) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, the library is sorted by size to include larger nucleotides. Random primed libraries are also preferred for identifying the 5 ′ and upstream regions of genes. Genomic libraries are preferred for introns and extended 5 ′ sequences.

For hybridization techniques, partial sequences can be labeled according to well-known techniques (eg, by nick translation or end labeling using ³² P). The bacterial or bacteriophage library is then screened by hybridizing a filter containing denatured bacterial colonies (or a flora containing phage plaques) with a labeled probe (see: Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). Hybridization colonies or plaques are selected, grown, and DNA is isolated for further analysis. For example, cDNA clones can be analyzed using primers from partial sequences and primers from vectors to determine the amount of additional sequences. Restriction maps and partial sequences can be generated to identify one or more overlapping clones. The complete sequence can then be determined according to standard techniques, which can involve the generation of a series of deletion clones. The resulting overlapping array is then assembled into a single adjacent array. Full length cDNA molecules can be generated by linking appropriate fragments according to well-known techniques.

  Alternatively, there are a number of amplification techniques that obtain a full-length coding sequence from a partial cDNA sequence. In such techniques, amplification is generally performed by PCR. A variety of commercially available kits can be used to carry out the amplification step. For example, the primers can be designed using software well known in the art. The primer is preferably 22-30 nucleotides long, has a GC content of at least 50%, and is annealed to the target sequence at a temperature of about 68 ° C to 72 ° C. The amplified region can be sequenced as described above and overlapping sequences can be assembled adjacent.

  One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 16: 8186, 1988), which uses restriction enzymes to generate fragments in known regions of the gene. The fragment is then cyclized by intramolecular linkage and used as a template for PCR using different primers derived from known regions. In another approach, sequences flanking the partial sequence can be recovered by amplification using primers for the linker sequence and primers specific for the known region. Typically, the amplified sequence is attached to the second round of amplification using the same linker primer and a second primer specific for the known region. A variation of this procedure using two primers that initiate an extension in the opposite direction from the known sequence is described in WO96 / 38591. Another such technique is known as “rapid amplification of cDNA ends” or RACE. In this technique, internal and external primers that hybridize to a poly A region or vector sequence are used to identify sequences that are 5 ′ and 3 ′ of known sequences. 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 using amplification can also be used to obtain a full-length cDNA sequence.

  In some cases, full length cDNA sequences can be obtained by analysis of sequences provided in an expressed sequence tag (EST) database, eg, a database available from a gene bank. Searches for overlapping ESTs are performed according to generally well-known programs (eg, NCBI BLAST searches), and such ESTs can be used to generate flanking full length sequences. Full-length DNA sequences can also be obtained by distribution of genomic fragments.

  Certain nucleic acid sequences of cDNA molecules encoding portions of prostate specific proteins are SEQ ID NOs: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332 -335, 340-375, 381, 382, 384-476, 524, 526, 530, 531, 533, 535, and 536. The isolation of these polynucleotides is described below.

  Polynucleotide variants can generally be produced by methods known in the art, including chemical synthesis, such as solid phase phosphoramidite chemical synthesis. Polynucleotide sequence modifications can also be introduced by standard mutagenesis techniques, such as site-directed mutagenesis directed at oligonucleotides (see Adelman et al., DNA 2: 183, 1983). Alternatively, by in vitro or in vivo transcription of a DNA sequence encoding a prostate specific protein, or part thereof, as long as the DNA is incorporated into the vector using an appropriate RNA polymerase promoter (eg, T7 or SP6) RNA molecules can be generated. Certain moieties can be used to produce the encoded polypeptides described herein. Further, optionally, a portion can be added to the patient so that the encoded polypeptide occurs in vivo (eg, antigen presenting cells, eg, dendritic cells with a cDNA construct encoding prostate specific polypeptide). By transfecting the cells and administering the transfected cells to the patient).

  The portion of the sequence that is complementary to the coding sequence (ie, an antisense polynucleotide) can also be used as a probe or to modulate the expression of a gene. A cDNA construct that can be transcribed into antisense RNA can also be introduced into cells of the tissue to promote the production of antisense RNA. As described herein, antisense polynucleotides can be used to inhibit protein expression. Antisense technology can be used to control gene expression by triple-helix formation, and such formation reduces the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. (See Gee et al., Huber and Carr, Molecular and Immunologic Apporoaches, Futura Publishing Co. (Mt. Kisco, NY; 1994)). Alternatively, to hybridize with the regulatory region of the gene (eg, promoter, enhancer or transcription start site) and block transcription of the gene or block translation by inhibiting the binding of the transcript to the ribosome Antisense molecules can be designed.

  A portion of the coding sequence or a portion of the complementary sequence can also be designed as a probe or primer to detect gene expression. Probes can be labeled with various reporter groups, such as radionuclides or enzymes, and such probes are preferably at least 10 nucleotides long, more preferably at least 20 nucleotides long, and even more preferably at least 30 nucleotides long. is there. As described above, the primer is preferably 22 to 30 nucleotides in length.

  Any polynucleotide can be further modified to increase in vivo stability. Possible modifications include, but are not limited to: addition of flanking sequences at the 5 ′ and / or 3 ′ ends; use of phosphorothioates or 2′O-methyl rather than phosphodiesterase linkages in the backbone; and And / or inclusion of non-traditional bases such as inosine, queosin and wivetocin, and acetyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

  The nucleotide sequences described herein can be linked to a variety of other nucleotide sequences by established recombinant DNA techniques. For example, polynucleotides can be cloned into various cloning vectors such as plasmids, phagemids, lambda phage derivatives and cosmids. Particularly problematic vectors include expression 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, a convenient restriction endonuclease site and one or more selectable markers. Other factors depend on the desired use and will be apparent to those skilled in the art.

  In certain embodiments, the polynucleotide can be formulated to allow entry into and expression in mammalian cells. Such formulations are particularly useful for therapeutic purposes, as described below. As those skilled in the art will appreciate, there are numerous ways to achieve expression of a polynucleotide in a target cell, and any suitable method can be used. For example, the polynucleotide can be incorporated into a viral vector, examples of such viral vectors are adenoviruses, adeno-associated viruses, retroviruses, or vaccinia or other poxviruses (eg, tripox viruses). However, it is not limited to these. Techniques for incorporating DNA into such vectors are well known to those skilled in the art. Retroviral vectors contain a gene for a selectable marker (to facilitate identification or selection of transduced cells) and / or a targeting moiety, eg, a gene encoding a receptor ligand on a specific target cell. Further transfer or incorporation can make the vector target specific. Targeting can also be accomplished using antibodies by methods known in the art.

  Other formulations for therapeutic purposes include colloidal dispersions such as polymer complexes, monocapsules, beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and Includes liposomes. A preferred colloidal system for use as a delivery system in vitro and in vivo is a liposome (ie, an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

Prostate Specific Polypeptide In the context of the present invention, a polypeptide can comprise at least an immunogenic portion of a prostate specific protein, or a variant thereof, as described herein. As mentioned above, a “prostate specific protein” is a protein expressed by normal prostate cells and / or prostate tumor cells. Proteins that are prostate specific proteins also detectably react with antisera from patients with prostate cancer in immunoassays (eg, ELISA). The polypeptides described herein can be of any length. There may be additional sequences and / or heterologous sequences derived from the native protein, and such sequences may have (but are not necessarily) more immunogenic or antigenic.

  An “immunogenic moiety”, as used herein, is a portion of a protein that is recognized (ie, 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, even more preferably at least 20 amino acid residues of the prostate specific protein or variant thereof. Certain preferred immunogenic portions include peptides in which the N-terminal leader sequence and / or transmembrane domain has been deleted. Other preferred immunogenic portions can contain small N-terminal and / or C-terminal deletions (eg, 1-30 amino acids, preferably 5-15 amino acids) with respect to the mature protein.

  Immunogenic portions can generally be identified by well-known techniques, such as those summarized in the following references 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 specifically bind to an antigen (ie, they react with a protein in an ELISA or other assay but do not detectably react to an irrelevant protein) “Antigen-specific”. Such antibodies can be produced according to well-known techniques as described herein. An immunogenic portion of a native prostate specific protein is a portion that reacts with such antisera and / or T cells at a level substantially lower than the reactivity of the full-length polypeptide (eg, ELISA and / or Or in T cell reactivity assays). Such immunogenic moieties can react at levels similar to or greater than the reactivity of full-length polypeptides in such assays. Such screening can generally be performed by methods well known to those skilled in the art, such as those described in the following literature: Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory 1988. For example, the polypeptide may be immobilized on a solid support and contacted with the patient's serum to allow antibodies in the serum to bind to the immobilized polypeptide. Unbound serum can then be removed and bound antibody can be detected with, for example, 125I-labeled protein A.

  As mentioned above, the composition can comprise various natural prostate specific proteins. A “variant” (variant) of a polypeptide, as used herein, is one or more substitutions, deletions, additions and / or such that the immunogenicity of the polypeptide is not substantially reduced. A polypeptide that differs from a native prostate-specific protein upon insertion. In other words, the ability to react with antigen-specific antisera can be enhanced or unchanged with respect to the native protein, or can be reduced with less than 50%, preferably less than 20% with respect to the native protein. Such variants generally modify one of the polypeptide sequences and evaluate the reactivity of the modified polypeptide with an antigen-specific antibody or antiserum, as described herein. Can be identified. Preferred variants include those in which one or more moieties have been removed, such as the N-terminal leader sequence or transmembrane domain. Other preferred variants include variants in which a small portion (eg, 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N-terminus and / or C-terminus of the mature protein. A variant of a polypeptide preferably exhibits at least about 70% identity, more preferably at least about 90%, and most preferably at least about 95% identity (measured as described below) to the identified polypeptide. .

  Preferably, the variant contains conservative substitutions. “Conservative substitutions” are other amino acids in which the amino acid has similar properties, such as those skilled in the art of peptide chemistry expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. A substitution that has been replaced. Amino acid substitutions can generally be performed based on the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilic nature of the residue. 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 with similar hydrophilicity values are: Includes leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that can represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, ser, thr; (2) cys, ser, tyr, thr (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Variants can also or alternatively include non-conservative changes. In a preferred embodiment, the variant polypeptide differs from the native sequence by 5 or fewer amino acid substitutions, deletions or additions. Variants can also (or alternatively) be modified, for example, by amino acid deletions or additions that have minimal effect on the immunogenicity, secondary structure and hydrophilicity of the polypeptide.

  As described above, the polypeptide can comprise a signal (or leader) sequence at the N-terminus of the protein that directs translocation of the protein either co-translationally or post-translationally. Polypeptides can also be linkers or other sequences (eg, poly-His) to facilitate polypeptide synthesis, purification or identification, or to enhance the binding of the polypeptide to a solid support. Can be combined. For example, the polypeptide can be linked to an immunoglobulin Fc region.

  A variety of well-known techniques can be used to produce polypeptides. As described above, the recombinant polypeptide encoded by the DNA sequence can be easily produced from the DNA sequence using various expression vectors known to those skilled in the art. Expression can be achieved in any appropriate host cell transformed or transfected with an expression vector containing a DNA molecule that encodes the recombinant polypeptide. Suitable host cells include prokaryotic cells, yeast cells and higher eukaryotic cells. Preferably, the host cell used is E. coli, yeast or a mammalian cell line such as COS or CHO. Supernatants from a suitable host / vector system that secretes the recombinant protein or polypeptide into the medium can be first concentrated with a commercially available filter. After concentration, the concentrate can be applied to a suitable purification matrix, such as an affinity matrix or an ion exchange resin. Finally, the recombinant polypeptide can be further purified using one or more reverse phase HPLC steps.

  Synthetic means can also be used to generate proteins and other variants having less than about 100 amino acids, generally less than about 50 amino acids, using techniques well known in the art. For example, such polypeptides can be synthesized by any commercially available solid phase technique, such as the Merrifield solid phase synthesis method, where amino acids are sequentially added to the growing amino acid chain. Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Polypeptide automated synthesizers are commercially available from suppliers such as Perkin Elmer / Applied BioSystems Division (Foster City, Calif.), And It can be operated according to the manufacturer's instructions.

  In certain specific embodiments, the polypeptide comprises a plurality of polypeptides described herein, or at least one polypeptide described herein and an unrelated sequence, such as a known It can be a fusion protein comprising a prostate specific protein. Fusion proteins, for example, help provide T helper epitopes (immunological fusion partners), preferably T helper epitopes recognized by humans, or help protein expression in higher yields than natural recombinant proteins (Expression enhancer). Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners can be selected to increase the solubility of the protein or allow the protein to be targeted to the desired intracellular compartment. Still further fusion partners include affinity tags that facilitate protein purification.

  Fusion proteins can generally be produced according to standard techniques, including chemical conjugation. Preferably, the fusion protein is expressed as a recombinant protein, allowing an increased level of production relative to the non-fusion protein in the expression system. Briefly, DNA sequences encoding polypeptide components can be assembled separately and ligated into an appropriate expression vector. With or without peptide linkers, the 3 ′ end of the DNA sequence encoding the polypeptide component is 5 of the sequence encoding the second polypeptide so that the reading frame of the sequence is in phase. 'Connected to the end. This allows translation into a single fusion protein that retains the biological activity of both component polypeptides.

  Peptide linker sequences can be used to separate the first and second polypeptide components at a distance sufficient to ensure that each polypeptide is folded into its secondary and ternary structure. . Such peptide linker sequences are incorporated into the fusion protein according to standard techniques well known in the art. Appropriate peptide linker sequences can be selected based on the following factors: (1) ability to adopt a flexible extended conformation; (2) can interact with epitopes on the first and second polypeptides. Ability to adopt secondary structure; and (3) lack of hydrophobic or charged residues capable of reacting with a functional epitope of a polypeptide. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other nearby neutral amino acids such as Thr and Ala can also be used in the linker sequence. Amino acid sequences that can typically be used as linkers include those described in the following literature: Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83; 8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence can generally be from 1 to about 50 amino acids in length. A linker sequence is unnecessary when the first and second polypeptides have a nonessential N-terminal amino acid residue that can be used to separate functional domains and prevent steric hindrance.

  The bound DNA sequence is operably linked to appropriate transcriptional or translational region factors. Regulators involved in DNA expression are located only 5 'to the DNA sequence encoding the first polypeptide. Similarly, the stop codon and transcription termination signal required to terminate translation are located only 3 'to the DNA sequence encoding the second polypeptide.

  Also provided is a fusion protein comprising a polypeptide of the invention together with an irrelevant immunogenic protein. Preferably, the immunogenic protein is capable of eliciting a regenerative response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, eg, Stoute et al., New England J. Med. 336: 86-91, 1997).

  In a preferred embodiment, the immunological fusion protein is derived from protein D, a surface protein of the Gram-negative Haemophilus influenzae (WO91 / 18926). Preferably, the protein D derivative comprises approximately 1/3 protein (eg, the first 100-110 amino acids at the N-terminus), and the protein D derivative can be lipidated. In certain preferred embodiments, the first 109 residues of the lipoprotein D fusion protein are included at the N-terminus to confer additional exogenous T cell epitopes on the polypeptide and increase expression levels in E. coli. (Thus functioning as an expression enhancer). Lipid tails ensure optimal presentation of antigen to antigen presenting cells. Other fusion partners include the nonstructural protein from influenza virus, NS1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, but different fragments encompassing the T helper epitope can be used.

  In other embodiments, the immunological fusion partner is a protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae and synthesizes N-acetyl-L-alanine amidase known as amylase 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 involved in affinity for choline or some choline analogs, such as DEAE. This property has been utilized for the generation of E. coli C-LYTA that expresses a plasmid useful for the expression of the fusion protein. Purification of hybrid proteins containing a C-LYTA fragment at the amino terminus has been described (see Biotechnology 10: 795-798, 1992). In a preferred embodiment, a repeat portion of LYTA can be incorporated into the fusion protein. The repeat is found in the C-terminal region starting at residue 178. A particularly preferred repeat moiety incorporates residues 188-305.

  In general, the polypeptides (including fusion proteins) and polynucleotides described herein are isolated. An “isolated” polypeptide or polynucleotide is one that is removed from its original environment. For example, a natural protein is isolated if it is separated from some or all of the coexisting substances 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 isolated if, for example, it is cloned into a vector that is not part of the natural environment.

Binding Factors The present invention further provides factors such as antibodies or antigen binding fragments thereof that specifically bind to prostate specific proteins. As used herein, an antibody or antigen-binding fragment thereof reacts with a prostate-specific protein at a detectable level (eg, in an ELISA) but does not detectably react with an irrelevant protein under similar conditions. In some cases, it is said to “specifically bind” to a prostate specific protein. As used herein, “bond” means a non-covalent association between two separate molecules such that a complex is formed. The ability to bind can be assessed, for example, by determining the binding constant for complex formation. The binding constant is a value obtained by dividing the complex concentration by the product of the component concentrations. In general, two components are said to be “bound” in the context of the present invention when the binding constant for complex formation is greater than about 10 ³ L / mol. The coupling constant can be determined by methods well known in the art.

  A binding agent can use a representative assay provided in the present invention to distinguish between a patient with cancer, eg, prostate cancer, and an individual without cancer. In other words, an antibody or other binding agent that binds to a prostate-specific protein generates a signal indicating the presence of cancer in at least about 20% of patients with the disease and at least about 90% of individuals without cancer Will generate a negative signal indicating the absence of disease in To determine whether the binding agent meets this requirement, biological samples (eg, blood, from patients with cancer and individuals without cancer (determined using standard clinical trials) Serum, urine and / or tumor biopsy) can be assayed for the presence of a polypeptide that binds to a binding agent, as described herein. It should be clear that a statistically significant number of samples with and without disease should be assayed. Each binding agent should meet the above criteria; however, as those skilled in the art will recognize, combinations of binding agents can be used to improve sensitivity.

  Any factor that satisfies the above requirements can be a binding factor. For example, the binding agent can be a ribosome, RNA molecule or polypeptide with or without a peptide component. In preferred embodiments, the binding agent is an antibody or antigen-binding fragment thereof. More preferably, the antibodies used in the methods of the invention are capable of inducing tumor cell lysis mediated by complement activation and antibody-dependent cytotoxicity (ADCC). Different classes and subclasses of antibodies differ in the above characteristics. For example, the IgG2a and IgG3 classes can activate serum complement during binding to target cells that express the antigen against which the antibody is raised, and can mediate ADCC.

  Antibodies can be produced by various techniques known to those skilled in the art. See, for example, the following literature: 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 described herein, or suitable bacteria or mammals to enable the production of recombinant antibodies. It can be produced by transfecting an antibody gene into a cellular host. In one technique, an immunogen comprising a polypeptide is first injected into a wide variety of mammals (eg, mice, rats, rabbits, sheep or goats). In this step, the polypeptide of the present invention can serve as an immunogen without modification. Alternatively, particularly for relatively short polypeptides, a better immune response can be elicited when the polypeptide is conjugated to a carrier protein such as bovine serum albumin or keyhole limpet hemocyanin. Preferably, the immunogen is injected into the animal host and periodically bled from the animal according to a predetermined schedule incorporating one or more boosts. Polyclonal antibodies specific for the polypeptide can then be purified from such serum, for example, by affinity chromatography using the polypeptide coupled to a suitable solid support.

