NO20023402L - Mixtures and Methods for the Treatment and Diagnosis of Prostate Cancer - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to the therapy and diagnosis of cancer, such as prostate cancer. The invention is more specifically related to polypeptides, comprising at least part of a prostate-specific protein and to polynucleotides encoding such polypeptides. Such polypeptides and polynucleotides are useful in pharmaceutical preparations, e.g. vaccines and other preparations for the diagnosis and treatment of prostate cancer.

BACKGROUND OF THE INVENTION

Cancer is a significant health problem worldwide. Although cancer is a significant health problem worldwide. Although progress has been made in the detection and therapy of cancer, no vaccine or other universally successful method of prevention or treatment is currently available. Current therapies, which are generally based on a combination of chemotherapy or surgery and radiation, continue to prove inadequate for many patients.

Prostate cancer is the most common form of cancer among men, with an estimated incidence of 30% in men over the age of 50. Overwhelming clinical evidence shows that human prostate cancer has a propensity to metastasize to bone, and the disease seems to inevitably progress from androgen-dependent to androgen-resistant status, leading to increased patient mortality. This widespread disease is currently the second leading cause of cancer death among men in the United States.

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

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

SUMMARY OF THE INVENTION

In one aspect, the present invention provides polynucleotide preparations comprising a sequence selected from the group consisting of: (a) sequences given in SEQ ID NOS: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; (b) complements of the sequences given in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; (c) sequences consisting of at least 20 consecutive residues of a sequence given in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335 , 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634 , 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; (d) sequences that hybridize to a sequence given in SEQ ID NO: 1-111,115-171,173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674 , 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788, under moderately stringent conditions; (e) sequences that have at least 75% identity with a sequence from SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; (f) sequences that have at least 90% identity to a sequence from SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326,328,330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639- 655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; and (g) degenerate variants of a sequence given in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375 , 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639 -655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788.

In one preferred embodiment, the polynucleotide preparations according to the present invention are expressed in at least approx. 20%, more preferably in at least approx. 30% and most preferred for at least approx. 50% of prostate tissue samples tested, at a level that is at least approx. 2 times, preferably at least approx. 5 times and most preferably at least approx. 10 times higher than that of other normal tissues.

The present invention provides, in another aspect, polypeptide preparations comprising an amino acid sequence encoded by a polynucleotide sequence described above.

The present invention further provides polypeptide preparations comprising an amino acid sequence selected from the group consisting of sequences indicated in SEQ ID NO: 112-114, 172, 176, 178,327, 329, 331,336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 627-629, 632, 633, 635, 637, 638, 656- 671, 675, 683, 684, 710, 712, 714, 715, 717-719, 723-734, 736, 740-750, 752, 754, 755, 766-772, 777-785 and 789-791.

In certain preferred embodiments, the polypeptides and/or polynucleotides according to the present invention are immunogenic, i.e. they can elicit an immune response, in particular a humoral and/or cellular immune response, as further described herein.

The present invention further provides fragments, variants and/or derivatives of the described polypeptide and/or polynucleotide sequences, where fragments, variants and/or derivatives preferably have a level of immunogenic activity of at least approx. 50%, preferably at least approx. 70% and more preferably at least approx. 90% of the level of immunogenic activity of a polypeptide sequence indicated in SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 627-629, 632, 633, 635, 637, 638, 656- or a polypeptide sequence coded for by a polynucleotide sequence set forth in SEQ ID NO: 1-111,115-171,173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476 , 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681 , 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788.

The present invention further provides polynucleotides which code for a polypeptide described above, expression vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.

In other aspects, the present invention provides pharmaceutical preparations comprising a polypeptide or polynucleotide as described above and a physiologically acceptable carrier.

In a related aspect of the present invention, pharmaceutical preparations, e.g. vaccine preparations, provided for prophylactic or therapeutic uses. Such preparations generally comprise an immunogenic polypeptide or polynucleotide according to the present invention and an immunostimulant, such as an adjuvant, together with a physiologically acceptable carrier.

The present invention further provides pharmaceutical preparations comprising: (a) an antibody or antigen-binding fragment thereof which specifically binds to a polypeptide according to the present invention or a fragment thereof; and (b) a physiologically acceptable carrier.

In further aspects, the present invention provides pharmaceutical compositions comprising: (a) an antigen presenting cell expressing a polypeptide as described above and (b) a pharmaceutically acceptable carrier or adjuvant. Illustrative antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B cells.

In related aspects, pharmaceutical compositions are provided comprising: (a) an antigen presenting cell expressing a polypeptide as described above and (b) an immunostimulant.

The present invention further provides, in other aspects, fusion proteins comprising at least one polypeptide as described above, as well as polynucleotides encoding such fusion proteins, typically in the form of pharmaceutical preparations, e.g. vaccine preparations, comprising a physiologically acceptable carrier and/or an immunostimulant. The fusion proteins may comprise multiple immunogenic polypeptides or parts/variants thereof, as described here and may further comprise one or more polypeptide segments to facilitate and/or improve expression, purification and/or immunogenicity of the polypeptide(s).

In further aspects, the present invention provides methods for stimulating an immune response in a patient, preferably a T-cell response in a human patient, comprising administering a pharmaceutical preparation described herein. The patient may be affected by prostate cancer, in which case the methods provide treatment for the disease, or a patient considered to be at risk of such a disease may be treated prophylactically.

In further aspects, the present invention provides methods of inhibiting the development of cancer in a patient, comprising administering to a patient a pharmaceutical composition as set forth above. The patient may be affected by prostate cancer, in which case the methods provide treatment for the disease, or a patient considered to be at risk of such a disease may be treated prophylactically.

The present invention further provides, in other aspects, methods for removing tumor cells from a biological sample, comprising bringing a

biological sample in contact with T cells that specifically react with a polypeptide according to the present invention, wherein the contact step is carried out under conditions and for a time sufficient to allow the removal of cells expressing the polypeptide from the sample.

In related aspects, provided are methods of inhibiting the development of cancer in a patient, comprising administering to a patient a biological sample processed as described above.

Methods are further provided, in other aspects, for stimulating and/or expanding T cells specific for a polypeptide of the present invention, comprising contacting T cells with one or more of: (i) a polypeptide as described above ; (ii) a polynucleotide encoding such a polypeptide; and (iii) an antigen presenting cell expressing such polypeptide; under conditions and for a time sufficient to allow stimulation and/or expansion of T cells. Isolated T cell populations comprising T cells prepared as described above are also provided.

In further aspects, the present invention provides methods of inhibiting the development of cancer in a patient, comprising administering to a patient an effective amount of a T-cell population as described above.

The present invention further provides methods for inhibiting the development of cancer in a patient, comprising the steps: (a) incubating CD4<+>and/or CD8<+>T cells isolated from a patient with one or more of: (i) a polypeptide comprising at least one immunogenic portion of the polypeptide described herein; (ii) a polynucleotide encoding such a polypeptide; and (iii) an antigen presenting cell expressing such polypeptide; and (b) administering to the patient an effective amount of the proliferated T cells, thereby inhibiting the development of cancer in the patient. Proliferated cells may, but need not, be cloned prior to administration to the patient.

Within further aspects the present invention provides

methods of determining the presence or absence of cancer, preferably prostate cancer, in a patient comprising: (a) contacting a biological sample taken from a patient with a binding agent that binds to a polypeptide as set forth above; (b)

detecting in the sample an amount of polypeptide that binds to the binding agent; and (c) comparing the amount of polypeptide to a predetermined cut-off value and thereby determining the presence or absence of cancer in the patient. In preferred embodiments, the binder is an antibody, more preferably a monoclonal antibody.

The present invention also provides, in other aspects, methods for monitoring the progression of cancer in a patient. Such methods comprise the steps of: (a) contacting a biological sample obtained from a patient at a first time point with a binding agent that binds to a polypeptide as set forth above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; (c) repeating steps (a) and (b) using a biological sample taken from the patient at a later time; and (d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b) and thereby monitoring the progression of the cancer in the patient.

The present invention further provides, in other aspects, methods for determining the presence or absence of cancer in a patient, comprising the steps of: (a) contacting a biological sample taken from a patient with an oligonucleotide that hybridizes to a polynucleotide of the present invention; (b) detecting in the sample a level of a polynucleotide, preferably mRNA, which hybridizes to the oligonucleotide; and (c) comparing the level of polynucleotide hybridizing to the oligonucleotide to a predetermined cut-off value and thereby determining the presence or absence of cancer in the patient. In certain embodiments, the amount of mRNA is detected via polymerase chain reaction using, for example, at least one oligonucleotide primer that hybridizes to a polynucleotide according to the present invention or a complement of such a polynucleotide. In other embodiments, the amount of mRNA is detected using a hybridization technique, using an oligonucleotide probe that hybridizes to a new polynucleotide or a complement of such a polynucleotide.

In related aspects, methods are provided for monitoring the progression of cancer in a patient, comprising the steps of: (a) contacting a biological sample taken from a patient with an oligonucleotide that hybridizes to a polynucleotide of the present invention; (b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; (c) repeating steps (a) and (b) using a biological sample taken from the patient at a later time; and (d) comparing the amount of polynucleotide detected in step (c) with the amount detected in step (b) and thereby monitoring the progression of the cancer in the patient.

In further aspects, the present invention provides antibodies, such as monoclonal antibodies, that bind to a polypeptide as described above, as well as diagnostic kits comprising such antibodies. Diagnostic kits comprising one or more oligonucleotide probes or primers as described above are also provided.

These and other aspects of the present invention will become clear with reference to the following detailed description and accompanying drawings. All references described herein are hereby incorporated by reference in their entirety as if each were incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS

Figure 1 illustrates the ability of T cells to kill fibroblasts expressing the representative prostate-specific polypeptide P502S, compared to control fibroblasts. The percentage light is shown as a series of effecton measure ratios, as indicated. Figures 2A and 2B illustrate the ability of T cells to recognize cells expressing the representative prostate-specific polypeptide P502S. In each case, the number of γ-interferon spots is shown for different numbers of responders. In Figure 2A, data are presented for fibroblasts pulsed with the P2S-12 peptide, compared to fibroblasts pulsed with a control E75 peptide. In Figure 2B, data are presented for fibroblasts expressing P502S, compared to fibroblasts expressing HER-2/neu. Figure 3 represents a peptide competitive binding assay showing that the P1S#10 peptide, derived from P501S, binds HLA-A2. Peptide P1S#10 inhibits HLA-A2-restricted presentation of fluM58 peptide to CTL clone D150M58 in the TNF release bioassay. D150M58 CTL is specific for the HLA-A2-binding influenza matrix peptide fluM58. Figure 4 illustrates the ability of T cell lines generated from P1S#10-immunized mice to specifically lyse P1S#10-pulsed Jurkat A2Kb targets and P501S-transduced Jurkat A2Kb targets, compared to EGFP-transduced Jurkat A2Kb. Percent lyse is shown as a series of effector to target ratios, as indicated. Figure 5 illustrates the ability of a T cell clone to recognize and specifically lyse Jurkat A2KB cells expressing the representative prostate-specific polypeptide P501S, thereby demonstrating that the P1S#10 peptide may be a naturally processed epitope of the P501S polypeptide . Figures 6A and 6B are graphs illustrating the specificity of a CD8<+>cell line (3A-1) for a representative prostate-specific antigen (P501S). Figure 6A shows the results of a<51>Cr release experiment. Percent specific lysis is shown as a series of effector-target ratios, as indicated. Figure 6B shows production of interferon-gamma by 3A-1 cells stimulated with autologous B-LCL transduced with P501S, with varying effector:target ratios as indicated. Figure 7 is a Western blot showing the expression of P501S in baculovirus. Figure 8 illustrates the results of epitope mapping studies on P501S. Figure 9 is a schematic representation of the P501S protein showing the location of transmembrane domains and predicted intracellular and extracellular domains. Figure 10 is a genomic map showing the location of prostate genes P775P, P704P, B305D, P712P and P774P in the cat's eye syndrome region of chromosome 22q11.2 Figure 11 shows the results of an ELISA assay to determine the specificity of rabbit polyclonal -antisera raised against P501S.

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

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

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

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

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

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

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

SEQ ID NO: 8 is the determined 3' cDNA sequence of J1-17 SEQ ID NO: 9 is the determined 5' cDNA sequence of J1-17 SEQ ID NO: 10 is the determined 3' cDNA sequence for L1-12 SEQ ID NO: 11 is the determined 5' cDNA sequence for L1-12 SEQ ID NO: 12 is the determined 3' cDNA sequence for N1-1862 SEQ ID NO: 13 is the determined 5' cDNA sequence for N1-1862 SEQ ID NO: 14 is the determined 3' cDNA sequence for J1-13 SEQ ID NO: 15 is the determined 5' cDNA sequence for J1-13 SEQ ID NO: 16 is the determined 3' cDNA sequence of J1-19 SEQ ID NO: 17 the determined 5' cDNA sequence of J1-19 SEQ ID NO: 18 the determined 3' cDNA sequence of J1-25 SEQ ID NO NO: 19 is the determined 5' cDNA sequence of J1-25 SEQ ID NO: 20 is the determined 5' cDNA sequence of J1-24 SEQ ID NO: 21 is the determined 3' cDNA sequence of J1 -24 SEQ ID NO: 22 is the determined 5' cDNA sequence of K1-58 SEQ ID NO: 23 is the determined 3' cDNA sequence of K1-58 SEQ ID NO: 24 is the determined 5' cDNA sequence for K1-63 SEQ ID NO: 25 is determined e 3' cDNA sequence for K1-63 SEQ ID NO: 26 is the determined 5' cDNA sequence for L1-4 SEQ ID NO: 27 is the determined 3' cDNA sequence for L1-4 SEQ ID NO : 28 is the determined 5' cDNA sequence of L1-14 SEQ ID NO: 29 is the determined 3' cDNA sequence of L1-14 SEQ ID NO: 30 is the determined 3' cDNA sequence of J1- 12 SEQ ID NO: 31 is the determined 3' cDNA sequence of J1-16 SEQ ID NO: 32 is the determined 3' cDNA sequence of J1-21 SEQ ID NO: 33 is the determined 3' cDNA sequence of K1-48 SEQ ID NO: 34 is the determined 3' cDNA sequence of K1-55 SEQ ID NO: 35 is the determined 3' cDNA sequence of L1-2 SEQ ID NO: 36 is the determined 3 ' cDNA sequence for L1-6 SEQ ID NO: 37 is the determined 3' cDNA sequence for N1-1858 SEQ ID NO: 38 is the determined 3 cDNA sequence for N1-1860 SEQ ID NO: 39 is the determined 3' cDNA sequence for N1-1861 SEQ ID NO: 40 is the determined 3' cDNA sequence for N1-1864

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

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

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

SEQ ID NO: 44 is the determined cDNA sequence of P18 SEQ ID NO: 45 is the determined cDNA sequence of P20 SEQ ID NO: 46 is the determined cDNA sequence of P29 SEQ ID NO: 47 is the determined cDNA sequence for P30 SEQ ID NO: 48 is the determined cDNA sequence for P34 SEQ ID NO: 49 is the determined cDNA sequence for P36 SEQ ID NO: 50 is the determined cDNA sequence for P38 SEQ ID NO: 51 is the determined cDNA sequence for P39 SEQ ID NO: 52 is the determined cDNA sequence for P42 SEQ ID NO: 53 is the determined cDNA sequence for P47 SEQ ID NO: 54 is the determined cDNA sequence for P49 SEQ ID NO: 55 is the determined cDNA sequence for P50 SEQ ID NO: 56 is the determined cDNA sequence for P53 SEQ ID NO: 57 is the determined cDNA sequence for P55 SEQ ID NO: 58 is the determined cDNA sequence for P60 SEQ ID NO: 59 is the determined cDNA sequence of P64 SEQ ID NO: 60 is the determined cDNA sequence of P65 SEQ ID NO: 61 is the determined cDNA sequence of P73 SEQ ID NO: 62 is the determined cDNA sequence of P75 SEQ ID NO : 63 is the determined cDNA sequence of P76 SEQ ID NO: 64 is the determined cDNA sequence of P79 SEQ ID NO: 65 is the determined cDNA sequence of P84 SEQ ID NO: 66 is the determined cDNA sequence of P68

SEQ ID NO: 67 is the determined cDNA sequence for P80 (also referred to as P704P)

SEQ ID NO: 68 is the determined cDNA sequence of P82 SEQ ID NO: 69 is the determined cDNA sequence of U1-3064 SEQ ID NO: 70 is the determined cDNA sequence of U1-3065 SEQ ID NO: 71 is the determined cDNA sequence of V1-3692 SEQ ID NO: 72 is the determined cDNA sequence of 1A-3905 SEQ ID NO: 73 is the determined cDNA sequence of V1-3686 SEQ ID NO: 74 is the determined cDNA sequence of R1-2330 SEQ ID NO: 75 is the determined cDNA sequence of 1B-3976 SEQ ID NO: 76 is the determined cDNA sequence of V1-3679 SEQ ID NO: 77 is the determined cDNA sequence of 1 G-4736 SEQ ID NO: 78 is the determined cDNA sequence of 1 G-4738 SEQ ID NO: 79 is the determined cDNA sequence of 1 G-4741 SEQ ID NO: 80 is the determined cDNA sequence of 1 G-4744 SEQ ID NO : 81 is the determined cDNA sequence of 1G-4734 SEQ ID NO: 82 is the determined cDNA sequence of 1H-4774 SEQ ID NO: 83 is the determined cDNA sequence of 1H-4781 SEQ ID NO: 84 is the determined cDNA sequence in 1H-4785 SEQ ID NO: 85 is the determined cDNA sequence in 1H-4787 SEQ ID NO: 8 6 is the determined cDNA sequence of 1H-4796 SEQ ID NO: 87 is the determined cDNA sequence of 11-4807 SEQ ID NO: 88 is the determined cDNA sequence of 11-4810 SEQ ID NO: 89 is the determined cDNA sequence in 11-4811 SEQ ID NO: 90 is the determined cDNA sequence in 1J-4876 SEQ ID NO: 91 is the determined cDNA sequence in 1K-4884 SEQ ID NO: 92 is the determined cDNA sequence in 1K- 4896 SEQ ID NO: 93 is the determined cDNA sequence of 1 G-4761 SEQ ID NO: 94 is the determined cDNA sequence of 1 G-4762 SEQ ID NO: 95 is the determined cDNA sequence of 1H-4766 SEQ ID NO: 96 is the determined cDNA sequence of 1H-4770 SEQ ID NO: 97 is the determined cDNA sequence of 1H-4771 SEQ ID NO: 98 is the determined cDNA sequence of 1H-4772 SEQ ID NO: 99 is the determined cDNA sequence in 1D-4297 SEQ ID NO: 100 is the determined cDNA sequence in 1D-4309 SEQ ID NO: 101 is the determined cDNA sequence in 1D.1-4278 SEQ ID NO: 102 is the determined cDNA sequence in 1D-4288 SEQ ID NO: 103 is the determined cDNA sequence in 1D-4283 SEQ ID NO: 104 is the determined c DNA sequence in 1D-4304 SEQ ID NO: 105 is the determined cDNA sequence in 1D-4296

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

SEQ ID NO: 107 is the determined full-length cDNA sequence for F1-12

(also referred to as P504S)

SEQ ID NO: 108 is the predicted amino acid sequence of F1-12 SEQ ID NO: 109 is the determined full-length cDNA sequence of J1-17

SEQ ID NO: 110 is the determined full-length cDNA sequence for L1-12

(also referred to as P501S)

SEQ ID NO: 111 is the determined full-length cDNA sequence 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 predicted amino acid sequence for L1-12 (also referred to as P501S)

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

SEQ ID NO: 115 is the determined cDNA sequence of P89 SEQ ID NO: 116 is the determined cDNA sequence of P90 SEQ ID NO: 117 is the determined cDNA sequence of P92 SEQ ID NO: 118 is the determined cDNA sequence for P95 SEQ ID NO: 119 is the determined cDNA sequence for P98 SEQ ID NO: 120 is the determined cDNA sequence for P102 SEQ ID NO: 121 is the determined cDNA sequence for P110 SEQ ID NO: 122 is the determined cDNA sequence for P111 SEQ ID NO: 123 is the determined cDNA sequence for P114 SEQ ID NO: 124 is the determined cDNA sequence for P115 SEQ ID NO: 125 is the determined cDNA sequence for P116 SEQ ID NO: 126 is the determined cDNA sequence for P124 SEQ ID NO: 127 is the determined cDNA sequence for P126 SEQ ID NO: 128 is the determined cDNA sequence for P130 SEQ ID NO: 129 is the determined cDNA sequence for P133 SEQ ID NO: 130 is the determined cDNA sequence of P138 SEQ ID NO: 131 is the determined cDNA sequence of P143 SEQ ID NO: 132 is the determined cDNA sequence of P151 SEQ ID NO: 133 is the determined c DNA sequence for P156 SEQ ID NO: 134 is the determined cDNA sequence for P157 SEQ ID NO: 135 is the determined cDNA sequence for P166 SEQ ID NO: 136 is the determined cDNA sequence for P176 SEQ ID NO: 137 is the determined cDNA sequence of P178 SEQ ID NO: 138 is the determined cDNA sequence of P179 SEQ ID NO: 139 is the determined cDNA sequence of P185 SEQ ID NO: 140 is the determined cDNA sequence of P192 SEQ ID NO: 141 is the determined cDNA sequence of P201 SEQ ID NO: 142 is the determined cDNA sequence of P204 SEQ ID NO: 143 is the determined cDNA sequence of P208 SEQ ID NO: 144 is the determined cDNA sequence of P211 SEQ ID NO NO: 145 is the determined cDNA sequence of P213 SEQ ID NO: 146 is the determined cDNA sequence of P219 SEQ ID NO: 147 is the determined cDNA sequence of P237 SEQ ID NO: 148 is the determined cDNA sequence of P239 SEQ ID NO: 149 is the determined cDNA sequence of P248 SEQ ID NO: 150 is the determined cDNA sequence of P251 SEQ ID NO: 151 is the determined cDNA sequence of P255 SEQ ID NO: NO: 152 is the determined cDNA sequence of P256 SEQ ID NO: 153 is the determined cDNA sequence of P259 SEQ ID NO: 154 is the determined cDNA sequence of P260 SEQ ID NO: 155 is the determined cDNA sequence of P263 SEQ ID NO: 156 is the determined cDNA sequence of P264 SEQ ID NO: 157 is the determined cDNA sequence of P266 SEQ ID NO: 158 is the determined cDNA sequence of P270 SEQ ID NO: 159 is the determined cDNA sequence for P272 SEQ ID NO: 160 is the determined cDNA sequence for P278 SEQ ID NO: 161 is the determined cDNA sequence for P105 SEQ ID NO: 162 is the determined cDNA sequence for P107 SEQ ID NO: 163 is the determined cDNA sequence for P137 SEQ ID NO: 164 is the determined cDNA sequence for P194 SEQ ID NO: 165 is the determined cDNA sequence for P195 SEQ ID NO: 166 is the determined cDNA sequence for P196

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

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

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

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

SEQ ID NO: 171 is the determined cDNA sequence of P703P-DE1 SEQ ID NO: 172 is the predicted amino acid sequence of P703P-DE1 SEQ ID NO: 173 is the determined cDNA sequence of P703P-DE2 SEQ ID NO: 174 is the determined cDNA sequence of P703P-DE6 SEQ ID NO: 175 is the determined cDNA sequence of P703P-DE13 SEQ ID NO: 176 is the predicted amino acid sequence of P703P-DE13 SEQ ID NO: 177 is the determined cDNA sequence of P703P- DE14 SEQ ID NO: 178 is the predicted amino acid sequence of P703P-DE14 SEQ ID NO: 179 is the determined extended cDNA sequence of 1G-4736 SEQ ID NO: 180 is the determined extended cDNA sequence of 1G-4738 SEQ ID NO: 181 is the determined extended cDNA sequence of 1G-4741 SEQ ID NO: 182 is the determined extended cDNA sequence of 1G-4744 SEQ ID NO: 183 is the determined extended cDNA sequence of 1H-4774 SEQ ID NO: 184 is the determined extended cDNA sequence in 1H-4781 SEQ ID NO: 185 is the determined extended cDNA sequence in 1H-4785 SEQ ID NO: 186 is the determined extended cDNA sequence in 1H-4787 V ID NO: 187 is the determined extended cDNA sequence of 1H-4796 SEQ ID NO: 188 is the determined extended cDNA sequence of 11-4807 SEQ ID NO: 189 is the determined 3' cDNA sequence of 11-4810 SEQ ID NO: 190 is the determined 3' cDNA sequence in 11-4811

SEQ ID NO: 191 is the determined extended cDNA sequence of 1J-4876 SEQ ID NO: 192 is the determined extended cDNA sequence of 1K-4884 SEQ ID NO: 193 is the determined extended cDNA sequence of 1K-4896 SEQ ID NO: NO: 194 is the determined extended cDNA sequence of 1 G-4761 SEQ ID NO: 195 is the determined extended cDNA sequence of 1 G-4762 SEQ ID NO: 196 is the determined extended cDNA sequence of 1H-4766 SEQ ID NO: 197 is the determined 3' cDNA sequence of 1H-4770 SEQ ID NO: 198 is the determined 3' cDNA sequence of 1H-4771 SEQ ID NO: 199 is the determined extended cDNA sequence of 1H-4772 SEQ ID NO: 200 is the determined extended cDNA sequence of 1D-4309 SEQ ID NO: 201 is the determined extended cDNA sequence of 1D,1-4278 SEQ ID NO: 202 is the determined extended cDNA sequence of 1D-4288 SEQ ID NO: 203 is the determined extended cDNA sequence of 1D-4283 SEQ ID NO: 204 is the determined extended cDNA sequence of 1D-4304 SEQ ID NO: 205 is the determined extended cDNA sequence of 1D-4296 SEQ ID NO: NO: 206 is the particular extended cDNA sequence sequence in 1D-4280 SEQ ID NO: 207 is the determined cDNA sequence in 10-d8fwd

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

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

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

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

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

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

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

SEQ ID NO: 215 is the determined cDNA sequence in 8-b6 rev

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

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

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

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

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

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

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

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

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

SEQ ID NO: 225 is the determined cDNA sequence of P703DE5 SEQ ID NO: 226 is the determined cDNA sequence of 9-A11

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

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

SEQ ID NO: 229 is the determined cDNA sequence of JPTPN13 SEQ ID NO: 230 is the determined cDNA sequence of JPTPN14 SEQ ID NO: 231 is the determined cDNA sequence of JPTPN23 SEQ ID NO: 232 is the determined cDNA sequence for JPTPN24 SEQ ID NO: 233 is the determined cDNA sequence for JPTPN25 SEQ ID NO: 234 is the determined cDNA sequence for JPTPN30 SEQ ID NO: 235 is the determined cDNA sequence for JPTPN34 SEQ ID NO: 236 is the determined cDNA sequence of PTPN35 SEQ ID NO: 237 is the determined cDNA sequence of JPTPN36 SEQ ID NO: 238 is the determined cDNA sequence of JPTPN38 SEQ ID NO: 239 is the determined cDNA sequence of JPTPN39 SEQ ID NO: 240 is the determined cDNA sequence of JPTPN40 SEQ ID NO: 241 is the determined cDNA sequence of JPTPN41 SEQ ID NO: 242 is the determined cDNA sequence of JPTPN42 SEQ ID NO: 243 is the determined cDNA sequence of JPTPN45 SEQ ID NO: 244 is the determined cDNA sequence of JPTPN46 SEQ ID NO: 245 is the determined cDNA sequence of JPTPN51 SEQ ID NO: 246 is the determined cDNA sequence for JPTPN56 SEQ ID NO: 247 is the determined cDNA sequence for PTPN64 SEQ ID NO: 248 is the determined cDNA sequence for JPTPN65 SEQ ID NO: 249 is the determined cDNA sequence for JPTPN67 SEQ ID NO: 250 is the determined cDNA sequence of JPTPN76 SEQ ID NO: 251 the determined cDNA sequence of JPTPN84 SEQ ID NO: 252 the determined cDNA sequence of JPTPN85 SEQ ID NO: 253 the determined cDNA sequence of JPTPN86 SEQ ID NO: 254 is the determined cDNA sequence of JPTPN87 SEQ ID NO: 255 is the determined cDNA sequence of JPTPN88 SEQ ID NO: 256 is the determined cDNA sequence of JP1F1 SEQ ID NO: 257 is the determined cDNA sequence of JP1F2 SEQ ID NO: NO: 258 is the determined cDNA sequence of JP1C2 SEQ ID NO: 259 is the determined cDNA sequence of JP1B1 SEQ ID NO: 260 is the determined cDNA sequence of JP1B2 SEQ ID NO: 261 is the determined cDNA sequence of JP1D3 SEQ ID NO: 262 is the determined cDNA sequence of JP1A4 SEQ ID NO: 263 is the determined cDNA sequence of JP1F5 SEQ ID NO: 264 is the determined cDNA sequence of JP1E6 SEQ ID NO: 265 is the determined cDNA sequence of JP1D6 SEQ ID NO: 266 is the determined cDNA sequence of JP1B5 SEQ ID NO: 267 is the determined cDNA sequence of JP1A6 SEQ ID NO: 268 is the determined cDNA sequence of JP1E8 SEQ ID NO: 269 is the determined cDNA sequence of JP1D7 SEQ ID NO: 270 is the determined cDNA sequence of JP1D9 SEQ ID NO: 271 is the determined cDNA sequence of JP1C10 SEQ ID NO : 272 is the determined cDNA sequence of JP1A9 SEQ ID NO: 273 is the determined cDNA sequence of JP1F12 SEQ ID NO: 274 is the determined cDNA sequence of JP1E12 SEQ ID NO: 275 is the determined cDNA sequence of JP1D11 SEQ ID NO: ID NO: 276 is the determined cDNA sequence of JP1C11 SEQ ID NO: 277 is the determined cDNA sequence of JP1C12 SEQ ID NO: 278 is the determined cDNA sequence of JP1B12 SEQ ID NO: 279 is the determined cDNA sequence of JP1A12 SEQ ID NO: 280 is the determined cDNA sequence of JP8 G2 SEQ ID NO: 281 is the determined cDNA sequence of JP8H1 SEQ ID NO: 282 is the b determined cDNA sequence for JP8H2 SEQ ID NO: 283 is the determined cDNA sequence for JP8A3 SEQ ID NO: 284 is the determined cDNA sequence for JP8A4 SEQ ID NO: 285 is the determined cDNA sequence for JP8C3 SEQ ID NO: 286 is the determined cDNA sequence of JP8 G4 SEQ ID NO: 287 is the determined cDNA sequence of JP8B6 SEQ ID NO: 288 is the determined cDNA sequence of JP8D6 SEQ ID NO: 289 is the determined cDNA sequence of JP8F5 SEQ ID NO: NO: 290 is the determined cDNA sequence of JP8A8 SEQ ID NO: 291 is the determined cDNA sequence of JP8C7 SEQ ID NO: 292 is the determined cDNA sequence of JP8D7 SEQ ID NO: 293 is the determined cDNA sequence of P8D8 SEQ ID NO: 294 is the determined cDNA sequence of JP8E7 SEQ ID NO: 295 is the determined cDNA sequence of JP8F8

SEQ ID NO: 296 is the determined cDNA sequence of JP8 G8 SEQ ID NO: 297 is the determined cDNA sequence of JP8B10 SEQ ID NO: 298 is the determined cDNA sequence of JP8C10 SEQ ID NO: 299 is the determined cDNA sequence of JP8E9 SEQ ID NO: 300 is the determined cDNA sequence of JP8E10 SEQ ID NO: 301 is the determined cDNA sequence of JP8F9 SEQ ID NO: 302 is the determined cDNA sequence of JP8H9 SEQ ID NO: 303 is the determined cDNA sequence of JP8C12 SEQ ID NO: 304 is the determined cDNA sequence of JP8E11 SEQ ID NO: 305 is the determined cDNA sequence of JP8E12 SEQ ID NO: 306 is the amino acid sequence of the peptide PS2#12 SEQ ID NO: 307 is the determined cDNA sequence for P711P SEQ ID NO: 308 is the determined cDNA sequence for P712P SEQ ID NO: 309 is the determined cDNA sequence for CLONE23 SEQ ID NO: 310 is the determined cDNA sequence for P774P SEQ ID NO: 311 is the determined cDNA sequence of P775P SEQ ID NO: 312 is the determined cDNA sequence of P715P SEQ ID NO: 313 is the determined cDNA sequence of P710 P SEQ ID NO: 314 is the determined cDNA sequence of P767P SEQ ID NO: 315 is the determined cDNA sequence of P768P

SEQ ID NO: 316-325 are the determined cDNA sequences of previously isolated genes

SEQ ID NO: 326 is the determined cDNA sequence of P703PDE5 SEQ ID NO: 327 is the predicted amino acid sequence of P703PDE5 SEQ ID NO: 328 is the determined cDNA sequence of P703P6.26 SEQ ID NO: 329 is the predicted amino acid sequence of P703P6 .26 SEQ ID NO: 330 is the determined cDNA sequence of P703PX-23 SEQ ID NO: 331 is the predicted amino acid sequence of P703PX-23 SEQ ID NO: 332 is the determined full-length cDNA sequence of P509S

SEQ ID NO: 333 is the determined extended cDNA sequence for P707P

(also referred to as 11-C9)

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

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

SEQ ID NO: 336 is the predicted amino acid sequence of P705P 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 of P509S

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

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

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

SEQ ID NO: 343 is the determined cDNA sequence of a clone showing homology to Homo sapiens MM46 mRNA

SEQ ID NO: 344 is the determined cDNA sequence of a clone showing homology to Homo sapiens TNF-alpha-stimulated ABC protein (ABC50) mRNA

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

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

SEQ ID NO: 347 is the determined cDNA sequence of a clone showing homology to Homo sapiens innate resistance-associated macrophage protein2

(NRAMP2)

SEQ ID NO: 348 is the determined cDNA sequence of a clone showing homology to Homo sapiens phosphoglucomutase-related protein (PGMRP)

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO: 360 is the determined cDNA sequence for unknown 17984 SEQ ID NO: 361 is the determined cDNA sequence for P787P

SEQ ID NO: 362 is the determined cDNA sequence of P788P SEQ ID NO: 363 is the determined cDNA sequence of unknown 17994 SEQ ID NO: 364 is the determined cDNA sequence of P781P SEQ ID NO: 365 is the determined cDNA sequence sequence for P785P

SEQ ID NO: 366-375 are the determined cDNA sequences for splice variants of B305D.