  Monoclonal antibodies specific for the antigenic polypeptide of interest are produced using, for example, the technique of Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976 and improvements of that technique. Can do. Briefly, these methods involve the preparation of immortalized cell lines that can produce antibodies having the desired specificity (ie, reactivity with the polypeptide of interest). Such a cell line can be produced, for example, from splenocytes obtained from an immunized animal as described above. The spleen cells are then immortalized, for example, by a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. Various fusion techniques can be used. For example, combining splenocytes and myeloma cells with a nonionic surfactant for several minutes and then plating at low density on selective media that supports hybrid cell growth but does not support myeloma cell growth. Can do. In a preferred selection technique, HAT (hypoxanthine, aminopterin, thymidine) selection is used. After sufficient time, usually about 1-2 weeks, hybrid colonies are obtained. Single colonies are selected and their culture supernatants are tested for binding to the polypeptide. Hybridomas with high reactivity and specificity are preferred.

  Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, the yield can be increased using various techniques, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, eg, a mouse. Monoclonal antibodies can then be collected from ascites or blood. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, sedimentation, and extraction. The polypeptides of the present invention can be used in purification methods, such as affinity chromatography steps.

  The preparation of mouse and rabbit monoclonal antibodies that specifically bind to the polypeptides of the invention is described in detail below. However, the antibody of the present invention is not limited to those derived from mice. Human antibodies can also be used in the present invention and can prove to be preferred. Such antibodies can be obtained using human hybridomas as described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Lisa, p. 77, 1985). The present invention relates to antibodies produced by recombinant means, such as chimeric antibodies derived from different species of variable and constant regions, and CDR-grafted antibodies derived from different species of complementarity determining regions, Also included are those described in patents 4,816,567 and 5,225,539. Chimeric antibodies are prepared by splicing a gene of a mouse antibody molecule with a desired antigen specificity together with a gene of a human antibody molecule that has the desired biological activity, eg, human complement activation and ADCC mediation. (Morrison et al. Proc. Natl. Acad. Sci. USA 81: 6851, 1984; Neuberger et al. Nature 312: 604, 1984; Takeda et al. Nature 314: 452, 1985).

  In certain embodiments, antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which can be produced using standard techniques. Briefly, immunoglobulins were purified from rabbit serum by affinity chromatography on a Protein A bead column (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988), digested with papain and Fab and Fc fragments can be generated. Fab and Fc fragments can be separated by affinity chromatography on protein A bead columns.

The monoclonal antibodies of the invention can be coupled to one or more therapeutic factors. Suitable therapeutic factors in this regard include radionuclides, differentiation inducing factors, drugs, toxins, and derivatives thereof. Preferred radionuclides include ⁹⁰ Y, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ²¹¹ At, and ²¹² Bi. Preferred agents include methotrexate and pyrimidine and purine analogs. Preferred differentiation inducing factors include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and American pokeweed antiviral protein.

  The therapeutic agent can be coupled (eg, covalently coupled) directly or indirectly (eg, via a linker group) to a suitable monoclonal antibody. When each has a substituent that can react with the other, a direct reaction between the agent and the antibody is possible. For example, a nucleophilic group present on one side, such as an amino or sulfhydryl group, may be present on a group containing a carbonyl present on the other side, such as an anhydride or acid halide, or an excellent leaving group containing an alkyl group (e.g. , Halides).

  Alternatively, it may be desirable to couple the therapeutic agent and the antibody via a linker group. The linker group can function as a spacer that prevents the antibody from separating from the factor and interfering with its binding ability. The linker group can also serve to increase the chemical reactivity of the substituent on the agent or antibody, thus increasing the coupling efficacy. Increasing chemical activity can also facilitate the use of factors, or the use of functional groups on factors, that would otherwise be impossible.

  As will be apparent to those skilled in the art, a variety of bifunctional or polyfunctional reagents that are homo- and heterofunctional (such as those described in the catalog of Pierce Chemical Co., Rockford, Ill.). It can be used as a linker group. Coupling can be performed through, for example, an amino group, a carboxyl group, a sulfhydryl group, or an oxidized carbohydrate residue. There are a number of references describing such methods, for example, US Pat. No. 4,671,958 (Rodwell et al.).

  It is desirable to use a linker group that is cleavable when released from the antibody portion of the immunoconjugate of the invention, when the therapeutic agent is more potent, during or during internalization into the cell. A number of different cleavable linker groups have been described. The mechanism of intracellular release of factors from these linker groups includes disulfide bond reduction (eg, US Pat. No. 4,489,710, Spitler), photolabile bond irradiation (eg, US Pat. No. 4,625,014, Senter et al.) Hydrolysis of derivatized amino acid side chains (eg, US Pat. No. 4,638,045, Kohn et al.), Serum complement mediated hydrolysis (eg, US Pat. No. 4,671,958, Rodwell et al.), And acid-catalyzed hydrolysis (eg, US Including cutting by Patent 4,569,789, Blattler et al.

  It may be desirable to couple more than one factor to the antibody. In one embodiment, multiple molecular factors are coupled to one antibody. In other embodiments, more than one type of factor is coupled to one antibody. Without being bound to a particular embodiment, immune complexes with two or more factors can be produced in various ways. For example, two or more factors can be coupled directly to the antibody molecule, or linkers that provide multiple binding sites can be used. Alternatively, a carrier can be used.

  The carrier can support the agent in a variety of ways, including covalent bonding, either directly or through a linker group. Suitable carriers include proteins such as albumin (eg, US Pat. No. 4,507,234, Kato et al.), Peptides and polysaccharides such as aminodextran (eg, US Pat. No. 4,699,784, Shih et al.). The carrier can support the agent by non-covalent bonding or by encapsulation, eg, encapsulation in liposome vesicles (eg, US Pat. No. 4,429,008 and US Pat. No. 4,873,088). Carriers specific for radionuclides include small radiohalogenated molecules and chelating compounds. For example, U.S. Pat. No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. Radionuclide chelates can be formed from chelating compounds. Such chelating compounds include metals or metal oxides, and compounds containing nitrogen and sulfur atoms as donor atoms that bind to the radionuclide. For example, US Pat. No. 4,673,562 (Davison et al.) Discloses representative radiochelating compounds and their synthesis.

  Various routes of administration of antibodies and immune complexes can be used. Typically, administration will be intravenous, intramuscular, subcutaneous, or in the excised tumor bed. Obviously, the exact dose of the antibody / immune complex will depend on the antibody used, the antigen density on the tumor, and the clearance rate of the antibody.

The T cell immunotherapy composition can also or alternatively comprise T cells specific for prostate specific protein. Such cells can generally be prepared according to standard procedures in vitro or ex vivo. For example, T cells can be obtained from a patient's bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood, commercially available cell separation systems such as ISOLEX ™ (available from Nexell Therapeutics Inc., Irvine, CA) ( Further, it can be isolated using US Pat. No. 5,240,856, US Pat. No. 5,215,926, WO89 / 06280, WO91 / 16116 and WO92 / 07243). Alternatively, T cells can be derived from related or unrelated humans, non-human mammals, cell lines or cultures.

  T cells can be stimulated with prostate specific polypeptides, polynucleotides encoding prostate specific polypeptides and / or antigen presenting cells (APCs) expressing such polypeptides. Such stimulation is performed under conditions that generate T cells specific for the polypeptide and for a sufficient period of time. Preferably, the prostate specific polypeptide or polynucleotide is present in a delivery vehicle, such as a microsphere, to promote the development of specific T cells.

A T cell is considered to be specific for a prostate-specific polypeptide if it kills a target cell that is coated with the polypeptide or that expresses a gene encoding the polypeptide. T cell specificity can be assessed according to various standard techniques. For example, in a chromium release assay or proliferation assay, an increased stimulation index that is greater than 2-fold in lysis and / or proliferation relative to a negative control indicates T cell specificity. Such assays can be performed, for example, as described in Chen et al., Cancer Res. 54: 1065-1070, 1994. Alternatively, detection of T cell proliferation can be achieved by various known techniques. For example, detection of T cell proliferation can be accomplished by measuring an increase in the rate of DNA synthesis (eg, pulse labeling a T cell culture with tritiated thymine and incorporating tritiated thymine incorporated into DNA). By measuring the amount of). Contact with a prostate specific polypeptide (100 ng / ml to 100 μg / ml, preferably 200 ng / ml to 25 μg / ml) for 3-7 days will increase T cell proliferation by at least 2-fold. Contact as described above over 2-3 hours activates T cells as measured by standard cytokine assays, where a 2-fold increase in the level of cytokines (eg, TNF or IFN-γ) (See Colgan et al., Current Protocols in Imunology, vol. 1, Wiley Interscince (Green 1998). In response to prostate-specific polypeptide, polynucleotide or polypeptide-expressing APC) Activated T cells can be CD4 ⁺ and / or CD8 ⁺ Prostate-specific protein-specific T cells can be expanded according to standard techniques In a preferred embodiment, the T cells are patients or It comes from a related or unrelated donor and is administered to the patient after stimulation and growth.

For therapeutic purposes, the number of CD4 ⁺ or CD8 ⁺ T cells that proliferate in response to prostate-specific polypeptide, polynucleotide or APC can be expanded in vitro or in vivo. For example, stimulating factors that synthesize T cell growth factors, such as interleukin-2 and / or prostate specific polypeptides, into T cells into prostate specific polypeptides, or short peptides corresponding to such polypeptides. It can be re-exposed with or without the addition of cells. Alternatively, the number of one or more T cells proliferating in the presence of prostate specific protein can be increased by cloning. Methods for cloning cells are well known in the art and include limiting dilution methods.

Pharmaceutical Compositions and Vaccines In certain embodiments, the polypeptides, T cells and / or binding agents disclosed herein can be incorporated into pharmaceutical compositions or immunogenic compositions (ie, vaccines). A pharmaceutical composition comprises one or more such compounds and a pharmaceutically acceptable carrier. A vaccine comprises one or more such compounds and an immunostimulant. An immunostimulatory agent can be any substance that enhances the immune response to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (eg, polylactic galactide) and liposomes (in which the compound is incorporated; see, eg, US Pat. No. 4,235,877). Vaccine preparations are generally described, for example, in the following literature: MF Powell and MJ Newman, edited by “Vaccine Design (the subunit and adjuvant approach)”, Plenum Press (NY, 1995). Pharmaceutical compositions and vaccines falling within the scope of the present invention can also contain other compounds that can be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens can be present in the composition or vaccine, incorporated into the fusion polypeptide, or as separate compounds.

  A pharmaceutical composition or vaccine can contain DNA encoding one or more of the aforementioned polypeptides, such that the polypeptide is generated in situ. As mentioned above, DNA can be present in a variety of delivery systems known to those skilled in the art, including nucleic acid expression systems, bacterial and viral expression systems. A number of gene delivery techniques are well known in the art and are described, for example, in Rolland, Crit. Rev. Therap. Durg Carrier Systems 15: 143-198, 1998, and references cited therein. Yes. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (eg, appropriate promoters and termination signals). Bacterial delivery systems include the administration of bacteria that express immunogenic portions of the polypeptide on the cell surface or secrete such epitopes (eg, Bacillus-Calmette-Guerrin). To do. In preferred embodiments, viral expression systems (eg, vaccinia or other poxviruses, retroviruses, or adenoviruses) can be used to introduce DNA, which is a non-pathogenic (defective), replication-competing virus. Use can be included. Suitable systems are disclosed, for example, in the following literature: Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86: 317-321, 1989; Flexner et al., Ann. NY Acad. Sci. Flexner et al., Vaccine 8: 17-21, 1990; US Pat. No. 4,603,112, US Pat. No. 4,769,330, and US Pat. No. 5,017,487; WO 89/01973; US Pat. No. 4,777,127; GB2, 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. Acad. 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., Circ. Res. 1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA can also be “naked”, for example, as described and reviewed in the following references: Ulmer et al., Science 259: 1745-1749, 1993 and Cohen, Science 259: 1691-11692. 1993. By coating the DNA on biodegradable beads that are efficiently transported into the cell, the absorption of naked DNA can be increased.

  Although any suitable carrier known to those skilled in the art can be used in the pharmaceutical compositions of the invention, the type of carrier will vary depending on the mode of administration. The compositions of the invention can be formulated for any suitable method of administration, including, for example, topical, oral, intranasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, eg subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the aforementioned carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, and magnesium carbonate can be used. Biodegradable microspheres (eg, polylactic acid polyglycolate) can also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in US Pat. No. 4,897,268 and US Pat. No. 5,075,109.

  Such compositions can also include buffers (eg, neutral buffered saline or phosphate buffers), carbohydrates (eg, glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids. For example, glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (eg aluminum hydroxide) and / or preservatives. Alternatively, the composition of the present invention can be formulated as a lyophilizate. The compounds can also be encapsulated in liposomes according to well-known techniques.

  A variety of immunostimulatory agents can be used in the vaccines of the invention. For example, an adjuvant can be added. Most adjuvants are substances designed to protect antigens from rapid catabolism, such as aluminum hydroxide or mineral oil, and stimulators of the immune response, such as lipid A, Bordetella pertussis or human forms Contains a protein derived from Mycobacterium tuberculosis. Suitable adjuvants are commercially available, for example: Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., New Jersey) State terway); aluminum salts, eg aluminum hydroxide gel (alum) or aluminum phosphate; calcium, iron or zinc salts; insoluble suspensions of acylated tyrosine; acylated sugars; cationically or anionically derivatized Polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quill A. Cytokines such as GM-CSF or interleukin-2, -7, or -12 can also be used as adjuvants.

  In the vaccine provided in the present invention, the adjuvant composition is preferably designed primarily to induce a Th1-type immune response. High levels of Th1-type cytokines (eg, IFN-γ, IL-2 and IL-12) tend to favor the induction of cell-mediated immune responses to the administered antigen. In contrast, high levels of Th2-type cytokines (eg, IL-4, IL-5, IL-6, IL-10 and TNF-β) tend to favor the induction of a human immune response. After application of the vaccine provided in the present invention, the patient will support an immune response including Th1-type and Th2-type responses. In a preferred embodiment where the response is predominantly Th1-type, the level of Th1-type cytokine will increase to a greater extent than the level of Th2-type cytokine. The levels of these cytokines can be easily assessed according to standard assays. For a review of the family of cytokines, see the following literature: Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.

  Preferred adjuvants primarily for use in eliciting a Th1-type response include, for example, combinations of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) and aluminum salts To do. MPL adjuvant is from Ribi ImmunoChem Research Inc. (Hamilton, Mont; US Pat. No. 4,436,727, US Pat. No. 4,877,611, US Pat. No. 4,866,034 and US Pat. No. 4,912,094) It is available. Oligonucleotides containing CpG (CpG dinucleotides are not methylated) also induce a predominantly Th1 response. Such oligonucleotides are well known in the art and are described, for example, in WO 96/02555. Another preferred adjuvant is saponin, preferably QS21, which can be used alone or in combination with other adjuvants. For example, enhanced systems are described in combinations of monophosphoryl lipid A and saponin derivatives, such as the combination of QS21 and 3D-MPL described in WO94 / 00153, or WO96 / 33739. Such as low reactogenic compositions in which QS21 is quenched with cholesterol. Another preferred composition comprises an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation comprising QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO95 / 17210. Any vaccine provided in the present invention can be produced by well-known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient.

  The compositions described herein can be administered as part of a sustained release formulation (ie, a formulation such as a capsule or sponge that slowly releases the compound after administration). Such formulations are generally prepared by well-known techniques and can be administered, for example, by oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained release formulations can contain polypeptides, polynucleotides or antibodies dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate controlling membrane. The carriers used in such formulations are biocompatible and can be biodegradable; preferably the formulation releases the active compound at a relatively constant level. The amount of active compound contained in the sustained release formulation will depend on the site of implantation, the rate of release and the expected duration and the nature of the condition to be treated or prevented.

  Various delivery vehicles can be used in pharmaceutical compositions and vaccines to facilitate the production of antigen-specific immune responses that target tumor cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be genetically engineered to be efficient APCs. Such cells are genetically modified to increase the ability to present antigens, improve activation and / or maintenance of T cell responses, have anti-tumor effects themselves and / or receptors Can be immunologically compatible (ie, HLA haplotype match), but this is not necessary. APCs can generally be isolated from a variety of biological fluids and organs, such as tumors and peritumoral tissues, and can be autologous, allogeneic, syngeneic or xenogeneic cells.

  In certain preferred embodiments of the invention, dendritic cells or their progeny are used as antigen presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392: 245-251, 1998) and are effective as physiological adjuvants to elicit prophylactic or therapeutic anti-tumor immunity (See Timmerman and Levy, Ann. Rev. Med. 50: 507-529, 1999). In general, dendritic cells take up, process, and present antigen with high efficiency based on their typical shape (astrocytes in situ, prominent cytoplasmic processes visible in vitro (dendrites)) Can be identified based on their ability to activate as well as their ability to activate a naive TS cell response. Dendritic cells can of course be engineered to express specific cell surface receptors or ligands not normally found in vivo or ex vivo, and such modified dendritic cells are Intended by the invention. As an alternative to dendritic cells, secreted vesicle antigen-loaded dendritic cells (referred to as exosomes) can be used in vaccines (see Zitvogel et al., Nature Med. 4: 594-600, 1998).

  Dendritic cells and progeny can be obtained from peripheral blood, bone marrow, tumor infiltrating cells, peritumoral tissue infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, differentiating dendritic cells ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and / or TNFα to monocyte cultures collected from peripheral blood Can do. Alternatively, peripheral blood may be added by adding GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and / or one or more other compounds that induce dendritic cell maturation and proliferation to the medium. CD34 positive cells collected from cord blood or bone marrow can be differentiated into dendritic cells.

  Dendritic cells have traditionally been categorized as “immature” and “mature” cells, which can be distinguished in a simple manner into two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APCs with high capacity for antigen absorption and processing, which correlates with high expression of Fcγ and mannanase receptors. While the mature phenotype is typically characterized by low expression of these markers, high expression of cell surface molecules is associated with T cell activation, eg, class I and class II MHC, adhesion molecules (eg, CD54 and CD11) and costimulatory molecules (eg, CD40, CD80, CD86, and 4-1BB).

  APC is generally transfected with a polynucleotide encoding a prostate-specific protein (or a portion or other variant thereof) so that the prostate-specific polypeptide, or an immunogenic portion thereof, is expressed on the cell surface be able to. Such transfection can be performed ex vivo and then a composition or vaccine comprising such transfected cells is used for therapeutic purposes as described herein. be able to. Alternatively, a gene delivery vehicle targeting dendritic cells or other antigen presenting cells can be administered to a patient and transfection performed in vivo. In vivo and ex vivo, for example, transfection of dendritic cells is generally performed using methods known in the art, such as those described in WO 97/24470, or Mahvi et al., Immunology and Cell Biology 75 Can be performed using the gene gun approach described in: 456-460, 1997. Incubate dendritic cells or progeny cells with prostate tumor polypeptide, DNA (naked or in plasmid vector) or RNA; or bacteria or virus expressing antigen (eg vaccinia, tripox, adenovirus or lentiviral vector) Thus, antigen loading of dendritic cells can be achieved. Prior to loading, the polypeptide can be covalently linked to an immunological partner (eg, a carrier molecule) that provides T cell help. Alternatively, dendritic cells can be pulsed with an unconjugated immunological partner, either separately or in the presence of a polypeptide.

Cancer Therapy In other embodiments of the invention, the compositions described herein can be used for immunotherapy of cancer, eg, prostate cancer. In such methods, pharmaceutical compositions and vaccines are typically administered to patients. As used herein, “patient” means a warm-blooded animal, preferably a human. Patients can have or have no cancer. Thus, the pharmaceutical compositions and vaccines described above can be used to prevent the development of cancer or to treat patients with cancer. Cancers can be diagnosed using criteria that include the presence of a malignant tumor generally accepted in the field. Pharmaceutical compositions and vaccines can be administered before or after treatment, such as surgical removal of the primary tumor and / or administration of radiation therapy or conventional chemotherapeutic agents.