SEQ ID NO: 376 is the putative amino acid sequence encoded by the sequence in SEQ ID NO: 366.

SEQ ID NO: 377 is the putative amino acid sequence encoded by the sequence in SEQ ID NO: 372.

SEQ ID NO: 378 is the putative amino acid sequence encoded by the sequence in SEQ ID NO: 373.

SEQ ID NO: 379 is the putative amino acid sequence encoded by the sequence in SEQ ID NO: 374.

SEQ ID NO: 380 is the putative amino acid sequence encoded by the sequence in SEQ ID NO: 375.

SEQ ID NO: 381 is the determined cDNA sequence for B716P. SEQ ID NO: 382 is the determined full-length cDNA sequence for P711P. SEQ ID NO: 383 is the predicted amino acid sequence for P711P. SEQ ID NO: 384 is the cDNA sequence for P1000C.

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

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

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

SEQ ID NO: 388 is the cDNA sequence for L-iditol-2-dehydrogenase. SEQ ID NO: 389 is the cDNA sequence in 23379.

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

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

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

SEQ ID NO: 393 is the cDNA sequence for a previously identified gene. SEQ ID NO: 394 is the cDNA sequence for HCLBP.

SEQ ID NO: 395 is the cDNA sequence for transglutaminase. SEQ ID NO: 396 is the cDNA sequence for a previously identified gene. SEQ ID NO: 397 is the cDNA sequence for PAP.

SEQ ID NO: 398 is the cDNA sequence for Ets transcription factor

PDEF.

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

SEQ ID NO: 400 is the cDNA sequence for KIAA0295. SEQ ID NO: 401 is the cDNA sequence in 22545.

SEQ ID NO: 402 is the cDNA sequence in 22547.

SEQ ID NO: 403 is the cDNA sequence in 22548.

SEQ ID NO: 404 is the cDNA sequence in 22550.

SEQ ID NO: 405 is the cDNA sequence in 22551.

SEQ ID NO: 406 is the cDNA sequence in 22552.

SEQ ID NO: 407 is the cDNA sequence of 22553 (also known as P1020C).

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

SEQ ID NO: 409 is the cDNA sequence in 22562.

SEQ ID NO: 410 is the cDNA sequence in 22565.

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

SEQ ID NO: 412 is the cDNA sequence in 22568.

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

SEQ ID NO: 414 is the cDNA sequence in 22571.

SEQ ID NO: 415 is the cDNA sequence in 22572.

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

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

SEQ ID NO: 418 is the cDNA sequence in 22575.

SEQ ID NO: 419 is the cDNA sequence of 22580.

SEQ ID NO: 420 is the cDNA sequence in 22581.

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

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

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

SEQ ID NO: 424 is the cDNA sequence in 22585.

SEQ ID NO: 425 is the cDNA sequence in 22586.

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

SEQ ID NO: 427 is the cDNA sequence in 22588.

SEQ ID NO: 428 is the cDNA sequence in 22589.

SEQ ID NO: 429 is the cDNA sequence in 22590.

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

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

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

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

SEQ ID NO: 434 is the cDNA sequence in 22595.

SEQ ID NO: 435 is the cDNA sequence in 22596.

SEQ ID NO: 436 is the cDNA sequence in 22847.

SEQ ID NO: 437 is the cDNA sequence in 22848.

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

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

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

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

SEQ ID NO: 442 is the cDNA sequence in 22854.

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

SEQ ID NO: 444 is the cDNA sequence in 22856.

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

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

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

SEQ ID NO: 448 is the cDNA sequence in 23605.

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

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

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

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

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

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

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

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

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

SEQ ID NO: 458 is the cDNA sequence for a previously identified gene. SEQ ID NO: 459 is the cDNA sequence in 23045.

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

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

SEQ ID NO: 462-467 are cDNA sequences for known genes. SEQ ID NO: 468-471 are cDNA sequences for P710P.

SEQ ID NO: 472 is a cDNA sequence for P1001C.

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

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

SEQ ID NO: 475 is the determined cDNA sequence for a third splice variant of P775P (referred to as 19941).

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

SEQ ID NO: 477 is a first putative amino acid sequence encoded by the sequence in SEQ ID NO: 474.

SEQ ID NO: 478 is a second putative amino acid sequence encoded by the sequence in SEQ ID NO: 474.

SEQ ID NO: 479 is the putative amino acid sequence encoded by the sequence in SEQ ID NO: 475.

SEQ ID NO: 480 is a first putative amino acid sequence encoded by the sequence in SEQ ID NO: 473.

SEQ ID NO: 481 is a second putative amino acid sequence encoded by the sequence in SEQ ID NO: 473.

SEQ ID NO: 482 is a third putative amino acid sequence encoded by the sequence in SEQ ID NO: 473.

SEQ ID NO: 483 is a fourth putative amino acid sequence encoded by the sequence in 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 the PCR primer AW003.

SEQ ID NO: 487 is the PCR primer AW027.

SEQ ID NO: 488 is the PCR primer AW026.

SEQ ID NO: 489-501 are peptides used in epitope mapping studies.

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

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

SEQ ID NO: 504 & 505 are peptides used in epitope mapping studies.

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

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

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

SEQ ID NO: 509-522 are peptides used in epitope mapping studies.

SEQ ID NO: 523 is a mature form of P703P used to raise antibodies against P703P.

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

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

SEQ ID NO: 526 is the full-length cDNA sequence for P790P. SEQ ID NO: 527 is the predicted amino acid sequence for P790P. SEQ ID NO: 528 & 529 are PCR primers.

SEQ ID NO: 530 is the cDNA sequence of a splice variant of SEQ ID NO: 366.

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

SEQ ID NO: 532 is the putative amino acid encoded by the sequence in SEQ ID NO: 531.

SEQ ID NO: 533 is the DNA sequence of a putative ORF for P775P.

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

SEQ ID NO: 535 is a first full-length cDNA sequence for P510S. SEQ ID NO: 536 is a second full-length cDNA sequence for P510S.

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

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

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

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

SEQ ID NO: 541-551 erepitoperav P501S.

SEQ ID NO: 552 is an extended cDNA sequence for P712P.

SEQ ID NO: 553-568 are the amino acid sequences encoded by predicted open reading frames in SEQ ID NO: 552.

SEQ ID NO: 569 is an extended cDNA sequence for P776P.

SEQ ID NO: 570 is the determined cDNA sequence for a splice variant of P776P referred to as contig 6.

SEQ ID NO: 571 is the determined cDNA sequence for a splice variant of P776P referred to as contig 7.

SEQ ID NO: 572 is the determined cDNA sequence for a splice variant of P776P referred to as contig 14.

SEQ ID NO: 573 is the amino acid sequence encoded by a first predicted ORF of SEQ ID NO: 570.

SEQ ID NO: 574 is the amino acid sequence encoded by a second predicted ORF of SEQ ID NO: 570.

SEQ ID NO: 575 is the amino acid sequence encoded by a predicted ORF of SEQ ID NO: 571.

SEQ ID NO: 576-586 are amino acid sequences encoded by the predicted ORF of SEQ ID NO: 569.

SEQ ID NO: 587 is a DNA consensus sequence of the sequences in P767P and P777P.

SEQ ID NO: 588-590 are amino acid sequences encoded by the predicted ORF of SEQ ID NO: 587.

SEQ ID NO: 591 is an extended cDNA sequence for P1020C.

SEQ ID NO: 592 is the putative amino acid sequence encoded by the sequence in SEQ ID NO: P1020C.

SEQ ID NO: 593 is a splice variant of P775P referred to as 50748. SEQ ID NO: 594 is a splice variant of P775P referred to as 50717. SEQ ID NO: 595 is a splice variant of P775P referred to as 45985. SEQ ID NO: 596 is a splice variant of P775P referred to as 38769. SEQ ID NO: 597 is a splice variant of P775P referred to as 37922. SEQ ID NO: 598 is a splice variant of P510S referred to as 49274. SEQ ID NO: 599 is a splice variant of P510S referred to as 39487.

SEQ ID NO: 600 is a splice variant of P504S referred to as 5167.16.

SEQ ID NO: 601 is a splice variant of P504S referred to as 5167.1.

SEQ ID NO: 602 is a splice variant of P504S referred to as 5163.46.

SEQ ID NO: 603 is a splice variant of P504S referred to as 5163.42.

SEQ ID NO: 604 is a splice variant of P504S referred to as 5163.34.

SEQ ID NO: 605 is a splice variant of P504S referred to as 5163.17.

SEQ ID NO: 606 is a splice variant of P501S referred to as 10640. SEQ ID NO: 607-615 are the sequences of PCR primers.

SEQ ID NO: 616 is the determined cDNA sequence of a fusion of P703P and PSA.

SEQ ID NO: 617 is the amino acid sequence of the fusion of P703P and

PSA.

SEQ ID NO: 618 is the cDNA sequence of the gene DD3.

SEQ ID NO: 619 is an extended cDNA sequence for P714P.

SEQ ID NO: 620-622 are cDNA sequences for splice variants of P704P.

SEQ ID NO: 623 is the cDNA sequence of a splice variant of P553S referred to as P553S-14.

SEQ ID NO: 624 is the cDNA sequence of a splice variant of P553S referred to as P553S-12.

SEQ ID NO: 625 is the cDNA sequence of a splice variant of P553S referred to as P553S-10.

SEQ ID NO: 626 is the cDNA sequence of a splice variant of P553S referred to as P553S-6.

SEQ ID NO: 627 is the amino acid sequence encoded by SEQ ID NO: 626.

SEQ ID NO: 628 is a first amino acid sequence encoded by SEQ ID NO: 623.

SEQ ID NO: 629 is a second amino acid sequence encoded by SEQ ID NO: 623.

SEQ ID NO: 630 is a first full-length cDNA sequence for the prostate-specific transglutaminase gene (also referred to herein as P558S).

SEQ ID NO: 631 is a second full-length prostate-specific transglutaminase gene cDNA sequence.

SEQ ID NO: 632 is the amino acid sequence encoded by the sequence in SEQ ID NO: 630.

SEQ ID NO: 633 is the amino acid sequence encoded by the sequence in SEQ ID NO: 631.

SEQ ID NO: 634 is the full-length cDNA sequence for P788P.

SEQ ID NO: 635 is the amino acid sequence encoded by SEQ ID NO: 634.

SEQ ID NO: 636 is the determined cDNA sequence for a polymorphic variant of P788P.

SEQ ID NO: 637 is the amino acid sequence encoded by SEQ ID NO: 636.

SEQ ID NO: 638 is the amino acid sequence of peptide 4 from P703P.

SEQ ID NO: 639 is the cDNA sequence encoding peptide 4 from P703P.

SEQ ID NO: 640-655 are cDNA sequences encoding epitopes of P703P.

SEQ ID NO: 656-671 are the amino acid sequences of epitopes of P703P. SEQ ID NO: 672 and 673 are PCR primers.

SEQ ID NO: 674 is the cDNA sequence encoding an N-terminal portion of P788P expressed in E. coli.

SEQ ID NO: 675 is the amino acid sequence of the N-terminal part of P788P expressed in E. coli.

SEQ ID NO: 676 is the amino acid sequence of the M. tuberculosis antigen Ra12.

SEQ ID NO: 677 and 678 are PCR primers.

SEQ ID NO: 679 is the cDNA sequence for the Ra12-P510S-C construct.

SEQ ID NO: 680 is the cDNA sequence for the P510S-C construct. SEQ ID NO: 681 is the cDNA sequence for the P510S-E3 construct.

SEQ ID NO: 682 is the amino acid sequence of the Ra12-P510S-C construct.

SEQ ID NO: 683 is the amino acid sequence of the P510S-C construct.

SEQ ID NO: 684 is the amino acid sequence of the P510S-E3 construct.

SEQ ID NO: 685-690 are PCR primers.

SEQ ID NO: 691 is the cDNA sequence of the construct Ra12-P775P-ORF3.

SEQ ID NO: 692 is the amino acid sequence of construct Ra12-P775P-ORF3.

SEQ ID NO: 693 and 694 are PCR primers.

SEQ ID NO: 695 is the determined amino acid sequence of a P703P His-tag fusion protein.

SEQ ID NO: 696 is the determined cDNA sequence for a P703P His-tag fusion protein.

SEQ ID NO: 697 and 698 are PCR primers.

SEQ ID NO: 699 is the determined amino acid sequence of the P705P His-tag fusion protein.

SEQ ID NO: 700 is the determined cDNA sequence for a P705P His-tag fusion protein.

SEQ ID NO: 701 and 702 are PCR primers.

SEQ ID NO: 703 is the determined amino acid sequence of a P711P His-tag fusion protein.

SEQ ID NO: 704 is the determined cDNA sequence for a P711P His-tag fusion protein.

SEQ ID NO: 705 is the amino acid sequence of M. tuberculosis antigen Ra12.

SEQ ID NO: 706 and 707 are PCR primers.

SEQ ID NO: 708 is the determined cDNA sequence for construct Ra12-P501S-E2.

SEQ ID NO: 709 is the determined amino acid sequence of construct Ra12-P501S-E2.

SEQ ID NO: 710 is the amino acid sequence of an epitope of P501S. SEQ ID NO: 711 is the DNA sequence encoding SEQ ID NO: 710. SEQ ID NO: 712 is the amino acid sequence of an epitope of P501S. SEQ ID NO: 713 is the DNA sequence encoding SEQ ID NO: 712.

SEQ ID NO: 714 is a peptide used in epitope mapping studies.

SEQ ID NO: 715 is the amino acid sequence of an epitope of P501S. SEQ ID NO: 716 is the DNA sequence encoding SEQ ID NO: 715.

SEQ ID NO: 717-719 are the amino acid sequences of CD4 epitopes of P501S.

SEQ ID NO: 720-722 are the DNA sequences that encode the sequences in SEQ ID NO: 717-719.

SEQ ID NO: 723-734 are the amino acid sequences of putative CTL epitopes of P703P.

SEQ ID NO: 735 is the full-length cDNA sequence for P789P.

SEQ ID NO: 736 is the amino acid sequence encoded by SEQ ID NO: 735.

SEQ ID NO: 737 is the determined full-length cDNA sequence for the splice variant of P776P referred to as contig 6.

SEQ ID NO: 738-739 are determined full-length cDNA sequences for the splice variant of P776P referred to as contig 7.

SEQ ID NO: 740-744 are amino acid sequences encoded by SEQ ID NO: 737.

SEQ ID NO: 745-750 are amino acid sequences encoded by the splice variant of P776P referred to as contig 7.

SEQ ID NO: 751 is the full-length cDNA sequence for human transmembrane protease serine 2.

SEQ ID NO: 752 is the amino acid sequence encoded by SEQ ID NO: 751.

SEQ ID NO: 753 is the cDNA sequence encoding the first 209 amino acids of human transmembrane protease serine 2.

SEQ ID NO: 754 is the first 209 amino acids of human transmembrane protease serine 2.

SEQ ID NO: 755 is the amino acid sequence of peptide 296-322 of P501S. SEQ ID NO: 756-759 are PCR primers.

SEQ ID NO: 760 is the determined cDNA sequence of the Vb chain of a T-cell receptor for the P501S-specific T-cell clone 4E5.

SEQ ID NO: 761 is the determined cDNA sequence of the Va chain of a T-cell receptor for the P501S-specific T-cell clone 4E5.

SEQ ID NO: 762 is the amino acid sequence encoded by SEQ ID NO: 760.

SEQ ID NO: 763 is the amino acid sequence encoded by SEQ ID NO: 761.

SEQ ID NO: 764 is the full-length open reading frame for P768P including the stop codon.

SEQ ID NO: 765 is the full-length open reading frame for P768P without a stop codon.

SEQ ID NO: 766 is the amino acid sequence encoded by SEQ ID NO: 765.

SEQ ID NO: 767-772 are the amino acid sequences of predicted domains of P768P.

SEQ ID NO: 773 is the full-length cDNA sequence of P835P.

SEQ ID NO: 774 is the cDNA sequence of the previously identified clone FLJ13581.

SEQ ID NO: 775 is the cDNA sequence of the open reading frame for P835P with a stop codon.

SEQ ID NO: 776 is the cDNA sequence of the open reading frame for P835P without a stop codon.

SEQ ID NO: 777 is the full-length amino acid sequence of P835P.

SEQ ID NO: 778-785 are the amino acid sequences of extracellular and intracellular domains of P835P.

SEQ ID NO: 786 is the full-length cDNA sequence for P1000C.

SEQ ID NO: 787 is the cDNA sequence of the open reading frame for P1000C, including the stop codon.

SEQ ID NO: 788 is the cDNA sequence of the open reading frame for P1000C, without a stop codon.

SEQ ID NO: 789 is the full-length amino acid sequence of P1000C. SEQ ID NO: 790 is amino acids 1-100 of SEQ ID NO: 789.

SEQ ID NO: 791 is amino acids 100-492 of SEQ ID NO: 789.

SEQ ID NO: 792 is the amino acid sequence of an α prepro-P501S recombinant protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to preparations and their use in therapy and diagnosis of cancer, especially prostate cancer. As described further below, illustrative preparations according to the present invention include, but are not limited to, polypeptides, especially immunogenic polypeptides, polynucleotides that code for such polypeptides, antibodies and other binders, antigen-presenting cells (APC) and immune system cells { e.g. T cells).

The practice of the present invention will employ, unless otherwise specifically indicated, conventional methods in virology, immunology, microbiology, molecular biology and recombinant DNA techniques known to those skilled in the art, many of which are described below for illustrative purposes. Such techniques are fully explained in the literature. See, e.g. Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd Ed., 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

All publications, patents and patent applications set forth herein, above or below are hereby incorporated by reference in their entirety.

As used in this specification and the following claims, the singular forms "a", "the" and "the" include plural forms unless the context clearly dictates otherwise.

Polypeptide preparations

As used herein, the term "polypeptide" is used in its conventional sense, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides and proteins are encompassed within the definition of polypeptide and such terms may are used interchangeably herein unless specifically indicated otherwise. This term does not refer to or exclude post-expression modifications of the polypeptide, such as glycosylations, acetylations, phosphorylations, and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein or a subsequence thereof. Particular polypeptides of interest in the context of the present invention are amino acid subsequences comprising epitopes, i.e. antigenic determinants mainly responsible for the immunogenic properties of a polypeptide and which can trigger an immune response.

Particularly illustrative polypeptides according to the present invention include those encoded by a polynucleotide sequence set forth in any of SEQ ID NOS: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328,330,332-335 , 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569- 572, 587, 591, 593-606, 618-626, 630, 631, 634 , 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788 or a sequence that hybridizes under moderately stringent conditions, or, alternatively, under very stringent conditions, to a polynucleotide sequence set forth in any of SEQ ID NOS: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788. In specific embodiments, the polypeptides of the present invention comprise amino acid sequences as set forth in any 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, 537-551, 553-568, 573-586, 588-590, 592, 627-629, 632, 633, 635, 637, 638, 656-671, 675, 683, 684, 710, 712, 714, 715, 717-719, 723-734, 736, 740-750, 752, 754, 755, 766-772, 777-785 and 789- 791.

The polypeptides of the present invention are sometimes referred to herein as prostate-specific proteins or prostate-specific polypeptides, as an indication that their identification is based at least in part on their increased levels of expression in prostate tissue samples. Thus, "prostate-specific polypeptide" or "prostate-specific protein," generally refers to a polypeptide sequence according to the present invention or a polynucleotide sequence encoding such a polypeptide, which is expressed in a substantial proportion of prostate tissue samples, for example preferably over approx. . 20%, more preferably over approx. 30% and most preferred over approx. 50% or more of prostate tissue samples tested, at a level that is at least two-fold, and preferably at least five-fold, greater than the level of expression in other normal tissues, as determined using a representative experiment set forth herein. A prostate-specific polypeptide sequence of the present invention, based on its increased level of expression in tumor cells, has particular utility both as a diagnostic marker as well as a therapeutic target, as further described below.

In certain preferred embodiments, the polypeptides of the present invention are immunogenic, i.e. they react detectably in an immunoassay (such as ELISA or T-cell stimulation assay) with antisera and/or T-cells from a patient with prostate cancer. Screening for immunogenic activity can be performed using techniques well known to those skilled in the art. For example, such screenings can be performed using methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one illustrative example, a polypeptide can be immobilized on a solid support and contacted with a patient -sera to allow binding of antibodies in the sera to the immobilized polypeptide. Unbound sera can then be removed and bound antibodies detected using, for example, 125l-labelled Protein A.

As will be understood by those skilled in the art, immunogenic parts of the polypeptides described herein are also encompassed by the present invention. An "immunogenic part" as used here is a fragment of an immunogenic polypeptide according to the present invention which is itself immunologically reactive (i.e. specifically binds) with B cells and/or T cell surface antigen receptors that recognize the polypeptide. Immunogenic moieties can generally be identified using well-known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are "antigen-specific" if they specifically bind to an antigen (ie, they react with the protein in an ELISA or other immunoassay and do not detectably react with unrelated proteins). Such antisera and antibodies can be prepared as described herein and using well-known techniques.

In one preferred embodiment, an immunogenic portion of a polypeptide according to the present invention is a portion that reacts with antisera and/or T cells at a level not substantially less than the reactivity of the full-length polypeptide (e.g. in ELISA and /or T-cell reactivity test). Preferably, the level of immunogenic activity of the immunogenic portion is at least about 50%, preferably at least approx. 70% and most preferred over approx. 90% of the immunogenicity of the full-length polypeptide. In some cases, preferred immunogenic portions will be identified that have a level of immunogenic activity greater than that of the corresponding full-length polypeptide, e.g. which has more than approx. 100% or 150% or more immunogenic activity.

In certain other embodiments, illustrative immunogenic moieties may comprise peptides in which an N-terminal leader sequence and/or transmembrane domain has been deleted. Other illustrative immunogenic parts will contain a small N- and/or C-terminal deletion (eg 1-30 amino acids, preferably 5-15 amino acids), relative to the mature protein.

In another embodiment, a polypeptide preparation according to the present invention may also comprise one or more polypeptides which are immunologically reactive with T cells and/or antibodies formed against a polypeptide according to the present invention, in particular a polypeptide which has an amino acid sequence described here or an immunogenic fragment or variant thereof.

In another embodiment of the invention, polypeptides are provided that comprise one or more polypeptides that can induce T cells and/or antibodies that are immunologically reactive with one or more polypeptides described here or one or more polypeptides encoded for by subsequent nucleic acid sequences contained in the polynucleotide sequences described here or immunogenic fragments or variants thereof or one or more nucleic acid sequences that hybridize to one or more of these sequences under conditions of moderate to high stringency.

The present invention provides in another aspect, polypeptide fragments comprising at least approx. 5, 10, 15, 20, 25, 50 or 100 consecutive amino acids or more, including all intermediate lengths, of a polypeptide preparation set forth herein, as set forth in 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, 537-551, 553-568, 573-586, 588 -590, 592, 627-629, 632, 633, 635, 637, 638, 656-671, 675, 683, 684, 710, 712, 714, 715, 717-719, 723-734, 736, 740-750 , 752, 754, 755, 766-772, 777-785 and 789-791 or those encoded by a polynucleotide sequence indicated in a sequence in SEQ ID NO: 1-111,115-171,173-175, 177, 179-305, 307 315, 326, 328, 330, 332-335, 340-375, 381, 382 and

384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788.

In another aspect, the present invention provides variants of the polypeptide preparations described herein. Polypeptide variants generally covered by the present invention will typically exhibit at least approx. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below) , along its length, to a polypeptide sequence set forth herein.

In one preferred embodiment, the polypeptide fragments and variants provided by the present invention are immunologically reactive with an antibody and/or T cell that reacts with a full-length polypeptide specifically set forth herein.

In another preferred embodiment, the polypeptide fragments and variants provided by the present invention exhibit a level of immunogenic activity of at least approx. 50%, preferably at least approx. 70% and most preferably at least approx. 90% or more of that shown by a full-length polypeptide sequence specifically set forth herein.

A polypeptide "variant," as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically described herein by one or more substitutions, deletions, additions, and/or insertions. Such variants may be naturally occurring or may be synthetically formed, for example by modification of one or more of the above polypeptide sequences of the present invention, and evaluation of their immunogenic activity as described herein may be performed using any of several techniques well known in the art .

For example, certain illustrative variants of the polypeptides of the present invention include those where one or more parts, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other illustrative variants include variants where a small part (eg 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminus of the mature protein.

In many cases, a variant will contain conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced by another amino acid having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications can be made in the structure of the polynucleotides and polypeptides according to the present invention while still obtaining a functional molecule which codes for a variant or derivative polypeptide with desirable characteristics, e.g. with immunogenic characteristics. When it is desired to change the amino acid sequence of a polypeptide to create an equivalent or even an improved, immunogenic variant or part of a polypeptide according to the present invention, those skilled in the art will typically change one or more of the codons in the coding DNA sequence according to Table 1.

For example, certain amino acids can be replaced by other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence and, of course, its underlying DNA coding sequence, still obtaining a protein with equal properties. It is thus assumed that various changes can be made in the peptide sequences of the described preparations or corresponding DNA sequences that code for said peptides without noticeable loss of their biological utility or activity.

When carrying out such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in transferring interactive biological function to a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relatively hydropathic character of the amino acid contributes to the secondary structure of the resulting protein, which then defines the interaction of the protein with other molecules, for example enzymes, substrates, receptors, DNA, antibodies, antigens and the like. Each amino acid is assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1,3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is known in the art that certain amino acids can be replaced with other amino acids that have a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. a biologically functionally equivalent protein is still obtained. When such changes are made, substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that substitution of like amino acids can be done efficiently on the basis of hydrophilicity. U.S. Patent 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Patent 4,554,101, they are as follows

hydrophilicity values assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1,3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2,3); phenylalanine (-2.5); tryptophan (-3,4). It will be understood that an amino acid can be replaced with another that has a similar hydrophilicity value, still obtaining a biological equivalent and, in particular, an immunologically equivalent protein. In such changes, substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred and those within +0.5 are even more particularly preferred.

As described above, amino acid substitutions are therefore generally based on the relative similarity of amino acid side chain substituents, for example their hydrophobicity, hydrophilicity, charge, size and the like. Examples of substitutions that take into account the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

In addition, any polynucleotide can be further modified to increase stability in vivo. Possible modifications include, but are not limited to, addition of flanking sequences at the 5' and/or 3' ends; use of phosphorothioate or 2' O -methyl instead of phosphodiesterase linkages in the backbone; and/or inclusion of non-traditional bases such as inosine, queosine and wybutosine, as well as acetyl-methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Amino acid substitutions can further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine, and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) light, angry, hiss; and (5) phe, bull, trp, his. A variant may also or alternatively contain non-conservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion, or addition of five or fewer amino acids. Variants can also (or alternatively) be modified by, for example, deletion or addition of amino acids that have minimal impact on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

As indicated above, polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide can also be conjugated to a linker or other sequence to facilitate synthesis, purification or identification of the polypeptide (eg poly-His) or to improve binding of the polypeptide to a solid support. For example, a polypeptide can be conjugated to an immunoglobulin Fc region.

When comparing polypeptide sequences, two sequences are said to be "identical" if the sequence of amino acids in the two sequences is the same when aligned for maximum agreement, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein denotes a segment of at least approx. 20 consecutive positions, usually 30 to approx. 75, 40 to approx. 50, where a sequence can be compared with a reference sequence with the same number of consecutive positions after the two sequences have been optimally arranged.

Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene series of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program includes many layout forms described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M.

(1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E.D. (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J. (1983) Proe. Nati. Acad., Sei. USA 80:726-730.

Alternatively, optimal arrangement of sequences for comparison can be performed by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity-formation algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by searching for the similarity methods of Pearson and Lipman (1988) Proe. Nati. Acad. Pollock. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wl) or by inspection.

A preferred example of algorithms suitable for determining percent sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described respectively in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST and BLAST 2.0 can, for example, be used with the parameters described here, to determine percent sequence identity for the polynucleotides and polypeptides according to the present invention. Software for performing BLAST analyzes is widely available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Expansion of the word hits in each direction is halted when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative scoring residual lineups; or the end of one of the sequences is reached. BLAST algorithm parameters W, T and X determine the sensitivity and speed of the array.

In one preferred method, "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polypeptide sequence in the comparison window may include additions or deletions [ie, gaps) of 20 percent or less, typically 5 to 15 percent or 10 to 12 percent, compared to the reference sequences (which do not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences, which gives the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100, which gives percent sequence identity.

In other illustrative embodiments, a polypeptide may be a fusion polypeptide comprising multiple polypeptides as described herein or comprising at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. For example, a fusion partner can assist in providing T-helper epitopes (an immunological fusion partner), preferably T-helper epitopes recognized by humans or can assist in expressing the protein (an expression enhancer) in higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners can be chosen to increase the solubility of the polypeptide or enable the polypeptide to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the polypeptide.

Fusion polypeptides can generally be prepared using

standard techniques, including chemical conjugation. Preferably, a fusion polypeptide is expressed as a recombinant polypeptide, which allows the production of increased levels, relative to a non-fused polypeptide, in an expression system. Short, DNA sequences that code for

the polypeptide components can be assembled separately and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the other polypeptide component so that the reading frames of the sequences are in phase . This allows translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.

A peptide linker sequence can be used to separate the first and

other polypeptide components at a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is introduced into the fusion polypeptide using standard techniques well known in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that could interact with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala can also be used in the binding sequences. Amino acid sequences that can suitably be used as linkers include those described in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proe. Nati. Acad. Pollock. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Pat. Patent No. 4,751,180. The linker sequences can generally be from 1 to approx. 50 amino acids in length. Linker sequences are not necessary when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

The ligated DNA sequences are operably linked to appropriate transcriptional or translational regulatory elements. The regulatory elements responsible for expression of the DNA are located just 5' to the DNA sequence that codes for the first polypeptides. Similarly, stop codons are necessary to terminate translation and transcription termination signals only present 3' to the DNA sequence encoding the second polypeptide.