  In certain embodiments, the immunotherapy can be active immunotherapy, in which the tumor is treated with the administration of immune response modifiers (eg, polypeptides and polynucleotides disclosed herein). To respond, treatment is performed by in vivo stimulation of the endogenous host immune system.

In other embodiments, the immunotherapy can be passive immunotherapy, in which the treatment includes delivery of a factor (eg, an effector cell or antibody) with established tumor immunoreactivity, wherein Such factors can directly or indirectly mediate anti-tumor effects and are not necessarily dependent on the intact host immune system. Examples of effector cells are as follows: T cells as described above, T lymphocytes (eg CD8 ⁺ cytotoxic T lymphocytes and CD4 ⁺ T helper infiltrating lymphocytes), killer cells (eg natural killer cells and lymphokines) Activated killer cells), B cells and antigen presenting cells (eg, dendritic cells and macrophages) that express the polypeptides provided herein. T cell receptors and antibody receptors specific for the polypeptides described herein can be cloned, expressed, and transferred into other vectors or effector cells for adaptive immunotherapy. The polypeptides provided in the present invention can also be used to generate antibodies for passive immunotherapy or anti-idiotype antibodies (as described above and described in US Pat. No. 4,918,164). like).

  Effector cells can generally be obtained in sufficient quantities for adaptive immunotherapy by in vitro growth, as described herein. Culture conditions that retain in vivo antigen recognition and allow the number of single antigen-specific effector cells to grow to billions are well known in the art. Such in vitro culture conditions typically involve intermittent stimulation with antigen, often in the presence of cytokines (eg, IL-2) and non-dividing feeder cells. As described above, an immunoreactive polypeptide provided in the present invention can be used to expand an antigen-specific T cell culture to generate a sufficient number of cells for immunotherapy. In particular, according to standard techniques well known in the art, antigen-presenting cells such as dendritic cells, macrophages, monocytes, fibroblasts or B cells are pulsed with immunoreactive polypeptides, or 1 or It can be transfected with more polynucleotides. For example, antigen presenting cells can be transfected with a polynucleotide having a suitable promoter to increase expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to proliferate, be widely distributed, and survive for long periods in vivo. Studies have shown that repeated stimulation using antigen supplemented with IL-2 can induce cultured effector cells to proliferate in vivo and survive for long periods in substantial numbers (eg, , Cheever et al., Immunological Reviews 157: 177, 1997).

  Alternatively, a vector expressing a polypeptide described herein can be introduced into antigen-presenting cells taken from a patient and grown clonal ex vivo for transplantation back into the same patient. . Transfected cells are reintroduced into the patient by means known in the art, preferably in sterile form, by intravenous, intracavitary, intraperitoneal or intratumoral administration be able to.

  The route and frequency of administration of the therapeutic compositions disclosed herein, as well as the dosage, will vary from individual to individual and can be readily established according to standard techniques. In general, pharmaceutical compositions and vaccines can be administered by injection (eg, intradermal, intramuscular, intravenous or subcutaneous), intranasally (eg, by inhalation) or orally. Preferably, 1-10 doses can be administered over a 52 week period. Preferably, 6 doses are administered at 1 month intervals and booster vaccinations are subsequently given periodically. A suitable dose is that amount of a compound capable of promoting an anti-tumor immune response when administered as described above, and at least 10-50% above the baseline (ie, untreated) level. Such a response can be monitored by measuring anti-tumor antibodies in the patient or by vaccine-dependent generation of cytolytic effector cells that can kill the patient's tumor cells in vitro. Such vaccines also have improved clinical outcomes in vaccinated patients compared to non-vaccinated patients (eg, more frequent remissions, complete or partial or longer disease-free survival). Can induce a immune response that leads. Generally, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in the dosage ranges from about 100 μg to 5 mg / kg host. Suitable dosages vary with the size of the patient, but typically range from about 0.1 ml to about 5 ml.

  In general, a suitable dosage and treatment regimen provides one or more active compounds in an amount sufficient to provide a therapeutic and / or prophylactic benefit. Such responses can be achieved by establishing improved clinical outcomes in treated patients compared to untreated patients (eg, more frequent remissions, complete or partial or longer disease-free survival) Can be monitored. An increase in pre-existing immune response to prostate-specific proteins generally correlates with improved clinical outcome. Such an immune response can generally be assessed by standard proliferation, cytotoxicity or cytokine assays, and such assays can be performed using samples obtained from patients before and after treatment. .

Methods for Detecting Cancer In general, cancer is one or more prostate-specific proteins and / or such proteins in a biological sample (eg, blood, serum, urine and / or tumor biopsy) obtained from a patient. Can be detected in a patient based on the presence of a polynucleotide encoding. In other words, such proteins can be used as markers that indicate the presence or absence of cancer, eg, prostate cancer. In addition, such proteins may be useful for the detection of other cancers. The binding agents provided in the present invention generally allow detection of the level of antigen that binds to the agent in a biological sample. Polynucleotide primers and probes can be used to detect the level of mRNA encoding the tumor protein, which also indicates the presence or absence of cancer. In general, a prostate tumor sequence will be present at a level at least 3 times higher in tumor tissue than in normal tissue.

  Various assay formats using binding agents that detect polypeptide markers in a sample are known to those skilled in the art. See, for example, the following literature: Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of cancer in a patient is (a) contacting a biological sample obtained from the patient with a binding agent; (b) detecting the level of polypeptide binding to the binding agent in the sample; And (c) can be determined by comparing the level of the polypeptide to a predetermined cut-off value.

  In a preferred embodiment, the assay involves binding and removing the polypeptide from the rest of the sample using a binding agent immobilized on a solid support. The bound polypeptide can then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent / polypeptide complex. Such detection reagents comprise, for example, a binding agent that specifically binds to a polypeptide or antibody, or specifically bind to a binding agent such as anti-immunoglobulin, protein G, protein A or lectin. Other factors can be included. Alternatively, a competitive assay can be utilized, wherein the polypeptide is labeled with a reporter group and the binding agent is incubated with the sample and then allowed to bind to the immobilized binding agent. The degree to which a component in the sample inhibits binding of the labeled polypeptide to the binding agent indicates the reactivity of the sample with the immobilized binding agent. Polypeptides suitable for use in such assays include full length prostate specific proteins and portions thereof to which the binding agent binds, as described above.

  The solid support can be any material known to those of skill in the art that can bind proteins. For example, the solid support can be a test well in a microtiter plate or nitrocellulose or other suitable membrane. Alternatively, the support can be a bead or disk, such as glass, glass fiber, latex or plastic material, such as polystyrene or polyvinyl chloride. The support can also be a magnetic particle or fiber optic sensor, such as those described in US Pat. No. 5,359,681. Binding agents can be immobilized on solid supports according to various techniques known to those skilled in the art, and these techniques are extensively described in the patient and scientific literature. In the present invention, the term “immobilization” refers to non-covalent associations, such as adsorption and covalent bonding (which can be direct bonding between a factor and a functional group on a support or bonding by a crosslinker. Can mean both). Immobilization by adsorption to a well or membrane in a microtiter plate is preferred. In such cases, adsorption can be achieved by contacting the binding agent in a suitable buffer with the solid support for a suitable time. Contact time varies with temperature, but is typically about 1 hour to about 1 day. In general, contacting the well of a plastic microtiter plate (eg, polystyrene or polyvinyl chloride) with an amount of binding agent in the range of about 10 ng to about 10 μg, preferably about 100 ng to about 1 μg Sufficient for the immobilization of the factors.

  Covalent binding of a binding agent to a solid support is generally accomplished by first reacting with a bifunctional reagent that reacts with both a support and a functional group on the binding agent, such as a hydroxyl group. . For example, the binding agent can be covalently bound to a support having a suitable polymer coating by using benzoquinone or by condensing an aldehyde group on the support with an amine and active hydrogen on the binding factor ( For example, see Pierce Immunotechnology Catalog and Handbook, 1991, A12-A13).

  In certain embodiments, the assay is a two antibody sandwich assay. This assay is performed by first contacting the sample with an antibody immobilized on a solid support, commonly a well of a microtiter plate, and thus allowing the polypeptide in the sample to bind to the immobilized antibody. can do. The unbound sample is then removed from the immobilized polypeptide-antibody complex and a detection reagent containing a reporter group (preferably a secondary antibody capable of binding to a different site on the polypeptide) is added. The amount of detection reagent that remains bound to the solid support is then measured by a method appropriate for the particular reporter group.

  More particularly, once the antibody is immobilized on a solid support as described above, it typically blocks the remaining protein binding sites. Suitable blocking agents, such as bovine serum albumin or Tween 20 ™ (Sigma Chemical Co., St. Louis, MO) are known to those skilled in the art. The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. Prior to incubation, the sample is diluted with a suitable buffer, eg, phosphate buffer (PBS). In general, a suitable contact time (ie, incubation time) is sufficient to detect the presence of the polypeptide in a sample obtained from an individual having prostate cancer. Preferably, the contact time is sufficient to achieve a level that is about 95% of the level achieved in equilibrium between bound and unbound polypeptide. As one skilled in the art will appreciate, by assaying the level of binding that occurs over a period of time, the time required to achieve equilibrium can be readily determined. At room temperature, an incubation time of about 30 minutes is generally sufficient.

  Unbound sample can then be removed by washing the solid support with a suitable buffer, eg, PBS containing 0.1% Tween 20 ™. A secondary antibody containing a reporter group is then added. Preferred reporter groups include those previously described.

  The detection reagent is then incubated with the immobilized antibody-polypeptide complex for a time sufficient to detect the bound polypeptide. In general, an appropriate time can be determined by assaying the level of binding that occurs over a period of time. The unbound detection reagent is then removed and the bound detection reagent is detected with a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectrophotometric methods can be used to detect dyes, luminescent groups and fluorescent groups. Avidin coupled to different reporter groups (usually radioactive or fluorescent groups or enzymes) can be used to detect biotin. The reporter group of the enzyme can generally be detected by adding a substrate (generally for a specific time) and then by spectroscopic measurement or other analysis of the reaction product.

  To determine the presence or absence of cancer, eg, prostate cancer, the signal detected from the reporter group bound to the solid support is generally compared to a predetermined cutoff value. In one preferred embodiment, the cutoff value for detecting cancer is the average signal obtained when incubating the immobilized antibody with a sample from a patient without cancer. In general, a sample that produces a signal that is 3 standard deviations greater than a predetermined cut-off value is considered positive for cancer. In another preferred embodiment, the cut-off value is determined using the Receiver Operator Curve according to the methods of the following literature: Sakett et al., Clinical Epidemiology: A Basic Science for Clinical Medicne, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, from a plot of a pair of true positive rate (ie sensitivity) and false positive rate (100% specificity) corresponding to each possible cut-off value for a diagnostic test result. The cut-off value can be determined. The cut-off value of the plot closest to the upper left corner (ie, the value surrounding the largest area) is the most accurate cut-off value, and samples that produce a signal higher than the cut-off value determined by this method are positive. You can think of it. Alternatively, the cutoff value can be shifted to the left along the plot to minimize the false positive rate, or shifted to the right to minimize the false negative rate. In general, a sample that produces a signal higher than the cut-off value determined by this method is considered positive for cancer.

  In related embodiments, the assay is performed in a flow-through or strip test format, where the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, the polypeptide in the sample binds to the immobilized binding agent as the sample passes through the membrane. Second, the labeled binding agent binds to the binding agent-polypeptide complex as the solution containing the second binding agent passes through the membrane. Detection of bound second binding agent can then be performed as described above. In the strip test, one end of the membrane with bound binding agent is immersed in a solution containing the sample. The sample moves along the membrane through the region containing the second binding agent to the region of immobilized binding agent. Concentration of the second binding factor in the region of immobilized antibody indicates the presence of cancer. Typically, the enrichment of the second binding agent at that site produces a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, in the aforementioned format, when a biological sample contains a level of polypeptide that is sufficient to generate a positive signal in a two-antibody sandwich assay, on the membrane to generate a visually distinguishable pattern. The amount of binding factor immobilized on is selected. 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 1 μg, more preferably from about 50 ng to about 500 ng. Typically, such a test can be performed using a very small amount of biological sample.

  Of course, there are numerous other assay protocols suitable for use with the proteins or binding agents of the invention. The above description is intended to be merely exemplary. For example, as will be apparent to those skilled in the art, the above protocol can be easily modified to use such polypeptides to detect antibodies that bind to prostate specific polypeptides in a biological sample. Can do. Detection of such prostate specific protein specific antibodies can be correlated with the presence of cancer.

Alternatively, or alternatively, cancer can be detected based on the presence of T cells that specifically react with prostate specific proteins in a biological sample. In certain methods, a biological sample comprising CD4 ⁺ and / or CD8 ⁺ T cells isolated from a patient is obtained from a prostate specific protein, a polynucleotide encoding such a peptide and / or Incubate with APCs that express at least the immunogenic portion of the polypeptide to detect the presence or absence of T cells. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be isolated from patients by routine techniques (eg, Ficoll / Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells can be incubated in vitro at 37 ° C. with a prostate specific polypeptide (eg, 5-25 μg / ml) for 2-9 days (typically 4 days). It may be desirable to incubate other aliquots of T cell samples to serve as controls in the absence of prostate tumor protein. For CD4 ⁺ T cells, activation is preferably detected by assessing T cell proliferation. For CD8 ⁺ T cells, activation is preferably detected by assessing cytolytic activity. A level of proliferation that is at least 2-fold greater and / or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of cancer in the patient.

  As described above, or alternatively, cancer can be detected based on the level of mRNA encoding a prostate specific protein in a biological sample. For example, in a polymerase chain reaction (PCR) -based assay, at least two oligonucleotide primers can be used to amplify a portion of prostate-specific cDNA from a biological sample, where the oligonucleotide primer At least one is specific for (ie, hybridizes to) a polynucleotide encoding a prostate specific protein. The amplified cDNA is then separated and detected using techniques well known in the art, such as electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to polynucleotides encoding prostate specific proteins are used in hybridization assays to detect the presence of polynucleotides encoding tumor proteins in biological samples. can do.

  In order to allow hybridization under assay conditions, the oligonucleotide primers and probes are at least for a portion of the polynucleotide encoding a prostate specific protein that is at least 10 nucleotides in length, preferably at least 20 nucleotides in length. It comprises an oligonucleotide sequence having about 60% identity, preferably at least about 75%, more preferably at least about 90% identity. Preferably, the 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 that can be routinely used in the diagnostic methods described herein are preferably at least 10 to 40 nucleotides in length. In preferred embodiments, the oligonucleotide primers are SEQ ID NOs: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382, 384-476, 524, 526, 530, 531, 533, 535, and 536, comprising at least 10 contiguous nucleotides of the DNA molecule, more preferably at least 15 contiguous nucleotides. Techniques for both PCR-based and hybridization assays are well known in the art (see, eg, the following literature: Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51: 263). 1987, edited by Erlich, PCR Technology, Stockton Press, NY, 1989).

  In one preferred assay, RT-PCR is used, where PCR is applied in combination with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and reverse transcribed to produce cDNA molecules. PCR amplification using at least one specific primer generates cDNA molecules, which can be separated and visualized, for example, by gel electrophoresis. Amplification can be performed on biological samples taken from the subject patient and from individuals who do not have cancer. Amplification reactions can be performed on several dilutions of cDNA spanning two orders of magnitude. A 2-fold or greater increase in expression in several dilutions of the sample of the subject patient compared to the same dilution of the non-cancer sample is typically considered positive.

  In other embodiments, the disclosed compositions can be used as markers of cancer progression. In this embodiment, the assays described above for the diagnosis of cancer can be performed over time and the change in the level of one or more reactive polypeptides or polynucleotides can be assessed. For example, the assay can be run every 24-72 hours for a period of 6 months to 1 year, and then run as needed. In general, cancer is progressing in patients whose levels of detected polypeptide or polynucleotide increase over time. In contrast, cancer is not progressing when the level of reactive polypeptide or polynucleotide stays constant or decreases with time.

  Certain in vivo diagnostic assays can be performed directly on the tumor. One such assay involves contacting tumor cells with a binding agent. The bound binding agent can then be detected directly or indirectly by the reporter group. Such binding factors can also be used in histological applications.

  As described above, a number of prostate specific protein markers can be assayed in a given sample to improve sensitivity. It will be apparent that binding agents specific for the different proteins provided in the present invention can be combined in a single assay. Protein markers are selected based on routine experimentation to determine the combination that yields optimal sensitivity. Additionally or alternatively, assays for proteins provided in the present invention can be combined with assays for other known tumor antigens.

Diagnostic Kit The present invention further provides a kit for use in the above diagnostic method. Such a kit comprises two or more components necessary to perform a diagnostic assay. The component can be a compound, reagent, container and / or device. For example, one container in the kit can contain a monoclonal antibody or fragment thereof that specifically binds to prostate specific protein. Such antibodies or fragments can be provided in a form bound to a support material as described above. One or more additional containers can surround the factors to be used in the assay. Such a kit can also or optionally contain a detection reagent as described above containing a reporter group suitable for direct or indirect detection of antibody binding.

  Alternatively, the kit can be designed to detect the level of mRNA encoding a prostate specific protein in a biological sample. Such kits generally comprise at least one probe or primer of the aforementioned oligonucleotide that hybridizes to a polynucleotide encoding a prostate specific protein. Such oligonucleotides can be used, for example, in PCR or hybridization assays. Additional components that can be present in such kits include a second oligonucleotide and / or a diagnostic reagent or container that facilitates detection of a polynucleotide encoding a prostate specific protein.

  The following examples are illustrative of the invention but should not be construed as limiting the invention.

Example 1
Isolation and Characterization of Prostate Specific Polypeptides In this example, the isolation of certain prostate specific polypeptides from a prostate tumor cDNA library is described.

Using the Superscript Plasmid System for DNA synthesis and the plasmid cloning kit (BRL Life Technologies, 20897 Gaithersburg, Md.) According to the manufacturer's protocol, polymorphize the human prostate tumor cDNA expression library. Constructed from A ⁺ RNA. Specifically, prostate tumor tissue was homogenized with Polytron (Kinematica, Switzerland) and total RNA was extracted using Trizol reagent (BRL Life Technologies) according to the manufacturer's instructions. Poly A ⁺ RNA was then purified using a Qiagen oligotex spin column mRNA purification kit (Qiagen, 91355 Santa Clarita, CA) according to the manufacturer's protocol. First strand cDNA was synthesized using NotI / oligo-dT18 primer. Double-stranded cDNA was synthesized, ligated with an EcoRI / BAXI adapter (Invitrogen, San Diego, Calif.) And digested with NotI. After size fractionation on a Chroma Spin-1000 column (Clontech, Palo Alto, Calif.), The cDNA was ligated into the EcoRI / NotI sites of pCDNA3.1 (Invitrogen) and electroMax E. coli ( E. coli) DH10B cells (BRL Life Technologies) were transformed by electroporation.