The fusion polypeptide may comprise a polypeptide as described herein together with an unrelated immunogenic protein, such as an immunogenic protein capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-derived Ra12 fragment. Ra12 compositions and methods of using them to improve the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences are described in U.S. Pat. patent application 60/158,585, the description therein being incorporated herein by reference in its entirety. Briefly, Ra12 denotes a polynucleotide region which is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is a serine protease with a molecular weight of 32 KD encoded by a gene in virulent and avirulent strains of M. tuberculosis. The nucleotide sequence and amino acid sequence of MTB32A have been described (for example, U.S. Patent Application 60/158,585; see also, Skeiky et al., Infection and Immun. (1999) 67:3998-4007, incorporated herein by reference). C-terminal fragments of the MTB32A coding sequence are expressed at high levels and remain as soluble polypeptides throughout the purification process. Furthermore, Ra12 can enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One preferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ra12 polynucleotides generally comprise at least about 15 consecutive nucleotides, at least approx. 30 nucleotides, at least approx. 60 nucleotides, at least approx. 100 nucleotides, at least approx. 200 nucleotides or at least approx. 300 nucleotides that encode part of a Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence (ie an endogenous sequence which codes for a Ra 12 polypeptide or part thereof) or may comprise a variant of such a sequence. Ra12 polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions so that the biological activity of the encoded fusion polypeptide is not significantly reduced, compared to a fusion polypeptide comprising a native Ra 12 polypeptide. Variants preferably exhibit at least approx. 70% identity, more preferably at least approx. 80% identity and most preferably at least approx. 90% identity to a polynucleotide sequence encoding a native Ra12 polypeptide or part thereof.

In other preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (eg the first N-terminal 100-110 amino acids) and a protein D derivative may be lipidated. In certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner are included at the N-terminus to provide a polypeptide with additional exogenous T-cell epitopes and to increase the level of expression in E. coli (thus acting as an expression -enhancer). The lipid tail ensures optimal presentation of the antigen to antigen-presenting cells. Other fusion partners include the non-structural protein of influenzae virus, NS1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, although various fragments comprising T-helper epitopes can be used.

In another embodiment, the immunological fusion partner is the protein known as LYTA or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292,1986). LYTA is an autolysin that specifically breaks down certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to choline or to some choline analogues such as DEAE. This property is exploited for the development of E. coli C-LYTA expressing plasmids useful for the expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 70:795-798,1992). In a preferred embodiment, a repetitive portion of LYTA can be introduced into a fusion polypeptide. A repetitive part is found in the C-terminal region starting at residue 178. A particularly preferred repeating part comprises residues 188-305.

Yet another illustrative embodiment comprises fusion polypeptides and the polynucleotides encoding them, wherein the fusion partner comprises a targeting signal capable of directing a polypeptide to the endosomal/lysosomal compartment, as described in U.S. Pat. Patent No. 5,633,234. An immunogenic polypeptide according to the present invention will, when condensed with this targeting signal, associate more effectively with MHC class II molecules and thereby provide improved in vivo stimulation of CD4<+>T cells specific for the polypeptide.

Polypeptides according to the present invention are produced using any of a number of well-known synthetic and/or recombinant techniques, the latter being further described below. Polypeptides, parts and other variants generally less than approx. 150 amino acids can be formed by synthetic methods, using techniques well known to those skilled in the art. In one illustrative example, such polypeptides are synthesized using any of the commercially available solid phase techniques, such as the Merrifield solid phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for the automated synthesis of polypeptides is commercially available from suppliers such as Perkin Eimer/Applied BioSystems Division (Foster City, CA) and can be operated according to the manufacturer's instructions.

In general, polypeptide preparations (including fusion polypeptides) according to the present invention are isolated. An "isolated" polypeptide is one that is removed from its original environment. For example, a naturally occurring protein or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are also purified, e.g. at least approx. 90% pure, more preferably at least approx. 95% pure and most preferably at least approx. 99% pure.

Polynucleotide Preparations

The present invention provides, in other aspects, polynucleotide preparations. The terms "DNA" and "polynucleotide" are used essentially interchangeably herein to refer to a DNA molecule isolated free of total genomic DNA from a particular species. "Isolated," as used herein, means that a polynucleotide is substantially absent from other coding sequences and that the DNA molecule does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional gene or polypeptide coding regions. Of course, this indicates the DNA molecule as originally isolated and does not exclude genes or coding regions later added to the segment by human hands.

As will be understood by those skilled in the art, the polynucleotide preparations according to the present invention may comprise genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or can be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated or modified synthetically by humans.

As will also be known to those skilled in the art, polynucleotides according to the present invention can be single-stranded (coding or antisense) or double-stranded and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules can include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequence genes may, but need not, be present in a polynucleotide according to the present invention and a polynucleotide may, but need not, be bound to other molecules and/or carrier materials.

Polynucleotides may comprise a native sequence (i.e. an endogenous sequence which codes for a polypeptide/protein according to the present invention or a part thereof) or may comprise a sequence which codes for a variant or derivative, preferably an immunogenic variant or derivative, of such sequence.

According to another aspect of the present invention, polynucleotide preparations are provided which comprise some or all of a polynucleotide sequence indicated in any of SEQ ID NOS: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326 , 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618 -626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786 -788, complements of a polynucleotide sequence set forth in any of SEQ ID NOS: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340 -375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636 , 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788 and degenerate variants of a polynucleotide sequence set forth in any of SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788. In certain preferred embodiments, the polynucleotide sequences set forth herein encode immunogenic polypeptides, as described above.

In other related embodiments, the present invention provides polynucleotide variants that have substantial identity to the sequences described herein in SEQ ID NOS: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330 , 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630 , 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788, for for example those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher, sequence identity compared to a polynucleotide sequence according to the present invention using the methods described here, (e.g. BLAST analysis using standard parameters, as described below). Those skilled in the art will appreciate that these values can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

Typically, polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably so that the immunogenicity of the polypeptide encoded by the variant polynucleotide is not significantly reduced compared to a polypeptide encoded by a polynucleotide sequence specifically indicated here). The term "variants" shall also be understood to include homologous genes of xenogenic origin.

In further embodiments, the present invention provides polynucleotide fragments comprising different lengths of consecutive stretches of sequences identical to, or complementary to, one or more of the sequences described herein. For example, polynucleotides are provided by the present invention which comprise at least approx. 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more consecutive nucleotides of one or more of the sequences described herein as well as all intermediate lengths therebetween. It will be readily understood that "intermediate lengths", in this context, means any length between the specified values, such as 16,17,18,19, etc; 21,22, 23, eat.; 30, 31,32, eat.; 50, 51,52, 53, eat.; 100, 101, 102, 103, ete.; 150, 151, 152,153, ete.; comprising all whole numbers from 200-500; 500-1000 and the like.

In another embodiment of the invention, polynucleotide preparations are provided which can hybridize under moderate to high stringency conditions to a polynucleotide sequence given here or a fragment thereof or a complementary sequence thereof. Hybridization techniques are well known in the field of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of the present invention with other polynucleotides include pre-washing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50°C-60°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes each with 2X, 0.5X and 0.2X SSC containing 0.1% SDS. Those skilled in the art will appreciate that the stringency of hybridization can be easily manipulated, such as by changing the salt content of the hybridization solution and/or the temperature at which the hybridization is performed. In another embodiment, for example, suitable very stringent hybridization conditions include those described above, with the exception that the temperature of the hybridization is increased, e.g. to 60-65°C or 65-70°C.

In certain preferred embodiments, the polynucleotides described above encode, e.g. polynucleotide variants, fragments and hybridization sequences, for polypeptides that are immunologically cross-reactive with a polypeptide sequence specifically set forth herein. In other preferred embodiments, such polynucleotides encode polypeptides having a level of immunogenic activity of at least about 50%, preferably at least approx. 70% and more preferably at least approx. 90% of that for a polypeptide sequence specifically set forth herein.

The polynucleotides according to the present invention or fragments thereof, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments and the like, so that their total length can vary considerably . It is therefore believed that a nucleic acid fragment of almost any length can be used, the total length being preferably limited by ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative polynucleotide segments with total lengths of about 10,000, approx. 5000, approx. 3000, approx. 2,000, approx. 1,000, approx. 500, approx. 200, approx. 100, approx. 50 base pairs in length and the like, (including all intermediate lengths) believed to be applicable in many implementations of the present invention.

When comparing polynucleotide sequences, two sequences are said to be "identical" if the sequence of nucleotides in the two sequences is the same when aligned for maximum agreement, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein denotes a segment of at least approx. 20 consecutive positions, usually 30 to approx. 75, preferably 40 to approx. 50, where a sequence can be compared with a reference sequence with the same number of consecutive positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene series of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program includes many layout forms described in the following references: Dayhoff, M.O. (1978) A model for evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O.

(ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignments and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M.

(1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E.D. (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J. (1983) Proe. Nati. Acad., Sei. USA 80:726-730.

Alternatively, optimal arrangement of sequences for comparison can be performed by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by identity the array algorithm of Needleman and Wunsch

(1970) J. Mol. Biol. 48:443, when searching for the similahtet methods of Pearson and Lipman (1988) Proe. Nati. Acad. Pollock. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wl) or by inspection.

A preferred example of algorithms suitable for determining percent sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described respectively in Altschul et al. (1977) A/wc/. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST and BLAST 2.0 can, for example, be used with the parameters described here, to determine percent sequence identity for the polynucleotides according to the present invention. Software for performing BLAST analyzes is publicly available from the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Expansion of the word hits in each direction is halted when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative scoring backstop positions; or the end of one of the sequences is reached. BLAST algorithm parameters W, T and X determine the sensitivity and speed of the array. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11 and expectation (E) of 10 and BLOSUM62 scoring matrix (see Henikoff and Henikoff

(1989) Proe. Nati. Acad. Pollock. USA 89:10915) lineups, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both threads.

Preferably, "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide sequence in the comparison window may include additions or deletions {ie, gaps) of 20 percent or less, typically 5 to 15 percent or 10 to 12 percent, compared to the reference sequences (which do not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which the identical nucleic acid bases occur in both sequences, which gives the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100, giving the percentage of sequence identity.

It will be understood by those skilled in the art that, as a result of degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of a native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically encompassed by the present invention. Furthermore, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that have been altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).

In another embodiment of the invention, a mutagenesis method, such as site-specific mutagenesis, is therefore used to produce immunogenic variants and/or derivatives of the polypeptides described here. By this method, specific modifications in a polypeptide sequence can be produced by mutagenesis of the underlying polynucleotides that code for them. These techniques provide a simple method for producing and testing sequence variants, for example comprising one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.

Site-specific mutagenesis allows the production of mutants using specific oligonucleotide sequences encoding the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations can be used in a selected polynucleotide sequence to improve, change, reduce, modify or otherwise change the properties of the polynucleotide itself and/or change the properties, activity, composition, stability or primary sequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventors contemplate mutagenesis of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide, such as the immunogenicity of a polypeptide vaccine. The techniques of site-specific mutagenesis are well known in the art and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to change a specific part of a DNA molecule. In such embodiments, a primer comprising typically approx. 14 to approx. 25 nucleotides or so in length used, with approx. 5 to approx. 10 residues on both sides of the junction of the sequence are changed.

As will be appreciated by those skilled in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as M13 phage. These subjects are readily commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely used in site-directed mutagenesis, which eliminates the step of transferring the relevant gene from a plasmid to a phage.

In general, site-directed mutagenesis in accordance with this is carried out by first obtaining a single-stranded vector or fusing two strands of a double-stranded vector which comprises in its sequence a DNA sequence encoding the desired peptide. An oligonucleotide primer carrying the desired mutated sequence is prepared, generally synthetically. This primer is then hybridized with the single-stranded vector and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, to completely synthesize the mutation-carrying strand. Thus, a heteroduplex is formed where one strand codes for the original non-mutated sequence and the other strand carries the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that comprise recombinant vectors carrying the mutated sequence arrangement.

Production of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides one method of producing potentially useful species and is not intended to be limiting, as there are other methods in which sequence variants of peptides and the DNA sequences encoding they can be achieved. For example, recombinant vectors encoding the desired peptide sequence can be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segel, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.

As used herein, the term "oligonucleotide-directed mutagenesis procedure" denotes template-dependent processes and vector-mediated propagation that result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide-directed mutagenesis procedure" shall refer to a procedure involving template-dependent extension of a primer molecule. The term template-dependent process denotes nucleic acid synthesis of an RNA or a DNA molecule where the sequence of the newly synthesized strand of the nucleic acid is dictated by the well-known rules for complementary base pairing (see for example Watson, 1987). Typically, vector-mediated methods involve the introduction of the nucleic acid fragment into a DNA or RNA vector, clonal amplification of the vector, and recovery of the amplified nucleic acid fragment. Examples of such methods are provided in U.S. Patent No. 4,237,224, specifically incorporated herein by reference in its entirety.

In another method for producing polypeptide variants according to the present invention, recursive sequence recombination, as described in U.S. Pat. patent no. 5,837,458, is used. In this method, repeated cycles of recombination and screening or selection are carried out to "develop" individual polynucleotide variants according to the present invention which, for example, have improved immunogenic activity.

In other embodiments of the present invention, the polynucleotide sequences given here can advantageously be used as probes or primers for nucleic acid hybridization. As such, it is assumed that nucleic acid segments comprising a sequence region of at least approx. 15 consecutive nucleotides having the same sequence as, or complementary to, a 15 nucleotide long contiguous sequence described herein will be of particular use. Longer consecutive identical or complementary sequences, e.g. those with approx. 20, 30, 40, 50,100, 200, 500,1000 (including all intermediate lengths) and even up to full-length sequences, will also be applicable in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to a sequence of interest will make them useful for detecting the presence of complementary sequences in a given sample. However, other uses are also envisaged, such as use of the sequence information for the production of mutant-species primers or primers for use in the production of other genetic constructs.

Polynucleotide molecules having sequence regions consisting of consecutive nucleotide stretches of 10-14, 15-20, 30, 50 or even 100-200 nucleotides or so (including intermediate lengths), identical or complementary to a polynucleotide sequence described herein, are particularly intended as hybridization probes for use in, e.g. Southern and Northern blotting. This would allow a gene product or fragment thereof to be analysed, both in various cell types and also in different bacterial cells. The overall size of the fragment, as well as the size of the complementary stretch(s), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments where the length of the subsequent complementary region can be varied, such as between about 15 and approx. 100 nucleotides, while larger consecutive complementary stretches can be used, according to the length of complementary sequences one wishes to detect.

Application of a hybridization probe of approx. 15-25 nucleotides in length allow the formation of a duplex molecule that is both stable and selective. Molecules which have consecutive complementary sequences over stretches greater than 15 bases in length are nevertheless generally preferred, in order to increase stability and selectivity of the hybrid and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 25 consecutive nucleotides or even longer if desired.

Hybridization probes can be selected from any portion of any of the sequences described herein. All that is necessary is to go through the sequences given here or any continuous part of the sequences, from approx. 15-25 nucleotides in length up to and including the full-length sequence, which one wishes to use as a probe or primer. The choice of probe and primer sequences can be controlled by various factors. For example, one may wish to use primers from towards the termini of the overall sequence.

Small polynucleotide segments or fragments can be easily prepared by, for example, direct synthesis of the fragment by chemical means, which is commonly practiced using an automated oligonucleotide synthesizer. Fragments can also be obtained using nucleic acid reproduction technology, such as the PCR™ technology of U.S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those skilled in the art in the field of molecular biology.

The nucleotide sequences according to the present invention can be used for their ability to selectively form duplex molecules with complementary stretches of whole genes or gene fragments of interest. Depending on the intended application, one will typically wish to use varying hybridization conditions to achieve varying degrees of selectivity of probe to target sequence. For applications that require high selectivity, one will typically want to use relatively stringent conditions to form the hybrids, e.g. will one choose relatively low-salt and/or high-temperature conditions, such as given by a salt concentration of from approx. 0.02 M to approx. 0.15 M salt at temperatures of from approx. 50°C to approx. 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand and will be particularly suitable for isolating related sequences.

Of course, for some applications, for example when one wishes to produce mutants using a mutant primer strand hybridized to an underlying template, less stringent (reduced stringency) hybridization conditions will typically be required to allow heteroduplex formation. Under these circumstances, one may wish to use salt conditions such as those on from approx. 0.15 M to approx. 0.9 M salt, at temperatures in the range from approx. 20°C to approx. 55°C. Cross-hybridization species can thereby be easily identified as positive hybridization signals with regard to control hybridizations. In all cases, it is generally understood that the conditions can be made more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same way as increased temperature. Thus, hybridization conditions can be easily manipulated and will thus generally be a method of choice depending on the desired results.

According to another embodiment of the invention, polynucleotide preparations comprising antisense oligonucleotides are provided. Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis and thus provide a therapeutic method by which a disease can be treated by inhibiting the synthesis of proteins that contribute to the disease. The effectiveness of antisense oligonucleotides in inhibiting protein synthesis is well established. For example, synthesis of polygalactauronase and muscarinic type 2 acetylcholine receptor is inhibited by antisense oligonucleotides directed against their respective mRNA sequences (U.S. Patent 5,739,119 and U.S. Patent 5,759,829). Furthermore, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptor and human EGF (Jaskulski et al., Science. June 10 1988;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun 15;57(2): 310-20; U.S. Patent 5,801,154; U.S. Patent 5,789,573; U.S. Patent 5,718,709 and U.S. Patent 5,610,288). Antisense constructs are also described as inhibiting and can be used to treat a variety of abnormal cellular proliferations, e.g. cancer (U.S. Patent 5,747,470; U.S. Patent 5,591,317 and U.S. Patent 5,783,683).

In certain embodiments, therefore, the present invention provides oligonucleotide sequences that comprise all or part of any sequence that can specifically bind to the polynucleotide sequence described herein or a complement thereof. In one embodiment, antisense oligonucleotides comprise DNA or derivatives thereof. In another embodiment, the oligonucleotides comprise RNA or derivatives thereof. In a third embodiment, the oligonucleotides are modified DNA comprising a phosphorothioated modified backbone. In a fourth embodiment, the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof. In each case, preferred compositions comprise a sequence region that is complementary, and more preferably substantially complementary, and even more preferably, completely complementary to one or more portions of the polynucleotides described herein. Selection of antisense preparations specific for a given gene sequence is based on analysis of the selected target sequence and determination of secondary structure, Tm, binding energy and relative stability. Antisense preparations may be selected based on their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prevent specific binding to target mRNA in a host cell. Highly preferred target regions of mRNA are those at or near the AUG translation initiation codon and those sequences that are substantially complementary to the 5' regions of the mRNA. These secondary structure analyzes and target-site selection considerations can be performed, for example, using v.4 of OLIGO primer analysis software and/or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. Sept. 1 .1997;25(17):3389-402).

Application of an antisense delivery method using a short peptide vector, designated MPG (27 residues), is also contemplated. The MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain from the nuclear localization sequence of SV40 T antigen (Morris et al., Nucleic Acids Res. Jul 15, 1997; 25(14):2730-6) . It has been demonstrated that many molecules of the MPG peptide coat antisense oligonucleotides and can be delivered to cultured mammalian cells in less than 1 hour with relatively high efficiency (90%). Furthermore, interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and the ability to cross the plasma membrane.

According to another embodiment of the invention, the polynucleotide preparations described here are used for designing and producing ribozyme molecules to inhibit expression of the tumor polypeptides and proteins according to the present invention in tumor cells. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have specific catalytic domains that have endonuclease activity (Kim and Cech, Proe Nati Acad Sei USA. Dec. 1987; 84(24):8788-92; Forster and Symons, Cell. Apr. 24, 1987; 49(2):211 -20). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of many phosphoesters in an oligonucleotide substrate (Cech et al., Cell. Dec. 1981; 27(3 Pt 2): 487-96; Michel and Westhof, J Mol Biol. 1990 Dec 5; 216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14; 357(6374): 173-6). This specificity is attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

Six basic variations of naturally occurring enzymatic RNA are now known. Each can catalyze hydrolysis of RNA phosphodiester bonds in transit (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids work by first binding to a target RNA. Such binding takes place through the target-binding part of an enzymatic nucleic acid which is kept in close proximity to an enzymatic part of the molecule which acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing and when bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to directly synthesize an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme required to effect a therapeutic treatment is lower than that of a antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule can cleave many molecules of target RNA. In addition, the ribozyme is a very specific inhibitor, where the specificity of inhibition depends not only on the base-pairing mechanism for binding to the target RNA, but also on the mechanism for target RNA cleavage. Simple mismatches or base substitutions near the cleavage site can completely eliminate the catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., Proe Nati Acad Sei U S A. Aug. 15, 1992; 89(16):7305-9). Thus, the specificity of the action of a ribozyme is greater than that of an antisense oligonucleotide that binds the same RNA site.

The enzymatic nucleic acid molecule can be formed in the hammerhead, hairpin, hepatitis 5 virus, group I intron or RNaseP RNA (along with an RNA guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are described by Rossi et al. Nucleic Acids Res. 11 September 1992; 20(17):4559-65. Examples of hairpin motifs are described by Hampel et al.

(Eur. Pat. Application Publ. No. EP 0360257), Hampel and Tritz, Biochemistry, June 13, 1989; 28(12):4929-33; Hampel et al., Nucleic Acids Res. 25 Jan 1990; 18(2):299-304 and U.S. Patent 5,631,359. An example of the hepatitis 8 virus motif is described by Perrotta and Been, Biochemistry. 1 Dec. 1992; 31 (47):11843-52; an example of the RNaseP motif is described by Guerrier-Takada et al., Cell. Dec. 1983; 35(3 Pt 2):849-57; The Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, Cell. May 18, 1990; 61(4):685-96; Saville and Collins, Proe Nati Acad Sei U S A. Oct. 1, 1991; 88 (19):8826-30; Collins and Olive, Biochemistry. 1993 Mar 23; 32(11):2795-9); and an example of a group I intron is described in (U.S. Patent 4,987,071). All that is important in an enzymatic nucleic acid molecule according to the present invention is that it has a specific substrate-binding site that is complementary to one or more of the target gene RNA regions and that it has nucleotide sequences within or surrounding the substrate-binding site that provide an RNA-cleaving activity of the molecule. Thus, the ribozyme constructs need not be limited to specific motifs mentioned herein.

Ribozymes can be designed as described in int. pat. application publ. No. WO 93/23569 and int. pat. application publ. No. WO 94/02595, each specifically incorporated herein by reference) and synthesized to be tested in vitro and in vivo, as described. Such ribozymes can also be optimized for delivery. While specific examples are provided, those skilled in the art will appreciate that equivalent RNA targets from other species can be used when necessary.

Ribozyme activity can be optimized by altering the length of ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see, e.g., int. pat. application, publ. no. WO 92/07065; int .Pat. Application Publ. No. WO 93/15187; Int. Pat. Application Pub. No. WO 91/03162; Eur. Publ. No. WO 94/13688, which describes various chemical modifications that can be introduced to the sugar groups in enzymatic RNA molecules), modifications that improve their efficiency in cells and removal of stem II bases to shorten RNA synthesis times and reduce chemical claim.

Sullivan et al. (int. pat. application publ. no. WO 94/02595) describes the general methods for the delivery of enzymatic RNA molecules. Ribozymes can be administered to cells by a variety of methods known to those skilled in the art, including, but not limited to, encapsulation in liposomes, by iontophoresis, or by introduction into other constituents, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes can be delivered directly ex vivo to cells or tissues with or without the above excipients. Alternatively, the RNA/constituent combination can be delivered locally by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in int. pat. application publ. No. WO 94/02595 and int. pat. application publ. No. WO 93/23569, each specifically incorporated herein by reference.

Another method for accumulating high concentrations of ribozyme(s) in cells is to introduce the ribozyme coding sequences into a DNA expression vector. Transcription of the ribozyme sequences is driven by a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II) or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the type of gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters can also be used, provided that the prokaryotic RNA polymerase enzyme is expressed in the cells in question. Ribozymes expressed from such promoters have been shown to act in mammalian cells. Such transcriptional units can be introduced into a variety of vectors for introduction into mammalian cells, including but not limited to plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors (such as retroviral, semliki forest virus, sindbis virus vectors).

In another embodiment of the invention, peptide nucleic acid (PNA) preparations are provided. PNA is a DNA mimic where the nucleobases are bound to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997 7(4) 431-37). PNA can be used by a number of methods that have traditionally used RNA or DNA. Often, PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have applications that are not real for RNA or DNA. An overview of PNA, including methods of preparation, characterization and methods of application, is provided by Corey (Trends Biotechnol, June 1997; 15(6):224-9). As such, in certain embodiments, PNA sequences that are complementary to one or more portions of the ACE mRNA sequence can be prepared, and such PNA preparations can be used to regulate, alter, down-regulate or reduce the translation of ACE-specific mRNA and thereby alter the level of ACE activity in a host cell to which such PNA preparations are administered.

PNA has 2-aminoethyl-glycine linkages that replace the normal one

phosphodiester backbone of DNA (Nielsen et al., Science, Dec. 6, 1991; 254(5037):1497-500; Hanvey et al., Science. Nov. 27, 1992; 258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 4 Jan 1996 (1): 5-23). This chemistry has three important consequences: first, unlike DNA or phosphorothioate oligonucleotides, PNA are neutral molecules; second, PNA is achiral, obviating the need to develop stereoselective synthesis; and third, PNA synthesis uses standard Boe or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used.

PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, MA). PNA syntheses by either Boe or Fmoc protocols are straightforward using manual or automated protocols (Norton et al., Bioorg Med Chem., April 1995; 3(4): 437-45). The manual protocol is suitable for the preparation of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesis will depend on the properties of the chosen sequence. For example, while in theory PNA can comprise any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. Anticipating this difficulty, it has been suggested that, in the production of PNA with adjacent purines, coupling of residues likely to be added inefficiently should be repeated. This should be followed by purification of PNA by reverse-phase high-pressure liquid chromatography, which gives yields and purity of product similar to those observed during synthesis of peptides.

Modifications of PNA for a given application can be achieved by coupling amino acids during solid phase synthesis or by attachment of compounds containing a carboxylic acid group to the exposed N-terminal amine. Alternatively, PNA can be modified after synthesis by linking to an introduced lysine or cysteine. The ease with which PNA can be modified facilitates optimization for better resolution or for specific functional requirements. Once synthesized, the identity of PNA and their derivatives can be confirmed by mass spectrometry. Many studies have performed and used modifications of PNA (for example, Norton et al., Bioorg Med Chem. April 1995; 3(4):437-45; Petersen et al., J Pept Sei. May-June 1995; 1(3 ): 175-83; Orum et al., Biotechniques. Sept. 1995; 19(3):472-80; Footeref a/., Biochemistry. 20 Aug. 1996; 35(33): 10673-9; Griffith et al. al., Nucleic Acids Res. 1995 Aug 11;23(15):3003-8; Pardridge et al., Proe Nati Acad Sei U S A. 1995 Jun 6;92(12):5592-6; Boffa et al. al., Proe Nati Acad Sei USA. 1995 Mar 14;92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 Aug 15;88(4):1411-7; Armitage et al. , Proe Nati Acad Sei USA. 1997 Nov 11;94(23):12320-5; Seeger et al., Biotechniques. 1997 Sep;23(3):512-7). U.S. patent No. 5,700,922 describes PNA-DNA-PNA chimeric molecules and their use in diagnostics, modulation of protein in organisms and treatment of disorders amenable to therapeutic agents.

Methods for characterizing the antisense binding properties of PNA are described in Rose (Anal Chem. Dec. 15, 1993; 65(24):3545-9) and Jensen et al. (Biochemistry. 1997 Apr 22;36(16):5072-7). Rose uses capillary gel electrophoresis to determine binding of PNA to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were carried out by Jensen et al. using BIAcore™ technology.

Other uses of PNA that are described and will be known to those skilled in the art include use in DNA strand invasion, antisense inhibition, mutation analysis, enhancer of transcription, nucleic acid purification, isolation of transcriptionally active genes, blocking of transcription factor binding, genome -cleavage, biosensors, in situ hybridization and the like.

Polvnucleotide identification, characterization and expression

Polynucleotide preparations of the present invention can be identified, prepared and/or manipulated using any of a number of well-established techniques (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989 and other similar references). For example, a polynucleotide can be identified, as described in more detail below, by screening a microarray of cDNA for tumor-associated expression (ie, expression that is at least twofold greater in a tumor than in normal tissue, as determined using a representative attempts listed here). Such screenings can be performed, for example, using the microarray technology of Affymetrix, Inc. (Santa Clara, CA) according to the manufacturer's instructions (and essentially as described by Schena et al., Proe. Nati. Acad. Sei. USA 93:10614-10619, 1996 and Heller et al., Proe. Nat. Acad. Sci. USA 94:2150-2155, 1997). Alternatively, polynucleotides can be amplified from cDNA prepared from cells expressing the proteins described herein, such as tumor cells.

Many template-dependent processes are available to amplify target sequences of interest present in a sample. One of the best known amplification methods is polymerase chain reaction (PCR™) which is described in detail in U.S. Pat. patent nos. 4,683,195, 4,683,202 and 4,800,159, each of which is hereby incorporated by reference in its entirety. Briefly, in PCR™ two primer sequences are produced that are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates is added to a reaction mixture together with a DNA polymerase (eg Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and polymerase will cause the primers to be extended along the target sequence by addition of nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated. Preferably, the reverse transcription and PCR™ amplification procedure can be performed to quantify the amount of mRNA amplified. Polymerase chain reaction methods are well known in the art.

Any of several other template-dependent processes, many of which are variations of the PCR™ amplification technique, are well known and available in the art. Illustratively, some such methods include ligase chain reaction (referred to as LCR), described for example in Eur. pat. application publ. No. 320,308 and U.S. Pat. Patent No. 4,883,750; Qbeta Replicase, described in PCT Int. pat. application publ. No. PCT/US87/00880; Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR). Still other amplification methods are described in British Pat. application no. 2 202 328 and in PCT Int. pat. application publ. No. PCT/US89/01025. Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (PCT Int. pat. application publ. no. WO 88/10315), comprehensive nucleic acid sequence-based amplification (NASBA) and 3SR. Eur. pat. application publ. No. 329,822 describes a nucleic acid amplification process involving the cyclic synthesis of single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA). PCT int. pat. application publ. No. WO 89/06700 describes a nucleic acid sequence amplification scheme based on hybridization of a promoter/primer sequence to a single-stranded DNA ("ssDNA") target followed by transcription of many RNA copies of the sequence. Other amplification methods such as "RACE" (Frohman, 1990) and "one-sided PCR"

(Ohara, 1989) is also well known to professionals in the field.

An amplified portion of a polynucleotide according to the present invention can be used to isolate a full-length gene from a suitable library (eg a tumor cDNA library) using well-known techniques. In such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Randomly primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred to obtain introns and extended 5' sequences.

For hybridization techniques, a partial sequence can be labeled { e.g. by nick-translation or end-labeling with <32>P) using well-known techniques. A bacterial or bacteriophage library is then generally screened by hybridization filters containing denatured bacterial colonies (or cloth containing phage plaque) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are selected and expanded and the DNA is isolated for further analysis. cDNA clones can be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences can be generated to identify one or more overlapping clones. The complete sequence can then be determined using standard techniques, which may involve the generation of a series of deletion clones. The resulting overlapping sequences can then be assembled into a single contiguous sequence. A full-length cDNA molecule can be formed by ligation of suitable fragments, using well-known techniques.

Alternatively, amplification techniques, such as those described above, may be applicable to obtain a full-length coding sequence from a partial cDNA sequence. One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 76:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. In an alternative method, sequences adjacent to a partial sequence can be recovered by amplification with a primer to a linker sequence and a primer specific for a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific for the known region. A variation of this procedure, using two primers which initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another such technique is known as "rapid amplification of cDNA ends" or RACE. This technique involves the use of an inner primer and an outer primer, which hybridize to a polyA region or vector sequence, to identify sequences that are 5" and 3' of a known sequence. Additional techniques include "capture"-PCR ( Lagerstrom et al., PCR Methods Applic. 7:111-19, 1991) and "walking" PCR (Parker et al., Nucl. Acids. Res. 79:3055-60, 1991). Other methods that use amplification can also used to obtain a full-length cDNA sequence.

In certain cases, it is possible to obtain a full-length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs can generally be performed using well-known programs (eg NCBI BLAST search) and such ESTs can be used to generate a subsequent full-length sequence. Full-length DNA sequences can also be obtained by analysis of genomic fragments.