Using the same procedure, a normal human pancreatic cDNA expression library was prepared from a pool of 6 tissue specimens (Clontech). The cDNA library was characterized by measuring the number of independent colonies, the percentage of clones carrying the insert, the average insert size and sequence analysis. The prostate tumor library contained 1.64 × 10 ⁷ independent colonies, 70% of the clones had inserts, and the average insert size was 1745 base pairs. The normal pancreatic cDNA library contained 3.3 × 10 ⁶ independent colonies, 69% of the clones had inserts, and the average insert size was 1120 base pairs. For both libraries, sequence analysis showed that most of the clones had full-length cDNA sequences and were synthesized from mRNA, with minimal contamination of rRNA and mitochondrial DNA.

Subtraction of the cDNA library was performed using the prostate tumor and normal pancreas cDNA library with slight modifications as described in the following literature: Hara et al., Blood 84: 189-199. 1994. Specifically, a prostate tumor-specific subtracted cDNA library was prepared as follows. A normal pancreatic cDNA library (70 μg) was digested with EcoRI, NotI, and SfuI, and then a filling-in reaction was performed with DNA polymerase Klenow fragment. After phenol-chloroform extraction and ethanol precipitation, the DNA was dissolved in 100 μl H ₂ O, heat denatured and mixed with 100 μl (100 μg) photoprobe biotin (Vector Laboratories, Burlingame, Calif.). The resulting mixture was irradiated with a 270 watt sunlamp for 20 minutes on ice as recommended by the manufacturer. Additional photoprobe biotin (50 μl) was added and the biotinylation reaction was repeated. After extraction 5 times with butanol, the DNA was ethanol precipitated and dissolved in 23 μl of H ₂ O to form driver DNA.

To form tracer DNA, 10 μg of prostate tumor cDNA library was digested with BamHI and XhoI, phenol-chloroform extracted and passed through a chroma spin-400 column (Clontech). After ethanol precipitation, the tracer DNA was dissolved in 5 μl of H ₂ O. Mix tracer DNA with 15 μl driver DNA and 20 μl 2 × hybridization buffer (1.5 M NaCl / 10 mM EDTA / 50 mM HEPES pH 7.5 / 0.2% sodium dodecyl sulfate), overlay with mineral oil, complete It was denatured by heating. Samples were immediately transferred into a 68 ° C. water bath and incubated for 20 hours (long hybridization [LH]). The reaction mixture was then treated with streptavidin and then extracted with phenol / chloroform. This method was repeated three more times. Subtracted DNA was precipitated, dissolved in 12 μl H ₂ O, mixed with 8 μl driver DNA and 20 μl 2 × hybridization buffer and hybridized for 2 hours at 68 ° C. (short hybridization [SH] ). After removal of the biotinylated double stranded DNA, the subtracted cDNA was ligated into the BamHI / XhoI site of chloramphenicol resistant pBCSK ⁺ (Stratagene, 92037 La Jolla, Calif.) And electroMax E. coli (E E. coli) DH10B cells were transformed by electroporation to produce 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 taken from the subtracted prostate tumor specific library and grouped based on the size of the insert. Representative cDNA clones were further characterized by DNA sequencing using the Perkin Elmer / Applied Biosystems Division automated sequencing device 373A (Foster City, Calif.) . Six cDNA clones (hereinafter referred to as F1-13, F1-12, F1-16, H1-1, H1-9 and H1-4) must be enriched in the subtracted prostate specific cDNA library. Indicated. The 3 ′ and 5 ′ cDNA sequences determined for F1-12 are set forth in SEQ ID NOs: 2 and 3, respectively, and were determined for F1-13, F1-16, H1-1, H1-9 and H1-4 The 3 ′ cDNA sequences are set forth in SEQ ID NOs: 1 and 4-7, respectively.

  In the gene bank, EMBL and GeneBank databases (Release 96) were used to compare the cDNA sequences of isolated clones with known sequences. Four of the prostate tumor cDNA clones, F1-13, F1-16, H1-1 and H1-4, were determined to encode the following previously identified proteins: prostate specific antigen (PSA), Human glandular kallikrein, human tumor expression enhancing gene, and mitochondrial cytochrome C oxidase subunit II. H1-9 was found to be identical to the previously identified human autonomously replicating sequence. No significant homology to the cDNA sequence of F1-12 was found.

  Subsequent work led to the isolation of the full-length cDNA sequence of F1-12. This sequence is set forth in SEQ ID NO: 107, and the corresponding predicted amino acid sequence is set forth in SEQ ID NO: 108.

  To clone low abundance prostate tumor-specific genes, the aforementioned prostate tumor cDNA library was subtracted with a normal pancreatic cDNA library, and the three most common of the previously subtracted prostate tumor-specific cDNA libraries. Abundant genes: Subtraction of the cDNA library was performed by subtracting with human gland kallikrein, prostate specific antigen (PSA), and mitochondrial cytochrome C oxidase subunit II. Specifically, 1 μg of human glandular kallikrein, PSA, and mitochondrial cytochrome C oxidase subunit II cDNA in pCDNA3.1 was added to the driver DNA, and subtraction was performed as described above to perform a second subtracted. A cDNA library (hereinafter referred to as a “subtracted prostate tumor-specific cDNA library with spikes”) was formed.

  Twenty-two cDNA clones were isolated from a subtracted prostate tumor-specific cDNA library with spikes. Determine clones (referred to as J1-17, L1-12, N1-1862, J1-13, J1-19, J1-25, J1-24, K1-58, K1-63, L1-4 and L1-14) The resulting 3 ′ and 5 ′ cDNA sequences are shown in SEQ ID NOs: 8-9, 10-11, 12-13, 14-15, 16-17, 18-19, 20-21, 22-23, 24-25, respectively. 26-27 and 28-29. Decided on clones (referred to as J1-12, J1-16, J1-21, K1-48, K1-55, L1-2, L1-6, N1-1858, N1-1860, N1-1861, N1-1864) The obtained 3 ′ cDNA sequences are described in SEQ ID NOs: 30 to 40, respectively. As described above, when these sequences are compared with sequences in the gene bank, three of the five most abundant DNA species (J1-17, L1-12 and N1-1862; SEQ ID NOs: 8-9, respectively) No significant homology was revealed for 10-11 and 12-13). Of the remaining two most abundant species, one (J1-12; SEQ ID NO: 30) is identical to the previously identified human lung surfactant associate protein and the other (K1-48; SEQ ID NO: 33) ) Was determined to have some homology to R. novbegicus mRNA for 2-arylpropionyl-CoA epimerase. Of the 17 low abundance cDNA clones isolated from spiked subtracted prostate tumor-specific cDNA libraries, 4 (J1-16, K1-55, L1-6 and N1-1864; , SEQ ID NOs: 31, 34, 36 and 40) are identical to previously identified sequences, and two (J1-21 and N1-1860, respectively; SEQ ID NOs: 32 and 38) are somewhat to non-human sequences. And two (L1-2 and N1-18601, respectively; SEQ ID NOs: 35 and 39, respectively) were found to show some homology to known human sequences. Polypeptides J1-13, J1-19, J1-24, J1-25, K1-58, K1-63, L1-4, L1-14 (SEQ ID NOs: 14-15, 16-17, 20-21, respectively) 18-19, 22-23, 24-25, 26-27, 28-29) no significant homology was found.

  Subsequent studies led to the isolation of full-length cDNA sequences for J1-17, L1-12 and N1-1862 (SEQ ID NOs: 109-111, respectively). The corresponding predicted amino acid sequence is set forth in SEQ ID NOs: 112-114. L1-12 is also referred to as P501S.

  In further experiments, 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 + RNAs (referred to as “prostate subtraction 2”). The cDNA sequences determined for three clones (hereinafter referred to as U1-3064, U1-3065, V13692 and 1A-3905) are set forth in SEQ ID NOs: 69-72, respectively. Comparing the determined sequence with that in the gene bank revealed no significant homology to U1-3065.

  A second subtraction with spikes (referred to as “prostate subtraction 2”) is performed by subtracting a prostate tumor-specific cDNA library with spikes with a normal pancreatic cDNA library, and then PSA, J1-17, lungs Spiked with detergent associate protein, mitochondrial DNA, cytochrome C oxidase subunit II, N1-1862, autonomously replicating sequence, L1-12 and tumor expression enhancing gene. Four additional clones (hereinafter referred to as V1-3686, R1-2330, 1B-3976 and V1-3679) were isolated. The cDNA sequences determined for these clones are set forth in SEQ ID NOs: 73-76, respectively. Comparison of the determined sequence with that in the gene bank revealed no significant homology to V1-3686 and R1-2330.

  In a further analysis of the three prostate subtractions described above (prostate subtraction 2, spiked subtracted prostate tumor-specific cDNA library, and spiked prostate subtraction 2), 16 additional clones (1G-4736, 1G- 4738, 1G-4471, 1G-4474, 1G-4474, 1H-4774, 1H-4781, 1H-4785, 1H-4787, 1H-4796, 1I-4810, 1I-4811, 1J-4876, 1K-4884 and 1K-4896) was identified. The cDNA sequences determined for these clones are set forth in SEQ ID NOs: 77-92. Comparing the sequence determined as described above with the sequence in the gene bank, 1G-4741, 1G-4734, 1I-4807, 1J-4876 and 1K-4896 (SEQ ID NOs: 79, 81, 87, 90 and 92) no significant homology was revealed. Further analysis of the isolated clones revealed that 1G-4376, 1G-4738, 1G-4741, 1G-4474, 1H-4744, 1H-4781, 1H-4785, 1H-4787, 1H-4479, 1I-4807, Extended cDNA sequences for 1J-4876, 1K-4484, and 1K-4896 were determined, which are described in SEQ ID NOs: 179-188 and 191-193, respectively, and additional portions for 1I-4810 and 1I-4811 Specific cDNA sequences were determined and are set forth in SEQ ID NOs: 189 and 190, respectively.

  In an additional study using prostate subtraction spike 2, three additional clones were isolated. Their sequences were determined as described above and compared against the latest GenBank. All three clones were found to have homology to known genes, these are cysteine-rich proteins, KIAA0242, and KIAA0280 (SEQ ID NOs: 317, 319, and 320, respectively). Further analysis of these clones by Synteni microarray (Synteni, Palo Alto, Calif.) Revealed that all three clones were overexpressed in most prostate tumors and prostate BPH, and most of the normal prostate tissues tested, but all The expression in other normal tissues was low.

  Additional subtraction was performed by subtracting a normal pancreatic cDNA library with normal pancreatic cDNA (“prostate subtraction 3”). This identified six additional clones (referred to as 1G-4761, 1G-4762, 1H-4766, 1H-4770, 1H-4771 and 1H-4772) (SEQ ID NOs: 93-98, respectively). Comparison of these sequences with the sequences in the gene bank revealed no significant homology to 1G-4761 and 1H-4771 (SEQ ID NOs: 93 and 97, respectively). Further analysis of the isolated clones determined extended cDNA sequences for 1G-4761, 1G-4762, 1H-4766, and 1H-4772, which are set forth in SEQ ID NOs: 194-196 and 199, respectively. An additional partial cDNA sequence was then determined for 1H-4770 and 1H-4771, which are set forth in SEQ ID NOs: 197 and 198, respectively.

  Subtracting a prostate tumor cDNA library prepared from a pool of poly A + RNA from 3 prostate cancer patients with a normal pancreatic cDNA library (prostate subtraction 4), 8 clones (1D-4297, 1D-4309, 1D.1-4278, 1D-4288, 1D-4283, 1D-4304, 1D-4296 and 1D-4280) (SEQ ID NOs: 99-107, respectively) were identified. These sequences were compared to sequences in the gene bank as described above. No significant homology was found for 1D-4283 and 1D-4304 (SEQ ID NOs: 103 and 104, respectively). Further analysis of the isolated clones determined extended cDNA sequences for 1D-4309, 1D.1-4278, 1D-4288, 1D-4283, 1D-4304, 1D-4296, and 1D-4280. Are described in SEQ ID NOs: 200-206, respectively.

  The cDNA clones isolated in the aforementioned prostate subtraction 1 and prostate subtraction 2 were colony PCR amplified and their microarray technology (Synteni, Palo Alto, Calif.) Allowed their clones in prostate tumors, normal prostate and various other normal tissues mRNA expression levels were measured. Briefly, PCR amplification products were dot arranged on the slide in an array format, and each product occupied a unique position in the array. mRNA was extracted from the tissue sample to be tested and reverse transcribed to produce a fluorescently labeled cDNA probe. The microarray was probed with a labeled cDNA probe, the slide was scanned, and the fluorescence intensity was measured. This intensity correlates with the hybridization intensity. Two clones (P509S and P510S) are overexpressed in prostate tumor and normal prostate, and all other normal tissues tested (liver, pancreas, skin, bone marrow, brain, breast, adrenal gland, bladder, testis, salivary gland, large intestine) , Kidney, ovary, lung, spinal cord, skeletal muscle and colon). The cDNA sequences determined for P509S and P510S are set forth in SEQ ID NOs: 223 and 224, respectively. As mentioned above, comparing these sequences with sequences in the gene bank revealed some homology for previously identified ESTs.

  In additional studies, a full-length cDNA sequence was isolated for the P509S clone. This sequence is set forth in SEQ ID NO: 332 and the corresponding predicted amino acid sequence is set forth in SEQ ID NO: 339. The corresponding deduced amino acid sequences that provide the two mutant full length cDNA sequences of P510S in SEQ ID NOs: 535 and 536 are provided in SEQ ID NOs: 537 and 538.

Example 2
Determination of Tissue Specificity of Prostate Specific Polypeptides Using gene specific primers, representative prostate specific polypeptides F1-16, H1-1, J1-17 (also referred to as P502S), L1-12 (Also referred to as P501S), F1-12 (also referred to as P504S) and N1-1862 (also referred to as P503S) were examined for mRNA expression levels in various normal and tumor tissues by RT-PCR.

  Briefly, total RNA was extracted from various normal and tumor tissues using Trizol reagent as described above. First strand synthesis was performed using 1-2 μg total RNA and Superscript II reverse transcriptase (BRL Life Technologies) at 42 ° C. for 1 hour. The cDNA was then amplified by PCR using gene specific primers. To ensure the semi-quantitative nature of RT-PCR, β-actin was used as an internal control for each of the examined tissues. First, serial dilutions of first strand cDNA were prepared and PCR assays were performed using β-actin specific primers. One dilution was selected that allows amplification of the linear range of β-actin template and is sensitive enough to reflect the initial copy number differences. Using these conditions, β-actin levels were measured for each reverse transcription reaction from each tissue. When using first strand cDNA prepared by DNase treatment and without the addition of reverse transcriptase, DNA contamination was minimized by confirming negative PCR results.

  4 different tumor tissues (prostate tumor from 2 patients, breast tumor, colon tumor, lung tumor from 3 patients) and prostate, colon, kidney, liver, lung, ovary, pancreas, skeletal muscle, skin MRNA expression levels were examined in 16 different normal tissues, including stomach, testis, bone marrow and brain. F1-16 is found to be expressed at high levels in prostate tumor tissue, colon tumors and normal prostate, and to be expressed at low levels in normal liver, skin and testis, and expression is detected in other tissues examined It was impossible. H1-1 is expressed at high levels in prostate tumor tissue, lung tumors, breast tumors, normal prostate, normal colon and normal brain, and expressed at very low levels in normal lung, pancreas, skeletal muscle, skin, small intestine, bone marrow And was not detected in other tissues. J1-17 (P502S) and L1-12 (P501S) appear to be specifically overexpressed in the prostate, both genes are expressed at high levels in prostate tumors and normal prostate, but all others tested Was expressed at low to undetectable levels in all tissues. N1-1862 (P503S) was overexpressed in 60% of prostate tumors and was found to be detectable in normal colon and kidney. Thus, as the RT-PCR results show, F1-16, H1-1, J1-17 (P502S), N1-1862 (P503S) and L1-12 (P501S) are prostate specific or prostate Is expressed at a significantly increased level.

  In addition, RT-PCR studies showed that F-12 (P504S) is overexpressed in 60% of prostate tumors and can be detected in normal kidneys but not in all other test tissues. It was. Similarly, R1-2330 was overexpressed in 40% of prostate tumors and was found to be detectable in normal kidney and liver but not in all other test tissues. U1-3064 was overexpressed in 60% of prostate tumors and was also expressed in breast and colon tumors, but was found not to be detected in normal tissues.

  Characterization of R1-2330, U1-3064 and ID-4279 by RT-PCR revealed that these three antigens were overexpressed in the prostate and / or prostate tumor.

  Northern analysis of 4 prostate tumors, 2 normal prostate samples, 2 BPH prostates, and normal colon, kidney, liver, lung, pancreas, skeletal muscle, brain, stomach, testis, small intestine and bone marrow, L1-12 (P501S) was found to be overexpressed in prostate tumors and normal prostate but not detected in other normal test tissues. J1-17 (P502S) was detected in two prostate tumors but not in other test tissues. N1-1862 (P503S) was found to be overexpressed in three prostate tumors and expressed in normal prostate, colon and kidney but not in other test tissues. F1-12 (P504S) was found to be highly expressed in two prostate tumors and undetectable in all other examined tissues.

  Using the microarray technique described above, prostate tumors, breast tumors and the following normal tissues of the representative antigens described herein: prostate, liver, pancreas, skin, bone marrow, brain, breast, Expression levels in the adrenal gland, bladder, testis, salivary gland, large intestine, kidney, ovary, lung, spinal cord, skeletal muscle and colon were measured. L1-12 (P501S) was found to be overexpressed in normal prostate and prostate tumor, and some expression was detected in normal skeletal muscle. Both J1-12 and F1-12 (P504S) were overexpressed in prostate tumors, and expression in all other test tissues was found to be low or undetectable. N1-1862 (P503S) expression was found to be high in prostate tumors and normal prostate, low in normal colon and normal colon, and undetectable in all other test tissues It was. R1-2330 was found to be overexpressed in prostate tumors and normal prostate and expressed at lower levels in all other test tissues. ID-4279 was found to be overexpressed in prostate tumors and normal prostate, expressed at lower levels in normal spinal cord, and undetectable in all other test tissues.

  As a result of further microarray analysis specifically examining the extent to which P501S (SEQ ID NO: 110) was expressed in breast tumors, moderate overexpression was made not only in breast tumors but also in metastatic breast tumors (2/31), It became clear that it was a negligible low expression in normal tissues. This data suggests that P501S is overexpressed not only in prostate tumors but also in various breast tumors.

  Vasmatzis et al., Proc. Natl. Acad. Sci. USA, 95: 300-304, 1998 32 EST (expression sequence tag) expression levels in various tumors and normal tissues were examined using the microarray technique described above. did. Two of these clones (referred to as P1000C and P1001C) are overexpressed in prostate tumors and normal prostate, and all other test tissues (normal aorta, thymus, quiescent and activated PBMC, epithelium) Low levels of expression in cells, spinal cord, adrenal gland, fetal tissue, skin, salivary gland, large intestine, bone marrow, liver, lung, dendritic cells, stomach, lymph nodes, brain, heart, small intestine, skeletal muscle, colon and kidney) It was found to be an undetectable level. The determined cDNA sequences of P1000C and P1001C are shown in SEQ ID NOs: 384 and 472, respectively. The sequence of P1001C was found to show some homology to the previously isolated human mRNA of the JM27 protein. There was no significant homology in the P1000C sequence.

  Expression of the polypeptide encoded by the full-length cDNA sequence of F1-12 (also referred to as P504S; SEQ ID NO: 108) was examined by immunohistochemical analysis. Rabbit anti-P504S polyclonal antibody was raised against the full length P504S protein by standard techniques. Subsequently, the polyclonal antibody was isolated and characterized by methods known in the art. As a result of immunohistochemical analysis, it was found that P504S polypeptide was expressed in 100% of prostate cancer test samples (n = 5).

  The rabbit anti-P504S polyclonal antibody appeared to label benign prostate cells with the same cytoplasmic granule staining but with light nuclear staining. Analysis of normal tissues has revealed that the encoded polypeptide is expressed in some normal human tissues but not in all normal tissues. Positive cytoplasmic staining with rabbit anti-P504S polyclonal antibody was found in macrophages present in normal human kidney, liver, brain, colon and lung, but heart and bone marrow were negative.

  This data indicates that P504S polypeptide is present in prostate cancer tissue and that there is a qualitative and quantitative difference in staining between benign prostatic hyperplastic tissue and prostate cancer tissue. Polypeptides can be selectively detected in prostate tumors, suggesting that they are useful for diagnosing prostate cancer.

Example 3
Isolation and characterization of prostate-specific polypeptide by PCR-based subtraction method
From normal prostate, subtracted with cDNA from 10 other normal tissues (brain, heart, kidney, liver, lung, ovary, placenta, skeletal muscle, spleen and thymus) and then subjected to the first round of PCR amplification A cDNA subtraction library containing cDNA was purchased from Clontech. This library was subjected to a second round of PCR amplification according to the manufacturer's protocol. The resulting cDNA fragment was subcloned into the vector pT7 Blue T vector (Novagen, Madison, WI, USA), and then XL-1 Blue MRF ′ E. coli (Stratagene) was transformed with the vector. DNA was isolated from independent clones and then sequenced using the Perkin Elmer / Applied Biosystems Division Automated Sequencer Model 373A.

  The sequence of 59 positive clones was determined. As a result of comparing the DNA sequences of these clones with the DNA sequences of the gene bank clones as described above, 25 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 cDNA sequences determined for these clones are shown in SEQ ID NOs: 41-45, 47-52 and 54-65, respectively. P29, P47, P68, P80 and P82 (SEQ ID NOs: 46, 53 and 66-68, respectively) were found to show some homology to previously isolated DNA sequences. To the best knowledge of the inventors of the present invention, it has never been reported that these sequences are present in the prostate.

  Further testing using the PCR-based method described above revealed that over 180 additional clones were isolated, of which 23 clones showed no significant homology to known sequences. It was issued. The determined cDNA sequences of these clones are shown in SEQ ID NOs: 115-123, 127, 131, 137, 145, 147-151, 153, 156-158 and 160. 23 clones (SEQ ID NOs 124-126, 128-130, 132-136, 138-144, 146, 152, 154, 155 and 159) show some homology to previously isolated ESTs It was found. An additional 10 clones (SEQ ID NOs 161-170) were found to have some homology to known genes. A larger cDNA clone containing the P20 sequence is an example of a splice variant of the gene designated P703P. The determined DNA sequences of these variants, designated DE1, DE13 and DE14 are shown in SEQ ID NOs: 171, 175 and 177, respectively, and the corresponding predicted amino acid sequences are shown in SEQ ID NOs: 172, 176 and 178, respectively. It is. The determined cDNA sequence of P703 in the extended spliced form is shown in SEQ ID NO: 225. The DNA sequences of the splice variants designated DE2 and DE6 are shown in SEQ ID NOs 173 and 174, respectively.

  Tumor tissue [prostate (n = 5), breast (n = 2), colon and lung], normal tissue [prostate (n = 5), colon, kidney, liver, lung (n = 2), ovary (n = 2) ), Skeletal muscle, skin, stomach, small intestine and brain], and representative clones in activated and non-activated PBMCs were measured for mRNA expression levels by RT-PCR as described above. Expression was tested on one sample of each tissue type unless otherwise stated.

  P9 was found to be highly expressed in normal prostate and prostate tumors compared to all normal test tissues except the normal colon that showed comparable expression. A portion of the P20 or P703P gene was found to be highly expressed in normal prostate and prostate tumors compared to all 12 normal test tissues. The increase in P20 expression seen in breast tumors (n = 2), colon tumors and lung tumors was moderate compared to all normal tissues except lung (1 of 2 samples). It was found that P18 expression was increased in normal prostate, prostate and breast tumors compared to other normal tissues except the lung and stomach. There was a modest increase in P5 expression in normal prostate compared to most other normal tissues. However, some elevated expression was seen in normal lung and PBMC. Increased expression of P5 was found in one sample of prostate tumor (2 of 5 samples), breast tumor and lung tumor. For P30, similar expression was seen in normal prostate and prostate tumors compared to 6 of 12 other normal test tissues. Increased expression was also seen in breast, lung and colon tumor samples as well as normal PMBC. P29 was found to be overexpressed in prostate tumors (5 of 5 samples) and normal prostate (5 samples in 5 samples) compared to the majority of normal tissues. However, significant expression of P29 was observed in normal colon and normal lung (2 of 2 samples). P80 was found to be overexpressed in prostate tumor (5 of 5 samples) and normal prostate (5 of 5 samples) compared to all other normal test tissues, and in colon tumors Increased expression was also observed.

  After further testing, 10-d8, 10-h10, 11-c8, 7-g6, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3, 8-h11, 9-f12 And 12 additional clones, designated 9-f3. The determined DNA sequences of 10-d8, 10-h10, 11-c8, 8-d4, 8-d9, 8-h11, 9-f12 and 9-f3 are respectively SEQ ID NOs: 207, 208, 209, 216, 217, 220, 221 and 222. The determined forward and reverse DNA sequences of 7-g6, 8-b5, 8-b6 and 8-g3 are shown in SEQ ID NOs: 210 and 211; 212 and 213; 214 and 215; and 218 and 219, respectively. . As a result of comparing these sequences with those of the gene bank, no significant homology to the 9-f3 sequence was found. Clones 10-d8, 11-c8 and 8-h11 were found to show some homology to previously isolated ESTs, but 10-h10, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3 and 9-f12 were found to show some homology to previously isolated genes. Further characterization of 7-G6 and 8-G3 showed identity to the known genes PAP and PSA, respectively.

  The mRNA expression level of these clones was measured using the microarray technique. Clone 7-G6, 8-G3, 8-B5, 8-B6, 8-D4, 8-D9, 9-F3, 9-F12, 9-H3, 10-A2, 10-A4, 11-C9 and 11 -F2 was found to be overexpressed in prostate tumors and normal prostate, but expression in other test tissues was low or undetectable. Increased expression of 8-F11 was seen in prostate tumors and normal prostate, bladder, skeletal muscle and colon. Increased expression of 10-H10 was seen in prostate tumors and normal prostate, bladder, lung, colon, brain and colon. Increased expression of 9-B1 was found in prostate tumor, breast tumor and normal prostate, salivary gland, large intestine and skin, while increased expression of 11-C8 was found in prostate tumor and normal prostate and large intestine.

  Additional cDNA fragments from the PCR-based normal prostate subtraction were found to be prostate specific by both microarray techniques and RT-PCR. The determined cDNA sequence of this clone (designated 9-A11) is shown in SEQ ID NO: 226. When this sequence was compared with the sequence in the public database, it was found to be 99% identical to the known gene HOXB13.

  Further testing resulted in the isolation of clones 8-C6 and 8-H7. The determined cDNA sequences of these clones are shown in SEQ ID NOs: 227 and 228, respectively. These sequences were found to show some homology to previously isolated ESTs.

  Using a method based on PCR and hybridization, a longer cDNA sequence (also referred to as P703P) was obtained for clone P20, and three additional cDNA fragments were obtained by sequentially extending the 5 ′ end of the gene. Obtained. These fragments are referred to as P703PDE5, P703P6.26 and P703 PX-23 (SEQ ID NOs: 326, 328 and 330; the expected corresponding amino acid sequences are shown in SEQ ID NOs: 327, 329 and 331, respectively) Contains an additional 5 'sequence. P703PDE5 was recovered by screening a cDNA library (# 141-26) using 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 a cDNA library (# 438-48). At the same time, these additional sequences contain all putative mature serine proteases along with a portion of the putative signal sequence. The predicted full length cDNA sequence of P703P is provided in SEQ ID NO: 524. This corresponds to the deduced amino acid sequence provided in SEQ ID NO: 525.

  Further testing with a PCR-based subtraction library (JR: PCR subtraction) of a prostate tumor pool subtracted against a pool of normal tissues resulted in the isolation of 13 additional clones, of which Seven of them did not share significant homology to known GenBank sequences. The determined cDNA sequences of these seven clones (P711P, P712P, Novell 23, P774P, P775P, P710P and P768P) are shown in SEQ ID NOs: 307-311, 313 and 315, respectively. The remaining 6 clones (SEQ ID NOs: 316 and 321-325) were found to share some homology to known genes. All 13 clones showed more than 3-fold overexpression in prostate tissues including prostate tumors, BPH and normal prostate by microarray analysis compared to normal non-prostate tissues. Clones P711P, P712P, Novell 23 and P768P showed overexpression in most prostate tumors tested and BPH tissues (n = 29) and most normal prostate tissues (n = 4), but all In normal tissues, there was a level of background expression to low expression. Clones P774P, P775P, and P710P were relatively low expressed and expressed in a few samples of prostate tumors and BPH, but negative to low expression in normal prostate.

  By adopting the partial sequence shown in SEQ ID NO: 307 and screening a prostate cDNA library, P711P full-length cDNA was obtained. Specifically, a directionally cloned prostate cDNA library was prepared using standard methods. One million colonies of this library were plated on LB / Amp plates. Nylon membrane filters were used to lift these colonies, and the cDNA collected by these filters was denatured by UV and cross-linked to the filters. The P711P cDNA fragment of SEQ ID NO: 307 was radiolabeled and used to hybridize with these filters. Positive clones were selected, cDNA was prepared and sequenced using an automated Perkin Elmer / Applied Biosystems sequencer. The determined full-length sequence of P711P is shown in SEQ ID NO: 382 and the corresponding predicted amino acid sequence is shown in SEQ ID NO: 383.

  Additional cDNA sequence information was obtained for the above two clones 11-C9 and 9-F3 using a method based on PCR and hybridization. These clones are hereinafter referred to as P707P and P714P (SEQ ID NOs: 333 and 334), respectively. As a result of comparison with the latest GenBank, P707P was found to be a splice variant of the known gene HoxB13. In contrast, there was no significant homology to P714P.

  Clones 8-B3, P89, P98, P130 and P201 (disclosed in US patent application Ser. No. 09 / 020,956 filed on Feb. 9, 1998) contain a single contiguous sequence designated P705P ( SEQ ID NO: 335, the predicted amino acid sequence was found to be contained within (SEQ ID NO: 336). P705P has been confirmed to be a splice variant of the known gene NKX3.1.

  Further testing for P775P resulted in four additional sequences (SEQ ID NOs: 473-476) that are all splice variants of the P775P gene. The sequence of SEQ ID NO: 474 was found to contain two open reading frames (ORF). The deduced amino acid sequences encoded by these ORFs are provided in SEQ ID NOs: 477 and 478. The cDNA sequence of SEQ ID NO: 475 was found to contain an ORF encoding the amino acid sequence of SEQ ID NO: 479. The cDNA sequence of SEQ ID NO: 473 was found to contain 4 ORFs. The deduced amino acid sequences encoded by these ORFs are provided in SEQ ID NOs: 480-483.

  Subsequent studies led to the identification of a genomic region on chromosome 22q11.2, known as the Cat Eye syndrome region, which contains five prostate genes P704P, P712P, P774P, P775P, and B305D. The relative position of each of these five genes within this genomic region is shown in FIG. Therefore, this region can be associated with malignant tumors and other potential oncogenes may be included within this region. The above test led to the identification of a potential open reading frame (ORF) for P775P (provided in SEQ ID NO: 533) encoding the amino acid sequence of SEQ ID NO: 534.

Example 4
Polypeptide Synthesis Polypeptide is a Perkin Elmer / Applied Biosystems 430A peptide utilizing FMOC chemistry that is activated with HPTU (O-benzotriazole-N, N, N ′, N′-tetramethyluronium hexafluorophosphate) It can be synthesized with a synthesizer. Methods are provided for attaching a Gly-Cys-Gly sequence to the amino terminus of a peptide for conjugation of the peptide, attachment to an immobilization surface or labeling. Cleavage of the peptide from the solid support can be carried out using the following cleavage mixture: trifluoroacetic acid: ethanedithiol: thioanisole: water: phenol (40: 1: 2: 2: 3). After this cleavage for 2 hours, the peptide can be precipitated with cold methyl-t-butyl-ether. The resulting peptide pellet is dissolved in water containing 0.1% trifluoroacetic acid (TFA), lyophilized and then purified by C18 reverse phase HPLC. The peptide is eluted using a gradient from 0% to 60% acetonitrile (containing 0.1% TFA) obtained by dissolving in water (containing 0.1% TFA). The lyophilized fraction is lyophilized and the characteristics of the peptide determined using electrospray or other types of mass spectrometry and amino acid analysis.

Example 5
Further isolation and characterization of prostate-specific polypeptides by PCR-based subtraction The cDNA library obtained from the prostate primary tumor mRNA described above was subtracted with cDNA from normal prostate. The subtraction was performed using a PCR-based protocol (Clontech). This protocol was modified to obtain larger fragments. According to the modified protocol, tester and driver double-stranded cDNAs are digested separately with five restriction enzymes (MluI, MscI, PvuII, SalI and StuI) that recognize restriction sites of 6 nucleotides. did. As a result of this digestion, cDNA having an average size of 600 bp was obtained instead of the average size of 300 bp obtained by digestion with RsaI according to the Clontech protocol. The modification did not affect the efficiency of subtraction. Next, two tester populations were made with different adapters and the driver library remained without adapters.

  The tester and driver library was then hybridized with excess driver cDNA. In the first hybridization step, the driver was hybridized separately with each of the two tester cDNA populations. As a result, (a) a tester cDNA that did not form a hybrid, (b) a tester cDNA that formed a hybrid with another tester cDNA, (c) a tester cDNA that formed a hybrid with a driver cDNA, and (d) a hybrid formed. A population of driver cDNAs that did not exist was generated. The above two separate hybrid reaction products were then mixed and re-hybridized in the presence of additional denatured driver cDNA. By this second hybridization reaction, in addition to the populations (a) to (d), the fifth population (e) in which the tester cDNA having one type of adapter forms a hybrid with the tester cDNA having the second adapter. ) Produced. Thus, the second hybridization step enriched for differentially expressed sequences that could be used as templates for PCR amplification with adapter-specific primers.

  The ends were then filled in and PCR amplification was then performed using adapter specific primers. Only the population (e) containing tester cDNA that did not hybridize with the driver cDNA was exponentially amplified. A second PCR amplification step was then performed to reduce the background and further enrich the sequences expressed with differences.

  This PCR-based subtraction method averages the specifically expressed cDNA so that rare transcripts that are overexpressed in prostate tumor tissue can be recovered. Such transcripts are difficult to recover by conventional subtraction methods.

  In addition to the genes known to be overexpressed in prostate tumors, 77 additional clones were identified. The sequences of these partial cDNAs are shown in SEQ ID NOs: 29-305. Most of these clones did not have significant homology to the database. The exceptions were: JPTPN23 (SEQ ID NO: 231; similar to a porcine valosin-containing protein), JPTPN30 (SEQ ID NO: 234; similar to rat mRNA for the proteasome subunit) JPTPN45 (SEQ ID NO: 234; rat norvegicus) Similar to cytoplasmic NADP-dependent isocitrate dehydrogenase], JPTPN46 (SEQ ID NO: 244; similar to the sequence of human subclone H8 4 d4 DNA), JP1D6 [SEQ ID NO: 265; G. gallus dynein JP8D6 (SEQ ID NO: 288; similar to human BAC clone RG016J04), JP8F5 (SEQ ID NO: 289; similar to human subclone H8 3 b5 DNA sequence) and JP8E9 (SEQ ID NO: 299; similar to human Alu sequence) is an exception.

  Three additional clones were obtained by additional testing using a PCR-based subtraction library consisting of a pool of prostate tumors subtracted against a pool of normal prostates (referred to as PT-PN PCR subtraction). . As a result of comparing the cDNA sequences of these clones with the latest publication of GenBank, no significant homology was found to two clones (SEQ ID NOs: 312 and 314) designated P715P and P767P. The remaining clones were found to show some homology to the known gene KIAA0056 (SEQ ID NO: 318). When the expression level of mRNA in various tissues was measured using a microarray analysis method, all the three clones were found to be overexpressed in prostate tumors and BPH tissues. Specifically, clone P715P was overexpressed in most prostate tumors and BPH tissues by more than 3 times the high expression found in the majority of normal prostate samples and in fetal tissues. Negative to low expression in all normal tissues. Clone P767P is overexpressed in several prostate tumors and BPH tissues, is moderately expressed in half of the normal prostate samples, and is background to low expressed in all other normal tissues tested. It was.

  By analyzing the PT-PN PCR subtraction library as described above, and a DNA subtraction library containing prostate tumor-derived cDNA subtracted with a pool of normal tissue cDNA, 27 additional samples were analyzed. Clones (SEQ ID NOs: 340-365 and 381) were isolated. These additional clones were confirmed to be overexpressed in prostate tumors. Also, clones of SEQ ID NOs: 341, 342, 345, 347, 348, 349, 351, 355-359, 361, 362 and 364 were found to be expressed in normal prostate. Expression of all 26 clones in various normal tissues was found to be low or undetectable, with the exception of P544S (SEQ ID NO: 356) found to be expressed in the small intestine. . Of the 26 clones, 10 (SEQ ID NOs: 340-349) were found to show some homology to previously identified sequences. No significant homology was found in the clones of SEQ ID NOs: 350, 351, and 353-365.

  Further testing on the clone of SEQ ID NO: 352 (also referred to as P790P) led to the isolation of the full length cDNA sequence of SEQ ID NO: 526. This corresponding deduced amino acid is provided in SEQ ID NO: 527. Data from two quantitative PCR experiments show that P790P is overexpressed in 11 of 15 tested prostate tumor samples and expressed at low levels in the spinal cord in all other normal samples tested. It was shown that it was not expressed. Data from further PCR experiments and microarray experiments showed overexpression in normal prostate and prostate tumors, but little or no expression in other tissues tested. P790P was subsequently discovered to show significant homology to previously identified G protein coupled prostate tissue receptors.

Example 6
Primary immunization of mice with peptides and growth of CTL lines
6.1
In this example, preparation of a CTL cell line specific for cells expressing the P502S gene is described.

Mice expressing the transgene of human HLA A2Kb (provided by Dr. L. Sherman of The Scripps Research Institute, La Jolla, CA, USA) were obtained from Theobald et al., Proc. Natl. Acad. Sci. USA, 92: 11993-11997, 1995. The method described in (1) was modified as follows and immunized with the P2S # 12 peptide (VLGWVAEL; SEQ ID NO: 306). This peptide is derived from the P502S gene (also referred to herein as J1-17, SEQ ID NO: 8). Mice, P2S # and 12 100 [mu] g, were immunized B types from hepatitis virus protein and I-A ^b binding peptide 120μg in that emulsified in incomplete Freund's adjuvant. Three weeks later, these mice were sacrificed and a single cell suspension was prepared using nylon mesh. The cells were then treated with 10% FCS, 2 mM glutamine (Gibco BRL), sodium pyruvate (Gibco BRL), non-essential amino acids (Gibco BRL), 2 × 10 ⁻⁵ M 2-mercaptoethanol, 50 U / ml penicillin. Resuspended in complete medium containing streptomycin (RPMI-1640; Gibco BRL, Gaithersburg, MD, USA) at a concentration of 6 × 10 ⁶ cells / ml and then irradiated (3000 rad) LPS blast pulsed with P2S # 12 (5 mg / ml P2S # 12 and 10 mg / ml β2-microglobulin) A2 cultured for 3 days in the presence of 7 μg / ml dextran sulfate and 25 μg / ml LPS The cells were cultured in the presence of transgenic spleen cells. After 6 days, cells (5 × 10 ⁵ / ml) were pulsed with 2.5 × 10 ⁶ / ml peptide and irradiated (20,000 rad) EL4A2Kb cells (Sherman et al., Science 258: 815-818, 1992) and 3 Restimulation was performed with × 10 ⁶ / ml A2 transgenic spleen feeder cells. Cells were cultured in the presence of 20 U / ml IL-2. In order to clone the cell line, cells were restimulated on a weekly basis as described above.