In other embodiments of the present invention, polynucleotide sequences or fragments thereof encoding polypeptides of the present invention or fusion proteins or functional equivalents thereof may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Because of the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or a functionally equivalent amino acid sequence can be produced and these sequences can be used to clone and express a given polypeptide.

As will be appreciated by those skilled in the art, it may be advantageous in some cases to produce polypeptide-encoding nucleotide sequences having non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the level of protein expression or to produce a recombinant RNA transcript that has desirable properties, such as a half-life longer than that of a transcript formed from it naturally occurring sequence.

Furthermore, the polynucleotide sequences of the present invention can be constructed using methods generally known in the art to change polypeptide coding sequences for a variety of reasons, including, but not limited to, changes that modify cloning, processing and/or expression of the gene product. For example, DNA "shuffling" by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to construct the nucleotide sequences. In addition, site-directed mutagenesis can be used to insert new restriction sites, change glycosylation patterns, change codon preference, produce splice variants or introduce mutations, etc.

In another embodiment of the invention, natural, modified or recombinant nucleic acid sequences can be ligated to a heterologous sequence to encode a fusion protein. For example, for screening peptide libraries for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognized by a commercially available antibody. A fusion protein can also be engineered to contain a cleavage site located between the polypeptide coding sequence and the heterologous protein sequence, so that the polypeptide can be cleaved and purified away from the heterologous group.

Sequences encoding a desired polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the protein itself can be produced using chemical methods to synthesize the amino acid sequence of a polypeptide or part thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge, J.Y. et al. (1995) Science 269:202-204) and automated synthesis can be performed, for example, using the ABI 431A Peptide Synthesizer (Perkin Eimer, Palo Alto, ABOUT).

A newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (eg, Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.) or other comparable techniques available in the art. The composition of the synthetic peptides can be confirmed by amino acid analysis or sequencing (e.g. Edman degradation procedure). In addition, the amino acid sequence of a polypeptide, or any portion thereof, may be altered during direct synthesis and/or combined, using chemical methods, with sequences from other proteins or any portion thereof, to produce a variant polypeptide.

To express a desired polypeptide, the nucleotide sequences encoding the polypeptide or functional equivalents can be inserted into a suitable expression vector, i.e. a vector containing the necessary elements for transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. Such techniques are described in, for example, Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. NEW.

A variety of expression vector/host systems can be used to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (eg, baculovirus); plant cell systems transformed with viral expression vectors (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (eg Ti or pBR322 plasmids); or animal cell systems.

"Control elements" or "regulatory sequences" present in an expression vector are the untranslated regions of vector enhancers, promoters, 5' and 3' untranslated regions that interact with host cellular proteins to effect transcription and translation. Such elements may vary in strength and specificity. Depending on the vector system and host used, any number of suitable transcriptional and

translation elements, including constitutive and inducible promoters, are used. For example, when cloning in bacterial systems, inducible promoters such as hybrid lacZ promoter of PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) and the like can be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to form a cell line containing multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV can advantageously be used with a suitable selectable marker.

In bacterial systems, any of several expression vectors may be selected depending on the intended use of the expressed polypeptide. When, for example, large amounts are required, for example for the induction of antibodies, vectors that direct high-level expression of fusion proteins that can be easily purified can be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), where the sequence encoding the polypeptide of interest can be ligated into the vector in frame with sequences for the amino-terminal Met and the following 7 residues of .beta.-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster

(1989) J. Biol. Chem. 264:5503-5509); and such. pGEX vectors (Promega, Madison, Wis.) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST group as desired.

In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used. For overviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression of sequences encoding polypeptides may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311. Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters, are used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 77:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in several generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system can also be used to express a polypeptide of interest. In one such system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide can be cloned into a non-essential region of the virus, such as the polyhedrin gene and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking the coat protein. The recombinant virus can then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae where the polypeptide of interest can be expressed (Engelhard, E. K. et al. (1994) Proe. Nati. Acad. Sei. 91 :3224-3227) .

In mammalian host cells, several viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest can be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion into a non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T.

(1984) Proe. Nati. Acad. Pollock. 87:3655-3659). In addition, transcriptional enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Specific initiation signals can also be used to achieve more efficient translation of sequences that code for a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals are necessary. However, in cases where only the coding sequence or part thereof is inserted, the exogenous translational control signals comprising the ATG initiation codon should be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure translation of the entire insertion. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be improved by the inclusion of enhancers appropriate for the particular cell system being used, such as those described in the literature (Scharf, D. et al.

(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain can be selected for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired manner. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves a "prepro" form of the protein can also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, COS, HeLa, MDCK, HEK293 and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, can be chosen to ensure correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines that stably express a polynucleotide of interest can be transformed using expression vectors that can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. After introduction of the vector, the cells can be allowed to grow for 1-2 days in an enriched medium before being transferred to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate for the cell type.

Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 77:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes that can be used for tk.sup.- or aprt.sup.- cells respectively. Antimetabolite, antibiotic or herbicide resistance can also be used as a basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proe. Nati. Acad. Sei. 77:3567-70); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al

(1981) J. Mol. Biol. 750:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinothricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example trpB which allows cells to use indole instead of tryptophan, or hisD which allows cells to use histinol instead of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proe. Nati. Acad. Sei. 85:8047- 51). The use of visible markers has gained popularity, with such markers as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin being widely used not only to identify transformants but also to quantify the amount of transient or stable protein expression that can be attributed to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression indicates that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a polypeptide is inserted into a marker gene sequence, recombinant cells containing the sequences can be identified by the absence of marker gene function. Alternatively, a marker gene may be located tandemly with a polypeptide coding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

Alternatively, host cells containing and expressing a desired polynucleotide sequence can be identified by a number of procedures known to those skilled in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include, for example, membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.

A number of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence-activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive for two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be used. These and other attempts are described, among others, in Hampton, R. et al.

(1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.)

and Maddox, D.E. et al. (1983; J. Exp. Med. 758:1211-1216).

A number of labeling and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences or any parts thereof can be cloned into a vector for production of an mRNA probe. Such vectors are known in the art, are commercially available and can be used to synthesize RNA probes in vitro by the addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. These procedures can be performed using a variety of commercially available kits. Suitable reporter molecules or labels that can be used include radionuclides, enzymes, fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.

Host cells transformed with a polynucleotide sequence of interest can be cultured under conditions suitable for expression and recovery of the protein from cell culture. The protein produced by a recombinant cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those skilled in the art, expression vectors containing polynucleotides according to the present invention can be designed to contain signal sequences that control secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to join sequences encoding a polypeptide of interest with a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins. Such purification-enhancing domains include, but are not limited to, metal-chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain used in the FLAGS extension/affinity purification system ( Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.) between the purification domain and the encoded polypeptide can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinase cleavage site provides a method for purifying the desired polypeptide from the fusion protein. A review of vectors containing fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 72:441-453).

In addition to recombinant production methods, polypeptides according to the present invention and fragments thereof can be produced by direct peptide synthesis using solid phase techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis can be carried out using manual techniques or by automation. Automated synthesis can be achieved, for example, using the Applied Biosystems 431A Peptide Synthesizer (Perkin Eimer). Alternatively, different fragments can be chemically synthesized separately and combined using chemical methods to produce the full-length molecule.

Antibody preparations, fragments thereof and other binders

According to another aspect, the present invention further provides binding agents, such as antibodies and antigen-binding fragments thereof, which show immunological binding to a tumor polypeptide described herein or to a part, variant or derivative thereof. An antibody or antigen-binding fragment thereof is said to "specifically bind," "immunogically bind," and/or is "immunologically reactive" with a polypeptide of the present invention if it reacts at a detectable level (in, for example, an ELISA experiment) with the polypeptide and does not detectably react with unrelated polypeptides under similar conditions.

Immunological binding, as used in this context, generally refers to the non-covalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of immunological binding interactions can be expressed as the dissociation constant (Kd) of the interaction, where a smaller Kdrepresents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method involves measuring the degree of antigen-binding site/antigen complex formation and dissociation, where the degrees depend on the concentrations of the complex partners, the affinity of interaction and geometric parameters that equally affect the degree in both directions. Thus both "on degree constant" (Kon) and "of degree constant" can

(Koff) is determined by calculating the concentrations and the relevant degrees of association and dissociation. The ratio of K0ff/Kon allows the cancellation of all parameters not related to affinity and is thus equal to the dissociation constant Kd. See generally Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

An "antigen-binding site," or "binding portion" of an antibody denotes the portion of the immunoglobulin molecule that participates in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as "hypervariable regions" that are sandwiched between more conserved flanking stretches known as "framework regions," or "FRs." Thus, the designation "FR" denotes amino acid sequences naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions," or "CDRs."

Binding agents may further be capable of differentiating between patients with and without cancer, such as prostate cancer, using the representative assays set forth herein. For example, antibodies or other binding agents that bind to a tumor protein will preferably generate a signal indicating the presence of cancer in at least approx. 20% of patients with the disease, more preferably at least approx. 30% of patients. Alternatively or additionally, the antibody will generate a negative signal indicating the absence of the disease for at least approx. 90% of individuals without the cancer. To determine whether a binder satisfies this requirement, biological samples (eg, blood, sera, sputum, urine and/or tumor biopsies) from patients with and without cancer (as determined using standard clinical tests) can be examined as described herein for the presence of polypeptides that bind to the binder. Preferably, a statistically significant number of samples with and without the disease will be examined. Each binder must satisfy the above criteria; however, those skilled in the art will appreciate that binders can be used in combination to improve sensitivity.

Any agent satisfying the above requirement can be a binder. For example, a binding agent can be a ribosome, with or without a peptide component, an RNA molecule or a polypeptide. In a preferred embodiment, the binding agent is an antibody or an antigen-binding fragment thereof. Antibodies can be prepared by any of a number of techniques known to those skilled in the art. See e.g. Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including generation of monoclonal antibodies as described herein or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, for to allow the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a variety of mammals (eg, mice, rats, rabbits, sheep, or goats). In this step, the polypeptides of the present invention can serve as the immunogen without modification. Alternatively, especially for relatively short polypeptides, a superior immune response can be elicited if the polypeptide is bound to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule comprising one or more booster immunizations and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide can then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interest can be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519,1976 and improvements thereof. Briefly, these methods involve the production of immortal cell lines capable of producing antibodies having the desired specificity (ie, reactivity with the polypeptide of interest). Such cell lines can be produced from, for example, spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one cognate to the immunized animal. A number of fusion techniques can be used. For example, the spleen and myeloma cells can be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. A preferred selection technique uses HAT-(hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually approx. 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity to the polypeptide. Hybridomas that have high reactivity and specificity are preferred.

Monoclonal antibodies can be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques can be used to improve the yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies can then be harvested from the ascites fluid or blood. Contaminants can be removed from the antibodies by conventional methods, such as chromatography, gel filtration, precipitation and extraction. The polypeptides according to the present invention can be used in the purification process in, for example, an affinity chromatography step.

Several therapeutically useful molecules are known in the field, which include antigen-binding sites that can have immunological binding properties to an antibody molecule. The proteolytic enzyme papain preferentially cleaves IgG molecules, yielding many fragments, two of which ("F(ab)" fragments) each comprise a covalent heterodimer comprising an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to yield many fragments, including the "F(ab')2" fragment that includes both antigen-binding sites. An "Fv" fragment can be produced by preferential proteolytic cleavage of an IgM and, rarely, an IgG or IgA immunoglobulin molecule. However, Fv fragments are more commonly derived using recombinant techniques known in the art. The Fv fragment comprises a non-covalent VH::VL heterodimer comprising an antigen binding site that retains much of the antigen recognition and binding capacities of the native antibody molecule. Inbar et al. (1972) Proe. Nat. Acad. Pollock. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv ("sFv") polypeptide is a covalently linked VH::VL heterodimer that is expressed from a gene fusion comprising VH and VL encoding genes linked by a peptide-encoding linker. Houston et al. (1988) Proe. Nat. Acad. Pollock. USA 85(16):5879-5883. Several methods have been described to separate chemical structures for converting the naturally aggregated - but chemically separated - light and heavy polypeptide chains from an antibody V region into an sFv molecule that will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g. U.S. pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and the U.S. pat. No. 4,946,778, to Ladner et al.

Each of the above described molecules comprises a heavy chain and a light chain CDR set, respectively inserted between a heavy chain and a light chain FR set which provides support to the CDRS and defines the spatial position of the CDRs in relation to each other. As used herein, the term "CDR set" denotes the three hypervariable regions of a heavy or light chain V region. Starting from the N-terminus of a heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3," respectively. An antigen-binding site therefore comprises six CDRs, comprising the set of CDRs from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (eg, a CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit." Crystal log crude analysis of several antigen-antibody complexes has demonstrated that the amino acid residues in the CDR form extensive contact with bound antigen, where the most extensive antigen contact is with heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.

As used herein, the term "FR set" denotes the four flanking amino acid sequences that frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, the FR is primarily responsible for folding the V region into an antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FR, certain amino residues and certain structural features are highly conserved. In this regard, all V-region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V regions fold into a binding site, the CDRs appear as protruding loop motifs that form an antigen-binding surface. It is generally known that there are conserved structural regions of FRs that influence the folded shape of CDR loops to certain "canonical" structures - regardless of the exact CDR amino acid sequence. Furthermore, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction of antibody heavy and light chains.

Several "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains (Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989) Proe. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown et al. ( 1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a human supporting FR prior to fusion with an appropriate human antibody constant domain (Riechmann et al.

(1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature 321:522-525) and rodent CDR supported by recombinant coated rodent FR (European Patent Publication No. 519,596, published Dec. 23, 1992). These "humanized" molecules are designed to minimize unwanted immunological responses to rodent anti-human antibody molecules that limit the duration and effectiveness of therapeutic applications of those groups in human recipients.

As used herein, the terms "grafted FR" and "recombinant grafted FR" refer to selective replacement of FR residues from, e.g. a rodent heavy or light chain V region, with human FR residues to provide a xenogeneic molecule comprising an antigen-binding site that retains substantially all of the native FR polypeptide folding structure. Coating techniques are based on the understanding that the ligand binding characteristics of an antigen binding site are determined primarily by the structure and relative arrangement of heavy and light chain CDR sets within the antigen binding surface. Davies et al. (1990) Ann. Fox. Biochem.

59:439-473. Thus, antigen binding specificity can be preserved in a humanized antibody only when the CDR structures, their interaction with each other and their interaction with the rest of the V region domains are carefully maintained. Using grafting techniques, external (eg, solvent-accessible) FR residues that are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule comprising either a weakly immunogenic or predominantly non-immunogenic coated surface.

The method of grafting uses the available sequence data for human antibody variable domains compiled by Kabat et al., in Sequences of Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Health and Human Services, U.S. Government Printing Office, 1987), updates to the Kabat database and other available U.S. and foreign databases (both nucleic acid and protein). Solvent accessibilities for V-region amino acids can be deduced from the known three-dimensional structure of human and murine antibody fragments. There are two general steps in mapping a murine antigen-binding site. Initially, the FR in the variable domains of an antibody molecule of interest is compared to corresponding FR sequences in human variable domains obtained from the sources indicated above. The most homologous human V regions are then compared residue by residue with corresponding murine amino acids. The residues in the murine FR that differ from the human counterpart are replaced with the residues present in the human group using recombinant techniques well known in the art. Residue switching is only performed with groups that are at least partially exposed (solvent-accessible) and caution is exercised when replacing amino acid residues that may have a significant effect on the tertiary structure of V-region domains, such as proline, glycine, and charged amino acids.

In this way, the resulting "grafted" murine antigen binding sites are thus designed to retain the murine CDR residues, the residues mainly adjacent to the CDRs, the residues identified as hidden or mostly hidden (solvent-inaccessible), the residues predicted to participate in non-covalent (eg, electrostatic and hydrophobic) contacts between heavy- and light-chain domains and the residues from conserved structural regions of the FRs that are thought to affect "canonical" tertiary structures of the CDR loops. These design criteria are then used to produce recombinant nucleotide sequences that combine CDRs from both heavy and light chains of a murine antigen-binding site into human-appearing FRs that can be used to transfect mammalian cells for the expression of recombinant human antibodies that display the antigen specificity to the murine antibody molecule.