Peptide pulsed EL4 A2Kb tumor cells (1 × 10 ⁴ cells / well) as stimulator cells and A2 transgenic spleen cells (5 × 10 ⁵ cells / well) as feeder cells grown in the presence of 30 U / ml IL-2 P2S # 12 cell line was cloned by limiting dilution method. On day 14, the cells were restimulated as described above. On day 21, the growing clones were isolated and continued to culture. Some of these clones were shown to be significantly more reactive (lytic) to human fibroblasts transduced with P502S (expressing HLA A2Kb) than to control fibroblasts . An example is shown in FIG.

  This data indicates that P2S # 12 is a natural epitope of the P502S protein expressed as a human HLA A2Kb molecule.

6.2
This example illustrates the preparation of a mouse CTL cell line and CTL clones specific for cells expressing the P501S gene.

  This series of tests was performed in the same manner as the above test. Mice were immunized with a P1S # 10 peptide (SEQ ID NO: 337) derived from the P501S gene (also referred to herein as LI-12, SEQ ID NO: 110). P1S # 10 peptide was used to identify the predicted polypeptide sequence of P501S for the potential HLA-A2 binding sequence defined by the published HLA-A2 binding motif (Parker, KC et al., J. Immunol., 152: 163, 1994). Obtained by analysis. P1S # 10 peptide was synthesized as described in Example 4 and then experimentally tested for HLA-A2 binding utilizing a T cell-based competition assay. The expected A2-binding peptides were tested for the ability to compete for the presentation of HLA-A2-specific peptides against the HLA-A2-restricted CTL clone (D150M58) specific for the HLA-A2-binding influenza matrix peptide flu M58. D150M58 CTL secretes TNF in response to self-presentation of the peptide flu M58. In this competition assay, 100-200 μg / ml of the test peptide was added to the CTL culture for binding to HLA-A2 on D150M58 CTL. After 30 minutes, CTL cultured with test or control peptides were tested for their antigen dose response to flu M58 peptides in a standard TNF bioassay. As shown in FIG. 3, peptide P1S # 10 competes with HLA-A2 restriction presentation of flu M58, indicating that peptide P1S # 10 binds to HLA-A2.

Mice expressing a transgene for human HLA 2Kb were immunized using the method described in Theobald et al., Proc. Natl. Acad. Sci. USA, 92: 11993-11997, 1995 as follows. And I-A ^b binding peptide from P1S # 10 and 120μg of hepatitis B virus proteins of 62.5μg mice were immunized with one emulsified in incomplete Freud adjuvant. Three weeks later, these mice were sacrificed and a single cell suspension was prepared using nylon mesh. The cells are then resuspended in complete medium (as above) at 6 × 10 ⁶ cells / ml and irradiated (3000 rad) P1S # 10 pulse (2 μg / ml P1S # 10 plus 10 mg / ml). β2-microglobulin) LPS blast (A2 transgenic spleen cells cultured for 3 days in the presence of 7 μg / ml dextran sulfate and 25 μg / ml LPS). After 6 days, re-stimulate cells with irradiated (20,000 rad) EL4 A2Kb cells pulsed with 2.5 × 10 ⁶ / ml peptide and 3 × 10 ⁶ / ml A2 transgenic spleen feeder cells as described above. did. Cells were cultured in the presence of 20 U / ml IL-2. In preparation for cloning, cells were restimulated on a weekly basis. After 3 rounds of stimulation in vitro, one cell line was generated that recognizes the P1S # 10 pulsed Jurkat A2Kb target and the P501S-transduced Jurkat target, as shown in FIG.

P1S # 10-specific CTL line was transformed into peptide-pulsed EL4 A2Kb tumor cells (1 × 10 ⁴ cells / well) as stimulating cells and A2 trans as support cells grown in the presence of 30 U / ml IL-2 The cells were cloned by limiting dilution method using transgenic spleen cells (5 × 10 ⁵ cells / well). On day 14, the cells were restimulated as described above. On day 21, viable clones were isolated and continued to culture. As shown in FIG. 5, five of these clones showed specific cytolytic reactivity against the P501S-transduced Jurkat A2Kb target. This data indicates that P1S # 10 is a naturally processed epitope of the P501S protein expressed as a human HLA-A2.1 molecule.

Example 7
In vivo primary immunization of CTL utilizing immunization of naked DNA with prostate antigen The prostate specific antigen L1-12 described above is also referred to as P501S. HLA A2Kb Tg mice (provided by Dr. L. Sherman of The Scripps Research Institute, La Jolla, CA, USA) were immunized by injecting 100 μg P501S in vector VR1012, intramuscularly or intradermally. These mice were immunized 3 times at 2-week intervals. Two weeks after the last immunization, immune spleen cells were cultured with stimulator cells transduced with Jurkat A2Kb-P501S. CTL lines were stimulated every week. Two weeks after stimulation in vitro, CTL activity against targets transduced with P501S was evaluated. Two out of eight mice developed strong anti-P501S CTL responses. These results indicate that P501S contains at least one naturally processed HLA-A2 restricted CTL epitope.

Example 8
Ability of human T cells to recognize prostate specific polypeptides This example illustrates the ability of T cells specific for prostate tumor peptides to recognize human tumors.

Human CD8 ⁺ T cells were obtained from P502S (also referred to as J1-17) P2S-12 peptide (SEQ ID NO :) using dendritic cells according to the protocol of Van Tsai et al., Critical Reviews in Immunology 18: 65-75, 1998. 306) were primed in vitro. The resulting CD8 ⁺ T cell microcultures were analyzed for the ability to recognize P2S-12 peptides presented by autologous fibroblasts or fibroblasts transduced to express the P502S gene. (See Lalvani et al., J. Exp. Med., 186: 859-865, 1997). Briefly, T cell titrating numbers against 10 ⁴ fibroblasts in the presence of 3 μg / ml human β ₂ -microglobulin and 1 μg / ml P2S-12 peptide or control E75 peptide. Were assayed in duplicate. In addition, T cells were simultaneously assayed for autologous fibroblasts transduced with the P502S gene, or fibroblasts transduced with HER-2 / neu as a control. Prior to performing this assay, fibroblasts were treated with 10 ng / ml gamma interferon for 48 hours to up-regulate class I MHC expression. One of the microcultures (# 5) showed strong recognition of both peptide-pulsed fibroblasts and transduced fibroblasts by γ-interferon ELISPOT assay. FIG. 2A shows that the number of gamma interferon spots increased strongly as the number of T cells on fibroblasts pulsed with P2S-12 peptide increased (black bars), but when the control E75 peptide was pulsed No increase (white bar). This indicates that these T cells can specifically recognize the P2S-12 peptide. As shown in FIG. 2B, this microculture showed that the number of gamma interferon spots increased as the number of T cells on the fibroblasts transduced to express the P502S gene increased. The HER-2 / neu gene did not show such a phenomenon. These results provide additional evidence confirming that the P2S-12 peptide is a naturally processed epitope of the P502S protein. Furthermore, this also indicates that there are high affinity T cells capable of recognizing this epitope in the human T cell repertoire. These T cells should also be able to recognize human tumors that express the P502S gene.

Example 9
Induction of prostate antigen-specific CTL response in human blood This example illustrates the ability of prostate-specific antigen to induce a CTL response in normal human blood.

Autologous dendritic cells (DC) were derived from normal donor PBMC by growing for 5 days in RPMI medium containing 10% human serum, 50 ng / ml GMCSF and 30 ng / ml IL-4. Differentiated from monocyte cultures. After incubation, DCs were infected overnight at 5M.OI with recombinant P501S expressing vaccinia virus and then matured for 8 hours by adding 2 μg / ml CD40 ligand. The virus was inactivated by UV irradiation. CD8 ⁺ cells were separated by positive selection using magnetic beads, and then the initial immunoculture was initiated in 24-well plates. Specificity of γ-interferon when stimulated with autologous P501S-transduced fibroblasts after 5 stimulation cycles using autologous fibroblasts retrovirally transduced to express P501S and CD80 The CD8 ⁺ strain produced was confirmed. The P501S specific activity of cell line 3A-1 could be maintained by adding a stimulation cycle to autologous B-LCL transduced with P501S. Cell line 3A-1 was found to specifically recognize autologous B-LCL transduced to express P501S but not EGFP-transduced autologous B-LCL. This is cytotoxicity assay ⁽⁵¹ Cr release) and gamma interferon production (gamma Interferon Elispot; supra and Lalvani et al., J Exp Med, 186:. .. 859-865, see 1997) was determined by. The results of these assays are shown in FIGS. 6A and 6B.

Example 10
Confirmation of naturally processed CTL epitope contained within prostate specific antigen A 9mer peptide p5 (SEQ ID NO: 338) was derived from the P703 antigen (also referred to as P20). This p5 peptide is immunogenic in the human HLA-A2 donor and is a natural epitope. Antigen-specific human CD8 + T cells can be primed with monocytes pulsed with p5 peptide by repeated stimulation in vitro. These CTLs specifically recognize p5 pulse and P703P transduced target cells in both ELISPOT assays (as described above) and chromium release assays. In addition, when HLA-A2Kb transgenic mice are immunized with p5, CTL lines that recognize various HLA-A2Kb or HLA-A2 transduced target cells expressing P703P are generated.

  An initial test demonstrating that p5 is a naturally processed epitope was performed using HLA-A2Kb transgenic mice. Immunization was performed by injecting Freund's incomplete adjuvant containing 100 µg of p5 peptide and 140 µg of hepatitis B virus core peptide (Th peptide) subcutaneously into the footpad of HLA-A2Kb transgenic mice. Three weeks after immunization, spleen cells from the immunized mice were stimulated in vitro with peptide pulse LPS blasts. Five days after the primary in vitro stimulation, CTL activity was assessed by a chromium release assay. Cells transfected with retrovirus expressing the control antigens P703P and HLA-A2Kb were used as targets. A CTL line that specifically recognizes both the p5 pulse target and the P703P expression target was confirmed.

  Human in vitro priming experiments have shown that the p5 peptide is immunogenic in humans. By culturing for 5 days in RPMI medium containing 10% human serum, 50 ng / ml human GM-CSF and 30 ng / ml human IL-4, dendritic cells (DC) were obtained from normal human donor PBMCs. Differentiated from derived monocyte cultures. After incubation, the DCs were pulsed with 1 μg / ml p5 peptide and then cultured with CD8 + T cell enriched PBMC. The CTL line was restimulated with p5 pulse monocytes every week. It was confirmed that CTL recognized p5 pulse target cells 5 to 6 weeks after the start of CTL culture. CTL was further shown to recognize human cells transduced to express P703P. This proves that p5 is a naturally processed epitope.

Example 11
Prostate Expression of Breast Tumor-Derived Antigens The antigen B305D is isolated from breast tumors by differential display in US patent application Ser. No. 08 / 700,014 filed Aug. 20, 1996. Are listed. Several different splice forms of the antigen were isolated. The cDNA sequences determined for these splice forms are shown in SEQ ID NOs: 366-375, and the predicted amino acid sequences corresponding to the sequences of SEQ ID NOs: 292, 298 and 301-303 are shown in SEQ ID NOs: 299-306. In further studies, a splice variant of the cDNA sequence of SEQ ID NO: 366 was isolated and was found to contain an additional guanine residue at position 884, leading to a frame shift of the open reading frame ( SEQ ID NO: 530). The determined DNA sequence of this ORF is provided in SEQ ID NO: 531. This frameshift creates a protein sequence consisting of 293 amino acids (provided in SEQ ID NO: 532) that contains a C-terminal domain common to other isoforms of B305D but whose N-terminal region is different.

  The expression level of B305D in various tumor tissues and normal tissues was examined by real-time PCR and Northern analysis. As a result, B305D is highly expressed in breast tumors, prostate tumors, normal prostate and normal testis, but all other test tissues (colon tumors, lung tumors, ovarian tumors, and normal bone marrow, colon, (Expression in kidney, liver, lung, ovary, skin, small intestine, stomach) was low or undetectable.

Example 12
Generation of human CTL in vitro using techniques of whole gene priming and stimulation with prostate specific antigen
The above-mentioned γ-interferon ELISPOT analysis method using the method of performing whole gene primary immunization in vitro with P501S vaccinia-infected DC (see, for example, Yee et al., The Journal of Immunology, 157 (9); 4079-4086, 1996) As can be seen, a human CTL line that specifically recognizes autologous fibroblasts transduced with P501S (also known as L1-12) was induced. A panel of HLA-incompatible B-LCL cell lines transduced with P501S was used to show that these CTL lines appear to be restricted to restricted HLAB class I alleles. Specifically, dendritic cells (DCs) were grown by growing for 5 days in RPMI medium containing 10% human serum, 50 ng / ml human GM-CSF and 30 ng / ml human IL-4. Differentiated from monocyte cultures derived from PBMC of normal human donors. After incubation, DCs were infected overnight with recombinant P501S vaccinia virus at a multiplicity of infection (MOI) of 5 and then matured overnight by adding 3 μg / ml of CD40 ligand. The virus was inactivated by UV irradiation. CD8 + T cells were isolated using a magnetic bead system and initial immunoculture was initiated using standard culture methods. Cultures were re-stimulated every 7-10 days using autologous primary fibroblasts transduced with retrovirus P501S and CD80. After four stimulation cycles, a CD8 + T cell line that specifically produced γ-interferon was identified when stimulated with autologous fibroblasts transduced with P501S and CD80. A panel of HLA-incompatible B-LCL cell lines transduced with P501S was generated to define restriction alleles of the response. By measuring γ interferon in an ELISPOT assay, it was found that the specific response of P501S appears to be limited by the HLAB allele. These results indicate that a CD8 + CTL response against P501S can be induced.

  In order to identify the recognized epitope, the cDNA encoding P501S was fragmented by various restriction digests and subcloned into the retroviral expression vector pBIB-KS. Retroviral supernatant was generated by transfection of helper packaging strain Phoenix-Ampho. The supernatant was then used to transduce Jurkat / A2Kb cells for CTL screening. CTL were screened in an IFN-gamma ELISPOT assay against an A2Kb target transduced with a “library” of P501S fragments. The first positive fragments P501S / H3 and P501S / F2 were sequenced and found to encode amino acids 106-553 and amino acids 136-547 of SEQ ID NO: 113, respectively. The cleavage of H3 was made to encode amino acid residues 106-351 of SEQ ID NO: 113. This cannot stimulate CTL and therefore the epitope is localized at amino acid residues 351-547. Additional fragments encoding amino acids 1-472 (fragment A) and amino acids 1-351 (fragment B) were also constructed. Fragment A but not fragment B stimulated CTL, which localized the epitope to amino acid residues 351-472. Overlapping 20mer and 18mer peptides representing this region were tested by pulsing Jurkat / A2Kb cells and CTL in an IFN-gamma assay. Only peptides P501S-369 (20) and P501S-369 (18) stimulated CTL. 9mer and 10mer peptides representing this region were synthesized and tested similarly. Peptide P501S-370 (SEQ ID NO: 539) was the smallest 9mer that gave a strong response. Peptide P501S-376 (SEQ ID NO: 540) also gave a weak response. This suggests that it may represent a cross-reactive epitope.

  Subsequent studies examined the ability of primary human B cells transduced with P501S to prime MHC class I restricted, P501S-specific, autologous CD8 T cells. Primary B cells are obtained from PBMCs of homozygous HLA-A2 donors by culture in CD40 ligand and IL-4, transduced frequently with recombinant P501S in the vector pBIB, and selected with blasticidin-S did. For in vitro priming, purified CD8 + T cells were cultured with autologous CD40 ligand + IL-4 derived, P501S-transduced B cells in a 96-well microculture format. These CTL microcultures were restimulated with P501S-transduced B cells and then assayed for specificity. After this initial screening, microcultures with significant signal above background were cloned on autologous EBV transformed B cells (BLCL) also transduced with P501S. Using IFN-gamma ELISPOT for detection, some of the above CD8 T cell clones were tested against P501S as evidenced by reactivity to BLCL / P501S, but not BLCL transduced with control antigen. It was discovered to be specific. It was further demonstrated that anti-P501S CD8 T cell specificity is HLA-A2 restricted. First, antibody blocking experiments using anti-HLA-A, B, C monoclonal antibody (W6.32), anti-HLA-A, B, C monoclonal antibody (B1.23.2), and control monoclonal antibody were performed using anti-HLA Only -A, B, C antibodies were shown to block recognition of P501S expressing autologous BLCL. Second, anti-P501S CTL also recognized HLA-A2-matched, heterologous BLCL transduced with P501S, but not the corresponding EGFP transduced control BLCL.

Example 13
Confirmation of Prostate Specific Antigens by Microarray Analysis This example describes the isolation of specific prostate specific polypeptides from a prostate tumor cDNA library.

The above human prostate tumor cDNA expression library is screened using microarray analysis to at least 3-fold excess in prostate tumors and / or normal prostate tissues compared to non-prostate normal tissues (excluding testis) A clone showing expression was identified. 372 clones were identified and 319 clones were successfully sequenced. Table I gives an overview 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 isolated sequences. The remaining sequences (SEQ ID NOs: 385, 387, 388, 391, 394, 395 and 397-400) correspond to known sequences as shown in Table I.

  CGI-82 overexpressed 4.06 times in prostate tissue compared to other normal tissues tested. It was overexpressed in 43% of prostate tumors and 25% of normal prostates, but was not detected in other normal tissues tested. L-Iditol-2 dehydrogenase was 4.94 fold overexpressed in prostate tissue compared to other normal tissues tested. It was overexpressed in 90% of prostate tumors and 100% of normal prostate, but was not detected in other normal tissues tested. The Ets transcription factor PDEF was 5.55 fold overexpressed in prostate tissue compared to other normal tissues tested. It was overexpressed in 47% of prostate tumors and 25% of normal prostates but was not detected in other normal tissues tested. hTGR1 was 9.11 times overexpressed in prostate tissue compared to other normal tissues tested. It was overexpressed in 63% of prostate tumors but was not detected in other normal tissues tested including normal prostate. KIAA0295 showed 5.59-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 47% of prostate tumors but was low to undetectable in other normal tissues tested including normal prostate tissue. Prostatic acid phosphatase showed a 9.14-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 67% of prostate tumors and 50% of normal prostates, but was not detected in other normal tissues tested. Transglutaminase showed a 14.84-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 30% of prostate tumors and 50% of normal prostates but was not detected in other normal tissues tested. High density lipoprotein binding protein (HDLBP) showed 28.06-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 97% of prostate tumors and 75% of normal prostates but was not detected in all other normal tissues tested. CGI-69 showed 3.56 fold overexpression in prostate tissue compared to other normal tissues tested. It was a minor gene detected in more than 90% of prostate tumors and 75% of normal prostate tissue. The expression of this gene in normal tissues was very low. KIAA0122 showed a 4.24-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 57% of prostate tumors but was undetectable in all normal tissues including normal prostate tissue. 19142.2 bangur showed a 23.25-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 97% of prostate tumors and 100% of normal prostate. It was not detectable in other normal tissues tested. 5566.1 Wang showed 3.31 fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 97% of prostate tumors and 75% of normal prostates, and was also overexpressed in normal bone marrow, pancreas and activated PBMC. The new clone 23379 showed a 4.86 fold overexpression in prostate tissue compared to other normal tissues tested. It was detectable in 97% of prostate tumors and 75% of normal prostates, but undetectable in all other normal tissues tested. New clone 23399 showed 4.09-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 27% of prostate tumors but was undetectable in all normal tissues tested, including normal prostate tissue. The new clone 23320 showed 3.15 fold overexpression in prostate tissue compared to other normal tissues tested. It was detectable in 50% of all prostate tumors and normal prostate tissue. It was also expressed in normal colon and trachea. Other normal tissues do not express this gene at high levels.

Example 14
Identification of Prostate Specific Antigen by Electronic Subtraction This example describes the identification of prostate specific antigen using the electronic subtraction method.

  Potential prostate specific genes present in the GenBank human EST database were identified by electronic subtraction (as described in Vasmatizis et al., Proc. Natl. Acad. Sci. USA 95: 300-304, 1998). The sequences of EST clones (43,482) derived from various prostate libraries were obtained from the GenBank public human EST database. Each prostate EST sequence was used as a query sequence for BLASTN (National Center for Biotechnology Information) to search the human EST database. All those determined to be identical (having a match sequence longer than 100 base pairs and a match density of the entire region of greater than 70%) were grouped (aligned) as clusters. Clusters containing more than 200 ESTs were discarded because they may represent repetitive elements, or may be highly expressed genes like ribosomal proteins. When two or more clusters have a common EST, these clusters were grouped as “superclusters”, resulting in 4,345 prostate superclusters.

We downloaded records of 479 human cDNA libraries shown in the GenBank release, and created a database of these cDNA library records. These 479 cDNA libraries are divided into 3 groups, plus (normal prostate and prostate tumor libraries, and breast cell lines where expression is desired), minus (libraries from other normal adult tissues where expression is not desired) and others (Library from fetal tissue considered to be unrelated to expression, infant tissue, tissue only found in women, non-prostate tumors and cell lines other than prostate cell lines). A summary of these library groups is shown in Table II.

Each supercluster was analyzed for ESTs in the supercluster. The tissues from the origin of each EST clone were recorded and used to classify the supercluster into 4 groups: EST clones found only in type 1-plus group libraries; no expression detected in minus or other group libraries; 2 EST clones derived only from type-plus and other group libraries; no expression is detected in the minus group; type 3 plus, minus and other group-derived EST clones, but the number of ESTs from the plus group is minus or others Higher than the number derived from the group; and type 4-plus, minus, and other group library ESTs, but the number of plus group derived ESTs is higher than the number derived from the minus group This analysis identified 4,345 mammary clusters (see Table III). From these clusters, 3,172 EST clones were ordered from Research Genetics and received as frozen glycerol stocks in 96-well plates.

  EST clone inserts were PCR amplified using amino-binding PCR primers suitable for Synteni microarray analysis. If more than one PCR product was obtained for a particular clone, the PCR product was not used for expression analysis. A total of 2528 clones from the electronic subtraction method were analyzed by microarray method to identify electronic subtraction breast clones with high tumor / normal tissue mRNA ratio. Such screening uses Synteny (Palo Alto, CA) microarrays and uses manufacturer instructions (and essentially 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). During these analyses, clones were sequenced on the chip and subsequently probed with fluorescent probes made from normal and tumor prostate cDNA and various other normal tissues. The slide was scanned and the fluorescence intensity was measured.

Clones with an expression ratio greater than 3 (ie, the levels of prostate tumor and normal prostate mRNA were at least 3 times that of other normal tissue mRNA) were identified as prostate tumor specific sequences (Table IV). The sequences of these clones are provided in SEQ ID NOs: 401-453, with some new sequences shown in SEQ ID NOs: 407, 413, 416-419, 422, 426, 427 and 450.

Example 15
Further Identification of Prostate Specific Antigens by Microarray Analysis This example describes the isolation of additional prostate specific polypeptides obtained from a prostate tumor cDNA library.

  The above-described human prostate tumor cDNA expression library was screened by microarray analysis, and clones showing at least a 3-fold excess expression in prostate tumor and / or normal prostate tissue compared to non-prostate normal tissue (excluding testis) Identified. 142 clones were identified and sequenced. Some of these clones are shown in SEQ ID NOs: 454-467. Of these, SEQ ID NOs: 459-461 are novel genes. The other (SEQ ID NOs: 454-458 and 461-467) correspond to known sequences.

Example 16
Further characterization of prostate specific antigen P710P This example describes the full length cloning of P710P.

  The prostate cDNA library was screened using the P710P fragment. One million clones were seeded on LB / ampicillin plates. These colonies were lifted using a nylon membrane filter, then the cDNA picked up by these filters was denatured and cross-linked onto the filter by UV light. The P710P fragment was radiolabeled and used for hybridization with the filter. Positive cDNA clones were selected and the cDNAs were recovered and sequenced by an automated Perkin Elmer / Applied Biosystems Division sequencer. Four types of sequences were obtained and are shown in SEQ ID NOs: 468-471. These sequences appear to represent different splice variants of the P710P gene.

Example 17
Protein expression of prostate specific antigen P501S . The expression and purification of the prostate specific antigen P501S in E. coli, baculovirus and mammalian cells is described.

(A) E.E. Expression of full-length P501S in E. coli was first performed by M. An attempt was made to clone P501S without the leader sequence (amino acids 36-553 of SEQ ID NO: 113) downstream of the first 30 amino acids of the tuberculosis antigen Ra12 (SEQ ID NO: 484). Specifically, PCR using primers AW025 (SEQ ID NO: 485) and AW003 (SEQ ID NO: 486) was performed using DNA of P501S. AW025 is a sense cloning primer containing a HindIII site. AW003 is an antisense cloning primer containing an EcoRI site. DNA amplification was 5 μl of 10 × Pfu buffer, 1 μl of 20 mM dNTP, 1 μl of 10 μM concentration PCR primer, 40 μl of water, 1 μl and 100 ng of Pfu DNA polymerase (Stratagene, La Jolla, Calif.). 1 μl of DNA / μl was used. Denaturation at 95 ° C for 30 seconds, 95 cycles for 30 seconds, 60 ° C for 1 minute, 72 ° C for 3 minutes, 10 cycles of 95 ° C for 30 seconds, 65 ° C for 1 minute, 72 ° C for 3 minutes, and finally One cycle at 72 ° C. for 10 minutes was performed. The PCR product was cloned into Ra12m / pET17b using HindIII and EcoRI. The sequence of the resulting fusion construct (referred to as Ra12-P510S-F) was confirmed by DNA sequencing.

  The fusion constructs were BL21 (DE3) pLysE, pLysS and CodonPlusE. E. coli (Stratagene) were transduced and grown overnight in LB broth with kanamycin. The resulting culture was induced using IPTG. The protein was transferred to a PVDF membrane, blocked with 5% non-fat milk (in PBS-Tween buffer), washed 3 times and then incubated with mouse anti-His tag antibody (Clontech) for 1 hour. The membrane was washed 3 times and then probed with HRP-Protein A (Zymed) for 30 minutes. Finally, the membrane was washed 3 times, and light was emitted with ECL (Amersham). No expression was detected by Western blot. Similarly, when expressed in BL21 CodonPlus by CE6 phage (Invitrogen) using Ra12-P501S-F fusion, expression was not detected by Western blot.

  The N-terminal fragment of P501S (amino acids 36-325 of SEQ ID NO: 113) was purified from M. p. It was cloned downstream of the first 30 amino acids of the tuberculosis antigen Ra12. PCR was performed using P501S DNA and primers AW025 (SEQ ID NO: 485) and AW027 (SEQ ID NO: 487). AW027 is an antisense cloning primer containing an EcoRI site and a stop codon. DNA amplification was performed essentially as described above. The resulting PCR product was cloned into the HindIII and EcoRI sites of Ra12 in pET17b. The fusion construct (designated Ra12-P501S-N) was confirmed by DNA sequencing.

  The Ra12-P501S-N fusion construct was used for expression in BL21 (DE3) pLysE, pLysS and CodonPlus essentially as described above. When Western blot analysis was used, a protein band was observed at an assumed molecular weight of 36 kDa. Several higher molecular bands were also observed, but these appear to be recombinant protein aggregates. When the Ra12-P501S-F fusion was used for expression on BL21 CodonPlus using CE6 phage, it was not detected by Western blot.

  The C-terminal part of P501S (amino acid 257-553 of SEQ ID NO: 113) was A fusion construct comprising the first 30 amino acids downstream of the tuberculosis antigen Ra12 (SEQ ID NO: 484) was prepared as follows. PCR was performed using P501S DNA and primers AW026 (SEQ ID NO: 488) and AW003 (SEQ ID NO: 486). AW026 is a sense cloning primer containing a HindIII site. DNA amplification was performed essentially as described above. The resulting PCR product was cloned into the HindIII and EcoRI sites of Ra12 in pE17b. The sequence of the fusion construct (referred to as Ra12-P501S-C) was confirmed.

  The Ra12-P501S-C fusion construct was used for expression in BL21 (DE3) pLysE, pLysS and CodonPlus as described above. A small amount of protein was detected in the Western blot, and several molecular weight aggregates were also observed at the same time. When the Ra12-P501S-C fusion was used for expression in BL21 CodonPlus induced with CE6 phage, expression was also detected by Western blot.

(B) Expression of P501S in baculovirus
P501S was expressed in insect cells using a Bac-to-Bac baculovirus expression system (BRL Life Technologies, Inc.). Full length P501S (SEQ ID NO: 113) was amplified by PCR and cloned into the XbaI site of the donor plasmid pFastBacI. Recombinant bacmid and baculovirus were prepared according to the manufacturer's instructions. Recombinant baculovirus was amplified into Sf9 cells and High Five cells (Invitrogen) were infected with high titer virus stock to produce recombinant protein. The full-length protein was confirmed by determining the N-terminal sequence of the recombinant protein and by Western blot analysis (FIG. 7). Specifically, 600,000 High Five cells in a 6-well plate were infected with irrelevant control virus BV / ECD_PD (lane 2), various amounts or MOI of recombinant baculovirus for P501S (lane). 4-8) or left uninfected (lane 3). Cell lysates were subjected to SDS-PAGE under reducing conditions and analyzed by Western blot using anti-P501S monoclonal antibody P501S-10E3-G4D3 (prepared as follows). Lane 1 is a biotinylated protein molecular weight marker (BioLabs).

  The localization of recombinant P501S in insect cells was examined as follows. Insect cells overexpressing P501S were fractionated into nucleus, mitochondria, membrane and cytoplasm. Each fraction of equal protein amount was analyzed by Western blot using a monoclonal antibody against P501S. According to the fractionation method, both the nuclear and mitochondrial fractions contain some plasma membrane components. However, the membrane fraction is essentially free of mitochondria and nuclei. P501S was found to be present in all fractions containing membrane components, suggesting that P501S appears to be associated with the plasma membrane of insect cells expressing the recombinant protein.

(C) Expression of P501S in mammalian cells Full-length P501S (553AA) is transformed into pCEP4 (Invitrogen), pVR1012 (Baikal, San Diego, CA (Vical, San Diego, Calif.)) And a retroviral vector pBMN called pBIB It was cloned into various mammalian cell expression vectors containing the above variants. Transfection of P501S / pCEP4 and P501S / pVR1012 into fibroblast HEK293 was performed using Fugene transfection reagent (Boehringer Mannheim). Briefly, 2 μl of Fugene reagent was diluted in 100 μl of serum-free medium and incubated at room temperature for 5-10 minutes. This mixed solution was added to 1 μg of P501S plasmid DNA, mixed briefly, and then incubated at room temperature for 30 minutes. The Fugene / DNA mixture was added to the cells and incubated for 24-48 hours. Expression of recombinant P501S in transfected HEK293 fibroblasts was detected by Western blot using a monoclonal antibody against P501S.

  Transfection of p501S / pCEP4 into CHO-K cells (American Type Culture Collection, Rockville, MD) was performed using GenePorter transfection reagent (Gene Therapy Systems (Gene Therapy Systems)). (Thermal Systems), San Diego, CA (CA)). Briefly, 15 μl GenePorter was diluted in 500 μl serum free medium and incubated at room temperature for 10 minutes. The GenePorter / medium mixture was added to 2 μg of plasmid DNA diluted in 500 μl of serum-free medium, mixed briefly and then incubated for 30 minutes at room temperature. CHO-K cells were rinsed with PBS to remove serum proteins, then a GenePorter / DNA mixture was added and incubated for 5 hours. The transfected cells were then given an equal volume of 2x medium and incubated for 24-48 hours.

  As a result of SFACS analysis of P501S of transiently infected CHO-K cells, it was shown that P501S was expressed on the surface. Expression was detected using rabbit polyclonal antisera raised against the P501S peptide as follows. Analysis by flow cytometry was performed using FaCSscan (Becton Dickinson), and data was analyzed using the Cell Quest program.

Example 18
Preparation and characterization of antibodies against prostate-specific polypeptides
(A) Preparation and characterization of antibody against P501S A mouse monoclonal antibody against the carboxy terminus of prostate specific antigen P501S was prepared as follows.

  A truncated fragment of P501S (amino acids 355-526 of SEQ ID NO: 113) was generated, cloned into the pET28b vector (Novagen), and E. coli as a histidine-tagged thioredoxin fusion protein. It was expressed in E. coli. The trx-P501S fusion protein was purified by nickel chromatography, digested with thrombin to remove the trx fragment, and precipitated with acid before further purification by reverse phase HPLC.

Mice were immunized with truncated P501S protein. Sera obtained from mice suspected of containing anti-P501S polyclonal sera were tested for P501S specific reactivity using an ELISA assay with purified P501S and trx-P501S proteins. Sera that appeared to react specifically with P501S were then screened for P501S reactivity by Western blot analysis. Mice containing P501S-specific antibody components were sacrificed, and spleen cells were used to produce hybridomas producing anti-P501S antibodies by standard methods. Hybridoma supernatants were first tested for P501S specific reactivity using ELISA, followed by FACS analysis for reactivity with P501S-introduced cells. Based on these results, a monoclonal hybridoma called 10E3 was obtained and further subcloned. A number of subclones were generated and tested for specific reactivity against P501S using ELISA and the IgG isotype was determined. The results of this analysis are shown in Table V below. Of the 16 subclones tested, monoclonal antibody 10E3-G4-D3 was selected for further study.

  The specificity of 10E3-G4-D3 for P501S was examined by FACS analysis. Specifically, cells were fixed (2% formaldehyde, 10 minutes), permeabilized (0.1% saponin, 10 minutes), and then stained with 0.5-1 μg / ml of 10E3-G4-D3. This was followed by incubation with FITC-labeled goat anti-mouse Ig secondary antibody (Pharmingen, San Diego, Calif. (CA)). The cells were then subjected to FITC fluorescence analysis using an Excalbur fluorescent cell sorter. In FACS analysis of transduced cells, P501S was transduced into B-LCL using retrovirus. For analysis of infected cells, B-LCL was infected with a vaccinia vector expressing P501S. To demonstrate the specificity of these assays, B-LCL transduced with another antigen (P703P) and uninfected B-LCL vector were used. 10E3-G4-D3 was shown not only to bind to P501S-transduced B-LCL but also to P501S-infected B-LCL cells, but not to uninfected cells or P703P-transduced cells.

  To determine whether the epitope recognized by 10E3-G4-D3 is on the surface of the cell or in the intracellular compartment, B-LCL was transduced with P501S or HLA-B8 as a control antigen, as above The sample was either fixed and permeabilized or stained directly with 10E3-G4-D3 and analyzed as described above. It was found that a specific permeation of P501S by 10E3-G4-D3 requires permeabilization, suggesting that the epitope recognized by this antibody is intracellular.

  The reactivity of 10E3-G4-D3 with three prostate tumor cell lines Lncap, PC-3 and DU-145, known to express P501S at high, medium and very low levels, respectively, The cells were permeabilized and processed and examined as described above. A higher reactivity of 10E3-G4-D3 was found with Lncap compared to PC-3, and a higher reactivity was found with PC-3 compared to DU-145. These results are consistent with the real-time PCR results, indicating that the antibody specifically recognizes P501S in these tumor cell lines and that the epitope recognized in the prostate tumor cell line is also present in the cells.

  The specificity of 10E3-G4-D3 for P501S was also shown by Western blot. Cell lysates were made from prostate tumor cell lines Lncap, DU-145 and PC-3, HEK293 cells transiently transfected with P501S, and non-transfected HEK293 cells. Western blot analysis with 10E3-G4-D3 on these lysates showed a 46 kDa immunoreactive band in Lncap, PC-3 and P501S transfected HEK cells, but for DU-145 or untransfected HEK293 cells No band was shown. Semi-quantitative PCR analysis showed that P501S mRNA was expressed in Lncap, which was lower in PC-3 cells but detectable in DU-145 cells. Therefore, the expression of P501S mRNA was also detected in these cells. It matches the result. Bacterial expressed and purified recombinant P501S (referred to as P501SStr2) is recognized by 10E3-G4-D3, as is full-length P501S transiently expressed in HEK293 cells using expression vector VR1012 or pCEP4 (24 kDa). The estimated molecular weight of P501S was 60.5 kDa, but both transfected and “native” P501S migrated with a mobility slightly lower than its hydrophobic nature.

  Immunohistochemical analysis was performed on panels of prostate tumors and normal tissue sections (prostate, adrenal gland, breast, cervix, colon, duodenum, gallbladder, ileum, kidney, ovary, pancreas, thyroid, skeletal muscle, spleen and testis) . Tissue samples were fixed in formalin for 24 hours, embedded in paraffin and then cut into 10 micron sections. Tissue sections were permeabilized and incubated with 10E3-G4-D3 for 1 hour. Incubation with HRP-labeled anti-mouse followed by DAB chromogen visualized the immunoreactivity of P501S. P501S was found to be strongly expressed in both normal prostate and prostate tumors, but was not detected in other tissues tested.

  In order to identify the epitope recognized by 10E3-G4-D3, an epitope mapping method was utilized. Thirteen overlapping 20-21mer series (5 amino acids overlap; SEQ ID NOs: 489-501) covering the entire P501S fragment used in 10E3-G4-D3 production were synthesized. The peptide or P501S fragment used to immunize mice in flat bottom 96-well microtiter plates was coated at 1 microgram / ml for 2 hours at 37 ° C. The wells were then aspirated and then blocked with phosphate buffered saline containing 1% (w / v) BSA for 2 hours at room temperature, followed by washing with PBS containing 0.1% Tween 20 (PBST). . Purified antibody 10E3-G4-D3 was added at a 2-fold dilution in PBST (1000 ng to 16 ng) and incubated at room temperature for 30 minutes. After subsequent 6 washes with PBST, a 1: 20000 HRP-labeled donkey anti-mouse IgG (H + L) Affinipure F (ab ′) fragment (Jackson Immunoresearch, West Grove) , Pennsylvania) for 30 minutes. The plates were then washed and incubated for 15 minutes in tetramethylbenzidine. The reaction was stopped by adding 1N sulfuric acid and the plate was measured at 450 nm using an ELISA plate reader. As shown in FIG. 8, reactivity was observed between the peptide of SEQ ID NO: 496 (corresponding to amino acids 439-459 of P501S) and the P501S fragment, but reactivity was recognized between the remaining peptides. First, it was shown that the epitope recognized by 10E3-G4-D3 is localized at amino acids 439-459 of SEQ ID NO: 113.

  To further investigate the tissue specificity of P501S, a multi-array immunohistochemical analysis was performed on approximately 4700 human tissues including major normal organs and neoplasms derived from these tissues. Of these human tissue samples, 65 were from the prostate. A tissue piece having a diameter of 0.6 mm was fixed in formalin and embedded in paraffin. Samples were pretreated with HIER using 10 mM citrate buffer, PH 6.0 and boiled for 10 minutes. Sections were stained with 10E3-G4-D3 and P501S immunoreactivity was visualized using HRP. All 65 prostate tissue samples (5 normal, 55 untreated prostate tumors, 5 hormone refractory prostate tumors) were positive and showed clear staining around each. All other tissues examined were negative for P501S expression.

(B) Preparation and characterization of antibody to P503S In substantially the same manner as P501S above, a fragment of P503S (amino acids 113-241 of SEQ ID NO: 114) was expressed in bacteria and purified for use in immunization of rabbits and mice It was. Mouse monoclonal antibodies were isolated using standard hybridoma technology as described above. Rabbit monoclonal antibodies were isolated using the selective lymphocyte antibody (SLAM) method at Imgenenics Pharmaceuticals (Vancouver, British Columbia, Canada). Table VI below lists monoclonal antibodies raised against P503S.

  DNA sequences encoding the complementarity determining regions (CDRs) for rabbit monoclonal antibodies 20D4 and JA1 were determined and are shown in SEQ ID NOs: 502 and 503, respectively.

  In order to better define the epitope binding region of each antibody, a series of overlapping peptides covering amino acids 109-213 of SEQ ID NO: 114 were generated. Using these peptides, an epitope map of the anti-P503S monoclonal antibody was prepared by the following ELISA. The recombinant fragment of P503S used as an immunogen was used as a positive control. A 96 well microtiter plate was coated overnight at 4 ° C. with a peptide or recombinant antigen at a concentration of 20 ng / well. The plate was aspirated, blocked with phosphate buffered saline containing 1% (w / v) BSA for 2 hours at room temperature, and then washed with PBS containing 0.1% Tween 20 (PBST). Rabbit monoclonal antibody, purified and diluted in PBST, was added to the wells and incubated for 30 minutes at room temperature. This was followed by 6 washes with PBST and a further 30 min incubation with 1: 2000 diluted protein A HPR conjugate. The plate was washed 6 times with PBST and further incubated with tetramethylbenzidine (TMB) substrate for 15 minutes. The reaction was stopped by adding 1N sulfuric acid and the plates were measured at 450 nm using an ELISA plate reader. An ELISA using a mouse monoclonal antibody was performed using a tissue culture supernatant that was successfully assayed.

  All antibodies bound to the recombinant P503S fragment except for the negative control SP2 supernatant. 20D4, JA1, and 1D12 strongly bound to peptide # 2101 (SEQ ID NO: 504) corresponding to amino acids 151-169 of SEQ ID NO: 114. 1C3 bound to peptide # 2102 (SEQ ID NO: 505) corresponding to amino acids 165-184 of SEQ ID NO: 114. 9C12 bound to peptide # 2099 (SEQ ID NO: 522) corresponding to amino acids 120-139 of SEQ ID NO: 114. Other antibodies bound to areas not examined in these studies.

  Following epitope mapping, the antibodies were tested by FACS analysis using a cell line stably expressing P503S to confirm that the antibodies bound to cell surface epitopes. Cells stably transfected with a control plasmid were used as a negative control. Cells were stained without fixation. 0.5 μg of anti-P503S monoclonal antibody was added and the cells were incubated on ice for 30 minutes before being washed twice, and then incubated with FITC-labeled goat anti-rabbit or mouse secondary antibody for 20 minutes. After washing twice, the cells were analyzed using an Excalibur fluorescent color cell sorter. Monoclonal antibodies 1C3, 1D12, 9C12, 20D4 and JA1 were found to bind to the cell surface epitope of P503S, but not 8D3.

  To determine which tissue is expressing P503S, a panel of normal tissues (prostate, adrenal gland, breast, cervix, colon, duodenum, gallbladder, ileum, kidney, ovary, substantially as described above) Immunohistochemical analysis was performed on pancreas, thyroid, skeletal muscle, spleen and testis. Following HRP-labeled anti-mouse or anti-rabbit antibody, incubation with TMB visualized the immunoreactivity of P503S. P503S is strong in prostate tissue and weakly expressed in the cervix, colon, ileum and kidney, but not in the adrenal gland, breast, duodenum, gallbladder, ovary, pancreas, thyroid, skeletal muscle, spleen and testis I understood that.

  Western blot analysis was used to examine the specificity of the anti-P503S monoclonal antibody. SDS-PAGE was performed on lysates from HEK293 cells transfected with recombinant (rec) P503S and full-length P503S expressed in bacteria and purified therefrom. The protein was transferred to a nitrocellulose membrane, and Western blotting was performed using anti-P503 monoclonal antibodies (20D4, JA1, 1D12, 6D12, and 9C12) having an antibody concentration of 1 μg / ml. Protein was detected using horseradish peroxidase (HRP) labeled goat anti-mouse monoclonal antibody or protein A sepharose. Monoclonal antibody 20D4 detected a 23.5 kDa protein species in HEK293 cell lysate transfected with recombinant P503S (amino acids 113-241) with a molecular weight of about 14 kDa and full-length P503S. Other anti-P503S monoclonal antibodies showed the same specificity as the Western blot.

(C) Preparation and characterization of antibody against P703P Recombinant of truncated (P703Ptr1; SEQ ID NO: 172) or full-length mature (P703Pf1; SEQ ID NO: 523) expressed in bacteria and purified therefrom as described above Rabbits were immunized with P703P protein. Affinity purified polyclonal antibodies were generated using immunogen P703Pf1 or P703Ptr1 bound to a solid support. The rabbit monoclonal antibody was isolated using Immunics Pharmaceuticals using SLAM method. Table VII below lists polyclonal and monoclonal antibodies raised against P703P.

  DNA sequences encoding the complementarity determining regions for rabbit monoclonal antibodies 8H2, 7H8 and 2D4 were determined and are shown in SEQ ID NOs: 506-508, respectively.

  Epitope mapping studies were performed as described above. Monoclonal antibodies 2D4 and 7H8 were found to specifically bind to SEQ ID NO: 509 (corresponding to amino acids 145-159 of SEQ ID NO: 172) and SEQ ID NO: 510 (corresponding to amino acids 11-25 of SEQ ID NO: 172), respectively. Polyclonal antibody 2594 was found to bind to the peptide of SEQ ID NO: 511-514 and polyclonal antibody 9427 to the peptide of SEQ ID NO: 515-517.

  The specificity of the anti-P703P antibody was determined by Western blot as follows. SDS-PAGE (1) Recombinant antigen expressed in bacteria; (2) Lysis of HEK293 cells and Ltk − / − cells not transfected or transfected with a plasmid expressing full length P703P And (3) supernatants isolated from these cell cultures. The protein was transferred to a nitrocellulose membrane and then Western blotted using anti-P703P polyclonal antibody # 2594 at an antibody concentration of 1 μg / ml. The protein was detected using an anti-rabbit antibody labeled with horseradish peroxidase (HRP). In the recombinant P703P, an immunoreactive band of 35 kDa was observed. Since the recombinant P703P has an epitope tag, it moves slightly as a high molecular weight. A 30 kDa band corresponding to P703P was observed in the lysates and supernatants of cells transfected with full length P703P. To confirm specificity, lysates from HEK293 cells stably transfected with the control plasmid were also tested, but no expression of P703P was observed. Other anti-P703P antibodies showed similar results.

  Immunohistochemistry studies were performed as described above using an anti-P703P monoclonal antibody. P703P was found to be expressed at high levels in normal prostate and prostate tumor tissues, but not detected in all other tissues tested (breast cancer, lung cancer and normal kidney).

Example 19
Characterization of cell surface expression and chromosomal localization of prostate-specific antigen P501S This example shows a study showing that prostate-specific antigen P501S is expressed in cell expression, and in conjunction with this, the possible chromosomal localization of P501S Explain the research to determine.

  The protein P501S (SEQ ID NO: 113) is estimated to have 11 transmembrane domains. Based on the discovery that the epitope recognized by the anti-P501S monoclonal antibody 10E3-G4-D3 (Example 17 above) exists in the cell, the extracellular domain of P501S can be inferred from the following transmembrane determinants. It was. FIG. 9 is a schematic diagram showing the predicted localization of the transmembrane domain and the intracellular epitope described in Example 17. The underlined sequence represents the putative transmembrane domain, the bold sequence represents the putative extracellular domain, and the italic sequence represents the putative intracellular domain. Polyclonal rabbit antisera was generated using bold and underlined sequences. The localization of the transmembrane domain was estimated in the same manner as reported by Tusnady and Simon using HHMTOP (Principles Governing of Amino Acid Composition of Integral Protein Protocol: Prediction), J. Mol. Biol, 283: 489-506, 1998).

  According to FIG. 9, the P501S domain connected to the transmembrane domain corresponding to amino acids 274-295 and 323-342 is presumed to be extracellular. The peptide of SEQ ID NO: 518 corresponds to amino acids 306-320 of P501S and is within the putative extracellular domain. The peptide of SEQ ID NO: 519, which was identical to the peptide of SEQ ID NO: 518 except that histidine was replaced by asparaginine, was synthesized as described above. Cys-Gly was added to the C-terminus of the peptide to facilitate binding to the carrier protein. Cleavage of the peptide from the solid support was performed using the following cleavage mixture: trifluoroacetic acid: ethanediol: thioanisole; water: phenol (40: 1: 2: 2: 3). After cleavage for 2 hours, the peptide was precipitated in cold ether. The peptide pellet was then dissolved in 10% v / v acetic acid and lyophilized prior to purification with C18 reverse phase hplc. Peptides were eluted using a gradient of 5-60% acetonitrile (with 0.05% TFA) in water (with 0.05% TFA). Peptide purity was confirmed by hplc and mass spectrometry and was found to be> 95%. A rabbit polyclonal antiserum was prepared using the purified peptide in the same manner as described above.

  The surface expression of P501S was verified by FACS analysis. Cells were stained with 10 μg / ml polyclonal anti-P501S peptide serum, washed, incubated with secondary FITC-labeled goat anti-rabbit Ig antibody (ICN), further washed, and then subjected to FITC fluorescence using an Excalibur fluorescence cell sorter. analyzed. In FACS analysis of the transduced cells, P501S was transduced into B-LCL using a retrovirus. To show the specificity of these assays, irrelevant antigen (P703P) or non-transduced B-LCL was also stained in parallel. Lncap, PC-3 and DU-145 were used for FACS analysis of prostate tumor cell lines. Prostate cell lines were detached from tissue culture plates using cell separation media and stained as described above. All samples were treated with propidium iodide (PI) before being subjected to FACS analysis, and data were obtained from PI-excluded (ie, live non-permeable) cells. Rabbit polyclonal serum generated against the peptide of SEQ ID NO: 519 has been shown to specifically recognize the surface of cells transduced and expressing P501S, and the epitope recognized by this polyclonal serum is extracellular. It was shown that.

  In order to determine biochemically whether P501S is expressed on the cell surface, the peripheral membrane of Lncap cells was isolated and subjected to Western blot analysis. Specifically, Lncap cells were dissolved in 5 ml of homogenization buffer (250 mM sucrose, 10 mM HEPES, 1 mM EDTA, pH 8.0, 1 complete protease inhibitor (Boehringer Mannheim)) using a Dounce homogenizer. did. The lysate sample was centrifuged at 1000 g for 5 minutes at 4 ° C. The supernatant was then centrifuged at 8000 g for 10 minutes at 4 ° C. The supernatant from 8000 g centrifugation was collected and centrifuged at 100,000 g for 30 minutes at 4 ° C. to recover the peripheral membrane. Samples were then separated by SDS-PAGE and subjected to Western blotting under the same conditions as described above using mouse monoclonal antibody 10E3-G4-D3 (described in Example 17 above). HEK293 cells transfected with and overexpressing recombinant purified P501S and P501S were included as positive controls for P501S detection. LCL cell lysate was included as a negative control. P501S could be detected not only in the Lncap whole cell lysate, 8000 g (inner membrane) fraction but also in the 100,000 g (plasma membrane) fraction. These results indicate that P501S is expressed and localized in the peripheral membrane.

  To show that the rabbit polyclonal antisera raised against the peptide of SEQ ID NO: 519 specifically recognizes not only this peptide but also the corresponding native peptide of SEQ ID NO: 518, an ELISA analysis was performed. In these analyses, flat-bottomed 96-well microtiter plates were prepared using 1 μg / ml of SEQ ID NO: 519 peptide, a longer peptide of SEQ ID NO: 520 covering the entire putative extracellular domain, 10E3-G4-D3, a P501S specific antibody. Was coated with either the peptide of SEQ ID NO: 521, which is the epitope recognized by, or a P501S fragment (corresponding to amino acids 355-526 of SEQ ID NO: 113) that does not contain the immunopeptide sequence for 2 hours at 37 ° C. The wells were aspirated and then blocked with phosphate buffered saline containing 1% (w / v) BSA for 2 hours at room temperature, followed by washing with PBS containing 0.1% Tween 20 (PBST). Purified anti-P501S polyclonal rabbit serum was diluted 2-fold into PBST (1000 ng to 125 ng) and incubated at room temperature for 30 minutes. Subsequently, it was washed 6 times with PBST and incubated with HRP-labeled goat anti-rabbit IgG (H + L) Affinipure F (ab ') fragment in a ratio of 1: 20000 for 30 minutes. The plates were then washed and incubated for 15 minutes in tetramethylbenzidine. The reaction was stopped by adding 1N sulfuric acid and the plate was measured at 450 nm using an ELISA plate reader. As shown in FIG. 11, the anti-P501S polyclonal rabbit serum specifically recognized not only the peptide of SEQ ID NO: 519 used for immunization but also the longer peptide of SEQ ID NO: 520, but the unrelated P501S-derived peptides and fragments were I did not recognize.

  In another study, rabbits were immunized with peptides derived from the P501S sequence and assumed to be extracellular or intracellular as shown in FIG. Polyclonal rabbit serum was isolated and the polyclonal antibody in the serum was purified as described above. To examine specific reactivity with P501S, FACS analysis was performed using either B-LCL transduced with P501S or an irrelevant antigen P703P, or B-LCL infected with vaccinia virus expressing P501S. It was. For surface expression, dead and incomplete cells were excluded from the analysis as above. For intracellular staining, cells were fixed and permeabilized as described above. Rabbit polyclonal serum generated against the peptide of SEQ ID NO: 548 corresponding to amino acids 181 to 198 of P501S was found to recognize the surface epitope of P501S. Rabbit polyclonal sera raised against the peptide of SEQ ID NO: 551 corresponding to amino acids 543-553 of P501S were found to be extracellular, since complete or permeabilized cells were recognized by this polyclonal antibody in another experiment. Or it was found to recognize an epitope that appears to be intracellular. For similar reasons, SEQ ID NOs corresponding to amino acids 109-122, 539-553, 509-520, 37-54, 342-359, 295-323, 217-274, 143-160 and 75-88 of P501S, respectively. The sequences 541-547, 549 and 550 can be considered to be potential surface epitopes of P501S recognized by the antibody.

  The chromosomal localization of P501S was determined using the GeneBridge 4 radiation hybrid panel (Research Genetics). In accordance with the manufacturer's instructions, PCR primers of SEQ ID NOs: 528 and 529 were used for PCR using the hybrid panel DNA pool. After 38 cycles of amplification, the reaction products were separated on a 1.2% agarose gel and the results were obtained from the Whitehead Institute / MIT Center for Genome Research web server (http: ///). analysis at www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) to determine possible chromosomal localization. Using this method, P501S was mapped to WI-9641 on the first chromosome long arm, between q32 and q42. This region of chromosome 1 is linked to prostate cancer susceptibility of hereditary prostate cancer (Smith et al., Science 274: 1371-1374, 1996 and Berthon et al., Am. J. Hum. Genet. 62: 1416-1424, 1998. ). These results suggest that P501S may play a role in prostate cancer malignancy.

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

Claims (20)

  An isolated antibody that specifically binds to the polypeptide set forth in SEQ ID NO: 113 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 496, 518, 519, and 541-551, or an antigen-binding fragment thereof.   2. The isolated antibody or antigen-binding fragment of claim 1 conjugated to a toxin.   The toxin is ricin toxin, abrin toxin, diphtheria toxin, cholera toxin, gelonin toxin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein The isolated antibody or antigen-binding fragment of claim 2, selected from the group consisting of:   2. The isolated antibody or antigen-binding fragment of claim 1 conjugated to a radionuclide. The isolated antibody or antigen-binding fragment according to claim 4, wherein the radionuclide is selected from the group consisting of ⁹⁰ Y, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At, and ²¹² Bi. .   The isolated antibody or antigen-binding fragment according to claim 1, which binds to the polypeptide of SEQ ID NO: 113 expressed on the cell surface of prostate tumor cells.   The isolated antibody or antigen-binding fragment according to any one of claims 1 to 6, which is a human isolated antibody or an antigen-binding fragment.   A composition comprising at least one antibody or antigen-binding fragment according to any one of claims 1 to 6 and a physiologically acceptable carrier.   A diagnostic kit comprising at least one antibody or antigen-binding fragment according to any one of claims 1 to 6, and a detection reagent.   The diagnostic kit according to claim 9, wherein the detection reagent contains a reporter group.   The diagnostic kit according to claim 10, wherein the antibody is immobilized on a solid support.   The diagnostic kit according to claim 10, wherein the detection reagent contains anti-immunoglobulin, protein G, protein A, or lectin.   The diagnostic kit according to claim 10, wherein the reporter group is selected from the group consisting of a radioisotope, a fluorescent group, a luminescent group, an enzyme, biotin, and dye particles.   7. Isolation according to any one of claims 1-6 in the manufacture of a medicament for killing or inhibiting the growth of prostate tumor cells expressing the polypeptide of SEQ ID NO: 113. Use of an antibody or antigen-binding fragment, thereby killing the tumor cell or suppressing its growth.   Use of an effective amount of the isolated antibody or antigen-binding fragment according to any one of claims 1 to 6 in the manufacture of a medicament for treating prostate cancer expressing the polypeptide of SEQ ID NO: 113. And thereby use the prostate cancer.   An isolated polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 496, 518, 519, and 541-551.   An isolated antibody, or antigen-binding fragment thereof, that specifically binds to the polypeptide of claim 16.   An isolated polynucleotide sequence encoding the polypeptide of claim 17. Physiologically acceptable carrier and the following:
(A) the polypeptide of claim 16,
(B) An isolated antibody or antigen-binding fragment according to any one of claims 1 to 6, or (c) a pharmaceutical composition comprising the polynucleotide according to claim 18. Nonspecific immune enhancers and the following:
(A) the polypeptide of claim 16,
(B) An isolated antibody or antigen-binding fragment according to any one of claims 1 to 6, or (c) a vaccine comprising the polynucleotide according to claim 18.

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