In another embodiment of the invention, monoclonal antibodies according to the present invention can be linked to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins and derivatives thereof. Preferred radionuclides include 90Y,1231,1251,1<3>11,18<6>Re,<188>Re,<2>1<1>At and<212>Bi. Preferred drugs include methotrexate and pyrimidine and purine analogs. Preferred differentiation inducers include porbole esters and butyric acid. Preferred toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and kermesberry antiviral protein.

A therapeutic agent can be linked (e.g. covalently bound) to a suitable monoclonal antibody either directly or indirectly (e.g. via a linker group). A direct reaction between an agent and an antibody is possible when each has a substituent that can react with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, may on one hand be able to react with a carbonyl-containing group, such as an anhydride or an acid halide or with an alkyl group containing a good leaving group (e.g. . a halide) on the other.

Alternatively, it may be desirable to connect a therapeutic agent and an antibody via a linker group. A linker group can act as a spacer to distance an antibody from an agent to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody and thus increase the coupling efficiency. An increase in chemical reactivity can also facilitate the application of agents or functional groups on agents, which would not otherwise be possible.

It will be apparent to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of Pierce Chemical Co., Rockford, IL), can be used as the linker group. Coupling can be carried out, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are a number of references that describe such methodology, e.g. U.S. Patent No. 4,671,958, to Rodwell et al.

When a therapeutic agent is more powerful when free of the antibody part of the immunoconjugates according to the present invention, it may be desirable to use a linker group which is cleavable during or after internalization in a cell. Several different cleavable linker groups have been described. The mechanisms for intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Patent No. 4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S. Patent No. 4,625,014 , to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Patent No. 4,638,045, to Kohn et al.), by serum complement-mediated hydrolysis (e.g., U.S. Patent No. 4,671,958, to Rodwell et al.) and acid-catalyzed hydrolysis (e.g., U.S. Patent No. 4,569,789, to Blattler et al.).

It may be desirable to link more than one agent to an antibody. In one embodiment, multiple molecules of an agent are linked to one antibody molecule. In another embodiment, more than one type of agent can be linked to one antibody. Regardless of the particular embodiment, immunoconjugates with more than one agent can be prepared in a variety of ways. For example, more than one agent can be linked directly to an antibody molecule, or linkers that provide multiple sites for binding can be used. Alternatively, a carrier may be used.

A carrier can carry the agents in a number of ways, including covalent attachment either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides, and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.). A carrier may also carry an agent by non-covalent binding or by encapsulation, such as in a liposomal vesicle (eg, U.S. Patent Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, the U.S. describes Patent No. 4,735,792 representative radiohalogenated small molecules and their synthesis. A radionuclide chelate can be formed from chelating compounds which include those containing nitrogen and sulfur atoms as donor atoms for the binding of metal or metal oxide radionuclide. For example, the U.S. describes Patent No. 4,673,562, to Davison et al. representative chelating compounds and their synthesis.

T-cell preparations

The present invention provides, in another aspect, T cells specific for a tumor polypeptide described herein or for a variant or derivative thereof. Such cells can generally be produced in vitro or ex vivo, using standard procedures. For example, T cells can be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood from a patient, using a commercially available cell separation system, such as the Isolex™ System, available from Nexell Therapeutics, Inc. (Irvine, CA; see also U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells can be derived from related or unrelated human, non-human mammals, cell lines or cultures.

T cells can be stimulated with a polypeptide, polynucleotide encoding a polypeptide and/or an antigen presenting cell (APC) expressing such a polypeptide. Such stimulation is performed under conditions and for a time sufficient to allow generation of T cells specific for the polypeptide of interest. Preferably, a tumor polypeptide or polynucleotide of the present invention is present in a delivery constituent, such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the present invention if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or express a gene encoding the polypeptide. T cell specificity can be evaluated using any of a number of standard techniques. For example, in a chromium release assay or proliferation assay, a stimulation index of greater than twofold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such experiments can be performed, for example, as described in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the proliferation of T cells can be achieved by a number of known techniques. For example, T-cell proliferation can be detected by measuring an increased degree of DNA synthesis (e.g. by pulse-labeling cultures of T-cells with tritiated thymidine and measuring the amount of tritiated thymidine introduced into DNA). Contact with a tumor polypeptide (100 ng/ml -100 ug/ml, preferably 200 ng/ml - 25 (ug/ml) for 3 - 7 days will typically result in at least a twofold increase in proliferation of the T cells. Contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays where a two-fold increase in the level of cytokine release (e.g. TNF or IFN-γ) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998)).T cells that are activated in response to a tumor polypeptide-, polynucleotide-, or polypeptide-expressing APC may be CD4 <+>and/or CD8<+>. Tumor polypeptide-specific T cells can be expanded using standard techniques. In preferred embodiments, the T cells are taken from a patient, a related donor, or an unrelated donor and are administered to the patient after stimulation and expansion.

For therapeutic purposes, CD4<+> or CD8<+> T cells that proliferate in response to a tumor polypeptide, polynucleotide, or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro can be carried out in a number of ways. For example, the T cells can be re-exposed to a tumor polypeptide or a short peptide corresponding to an immunogenic portion of such a polypeptide, with or without the addition of T cell growth factors, such as interleukin-2 and/or stimulator cells that synthesize a tumor polypeptide. Alternatively, one or more T cells that proliferate in the presence of the tumor polypeptide can be expanded in number by cloning. Methods for cloning cells are well known in the art and include limiting dilution.

Pharmaceutical preparations

In further embodiments, the present invention relates to a preparation of one or more of the polynucleotide, polypeptide, T-cell and/or antibody preparations described herein in pharmaceutically acceptable carriers for administration to a cell or an animal, either alone or in combination with one or more other therapy modalities.

It will be understood that, if desired, a preparation as described here can be administered in combination with other agents as well, such as, e.g. other proteins or polypeptides or various pharmaceutically active agents. Indeed, there is virtually no limit to the other components that may also be included, provided that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The preparations can thus be delivered together with various other means as necessary in the particular case. Such preparations can be purified from host cells or other biological sources or can alternatively be synthesized chemically as described here. Likewise, such preparations can further include substituted or derivatized RNA or DNA preparations.

In another aspect of the present invention, pharmaceutical preparations comprising one or more of the polynucleotide, polypeptide, antibody and/or T-cell preparations described here are therefore provided in combination with a physiologically acceptable carrier. In certain preferred embodiments, the pharmaceutical preparations according to the present invention comprise immunogenic polynucleotide and/or polypeptide preparations according to the present invention for use in prophylactic and therapeutic vaccine applications. Vaccine production is generally described in, for example, M.F. Powell and M.J. Newman, ed., "Vaccine Design (the subunit and adjuvant approach)," Plenum Press (NY, 1995). In general, such preparations will comprise one or more polynucleotide and/or polypeptide preparations according to the present invention in combination with one or more immunostimulants.

It will be clear that any of the pharmaceutical preparations described herein may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides according to the present invention. Such salts can be prepared, for example, from pharmaceutically acceptable non-toxic bases, including organic bases (eg, salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (eg, sodium, potassium, lithium -, ammonium, calcium and magnesium salts).

In another embodiment, illustrative immunogenic preparations, e.g. vaccine preparations according to the present invention, DNA which codes for one or more of the polypeptides as described above, so that the polypeptide is formed in situ. As noted above, the polynucleotide can be administered in any of a variety of delivery systems known to those skilled in the art. Indeed, a number of gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Fox. Therapy. Drug Carrier Systems 75:143-198, 1998 and references therein. Appropriate polynucleotide expression systems will of course contain the necessary regulatory DNA regulatory sequences for expression in a patient (such as a suitable promoter and termination signal). Alternatively, bacterial delivery systems may involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.

Therefore, in certain embodiments, polynucleotides encoding immunogenic polypeptides described herein are introduced into suitable mammalian host cells for expression using any of several known viral-based systems. In one illustrative embodiment, retroviruses provide a convenient and efficient platform for gene delivery systems. A selected nucleotide sequence encoding a polypeptide according to the present invention can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to an individual. Several illustrative retroviral systems are described { e.g. U.S. pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A.D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al.

(1993) Proe. Nati. Acad. Pollock. USA 90:8033-8037; and Boris-Lawrie and Temin

(1993) Curr. Opinion. The gene. Develop. 3:102-109.

In addition, several illustrative adenovirus-based systems are also described.

Unlike retroviruses that integrate into the host genome, adenoviruses remain extrachromosomal and thus minimize the risks associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol 67:5911-5921 Mitterederet et al (1994) Human Gene Therapy 5:717-729 Seth et al (1994) J Virol 68:933-940 Barr et al (1994) Gene Therapy 1: 51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476).

Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g. U.S. pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B.J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R.M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

Additional viral vectors useful for delivery of the polynucleotides encoding polypeptides of the present invention by gene transfer include those derived from the pox family of viruses, such as vaccinia virus and avian pox virus. For example, vaccinia virus recombinants expressing the new molecules can be constructed as follows. DNA encoding a polypeptide is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanks vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells that are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome. The resulting TK.sup.(-) recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and selecting viral plaque resistant to this.

A vaccinia-based infection/transfection system can conveniently be used to provide for inducible, transient expression or co-expression of one or more polypeptides described herein in host cells of an organism. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant encoding the bacteriophage T7 RNA polymerase. This polymerase shows excellent specificity in that it only transcribes templates carrying T7 promoters. After infection, cells are transfected with the polynucleotide or polynucleotides of interest, driven by a T7 promoter. Polymerase expressed in the cytoplasm of the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into polypeptide by the host translational machinery. The method provides high-level, transient, cytoplasmic production of large amounts of RNA and its translation products. See e.g. Elroy-Stein and Moss, Proe. Nati. Acad. Pollock. USA (1990) 87:6743-6747; Fuerst et al. Pro. Nati. Acad. Pollock. USA (1986) 83:8122-8126.

Alternatively, avipox viruses, such as fowl pox and canary pox viruses, can also be used to deliver the coding sequences of interest. Recombinant avipox viruses, which express immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. Use of an Avipox vector is particularly desirable for human and other mammalian species since members of the Avipox genus can only replicate productively in susceptible avian species and are therefore non-infectious in mammalian cells. Methods for producing recombinant Avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia virus. See e.g. WO 91/12882; WO 89/03429; and WO 92/03545.

Any of several alphavirus vectors may also be used for delivery of polynucleotide preparations according to the present invention, such as the vectors described in U.S. Pat. Patent No. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE) can also be used, illustrative examples of which can be found in U.S. Pat. Patent Nos. 5,505,947 and 5,643,576.

Furthermore, molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al. Pro. Nati. Acad. Pollock. USA (1992) 89:6099-6103, also used for gene delivery in the invention.

Additional illustrative information on these and other known viral-based delivery systems can be found, for example, in Fisher-Hoch et al., Proe. Nati. Acad. Pollock. USA 86:317-321, 1989; Flexner et al., Ann. N. Y. Acad. Pollock. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proe. Nati. Acad. Pollock. USA 97:215-219, 1994; Kass-Eisler et al., Proe. Nati. Acad. Pollock. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993.

In certain embodiments, a polynucleotide can be integrated into the genome of a target cell. This integration can occur in a specific location and orientation via homologous recombination (gene replacement) or it can be integrated in a random, non-specific location (gene gain). In still further embodiments, the polynucleotide may be stably maintained in the cell as a separate, episomal segment of DNA. Such polynucleotide segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or synchronized with the host cell cycle. The method by which the expression construct is delivered to a cell and where in the cell the polynucleotide remains depends on the type of expression construct used.

In another embodiment of the invention, a polynucleotide is administered/delivered as "naked" DNA, for example as described in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA can be increased by coating the DNA on biodegradable beads, which are efficiently transported into the cells.

In yet another embodiment, a preparation according to the present invention can be delivered via a particle bombardment method, many of which have been described. In one illustrative example, gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, WI), some examples of which are described in U.S. Pat. Patent No. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799. This method provides a needle-free delivery method in which a dry powder preparation of microscopic particles, such as polynucleotide or polypeptide particles, is accelerated to high velocity in a helium gas jet formed with a hand-held device, which drives the particles into a relevant target tissue.

In a related embodiment, other devices and methods that may be useful for gas-powered needle-free injection of compositions of the present invention include those provided by Bioject, Inc. (Portland, OR), some examples of which are described in U.S. Pat. Patent No. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

According to another embodiment, the pharmaceutical preparations described here will comprise one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T-cell and/or APC preparations according to the present invention. An immunostimulant essentially refers to any substance that enhances or enhances an immune response (antibody and/or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A-, Bortadella pertussis-, or Mycobacterium tuberculosis-derived proteins. Certain adjuvants are commercially available, such as Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12 and other similar growth factors, can also be used as adjuvants.

In certain embodiments of the present invention, the adjuvant preparation is preferably one that induces an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (eg, IFN-γ, TNFα, IL-2 and IL-12) tend to favor the induction of cell-mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (eg, IL-4, IL-5, IL-6, and IL-10) tend to favor the induction of humoral immune responses. Following administration of a vaccine as provided herein, a patient will mount an immune response comprising Th1 and Th2 type responses. In a preferred embodiment, where a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines can be easily assessed using standard assays. For an overview of the cytokine families, see Mosmann and Coffman, Ann. Fox. Immunol. 7:145-173, 1989.

Certain preferred adjuvants for producing a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt. MPL<®> adjuvants are available from Corixa Corporation (Seattle, WA; see, for example, US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (where the CpG dinucleotide is unmethylated) also elicit a predominantly Th1 response. Such oligonucleotides are well known and are described in, for example, WO 96/02555, WO 99/33488 and U.S. Pat. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences have also been described, for example, by Sato et al., Science 273:352,1996. Another preferred adjuvant comprises a saponin, such as Quil A or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins. Other preferred preparations comprise more than one saponin in the adjuvant combinations according to the present invention, for example combinations of at least two of the following groups comprising QS21, QS7, Quil A, p-escin or digitonin.

Alternatively, saponin preparations can be combined with vaccine constituents composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid- based particles, particles composed of glycerol monoesters, etc. The saponins can also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOM. Furthermore, the saponins can be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension or in a particulate structure such as a paucilamellar liposome or ISCOM. The saponins can also be formulated with adjuvants such as Carbopol<R> to increase viscosity or can be formulated in a dry powder form with a powder adjuvant such as lactose.

In one preferred embodiment, the adjuvant system comprises the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL<®>adjuvant, as described in WO 94/00153 or a less reactogenic preparation where QS21 becomes treated with cholesterol, as described in WO 96/33739. Other preferred preparations include an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant preparation using QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

Another improved adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative, in particular the combination of CpG and QS21 is described in WO 00/09159. Preferably, the preparation additionally comprises an oil-in-water emulsion and tocopherol.

Further illustrative adjuvants for use in the pharmaceutical preparations according to the present invention include Montanid ISA 720 (Seppic, France), SAF (Chiron, California, USA), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants {f e.g. SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhanzyn<®>; Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT), and other aminoalkyl-glucosaminide-4- phosphates (AGP), such as those described in current U.S. patent application nos. 08/853,826 and 09/074,720, the descriptions therein being incorporated herein by reference in their entirety and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the general formula

(I): HO(CH2CH20)n-A-R,

where, n is 1-50, A is a bond or -C(O)-, R is C1-50 alkyl or phenyl C1-50 alkyl.

An embodiment of the invention consists of a vaccine preparation comprising a polyoxyethylene ether of the general formula (I), where n is between 1 and 50, preferably 4-24, most preferably 9; The R component is C1.50, preferably C4-C20 alkyl and most preferably C12 alkyl and A is a bond. The concentration of polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10% and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck Index (12th ed.: entry 7717). These adjuvant molecules are described in WO 99/52549. The polyoxyethylene ether according to the general formula (I) above can, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in current UK patent application GB 9820956,2.

According to another embodiment of the invention, an immunogenic preparation described herein is delivered to a host via antigen presenting cells (APC), such as dendritic cells, macrophages, B cells, monocytes and other cells that can be engineered to be effective APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to enhance activation and/or maintenance of the T-cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the recipient (ie matched HLA haplotype). APC can generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissue and can be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention employ dendritic cells or progenitors thereof as antigen presenting cells. Dendritic cells are very potent APCs (Banchereau and Steinman, Nature 392:245-251,1998) and have been shown to be effective as a physiological adjuvant to produce prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med .50:507-529, 1999). In general, dendritic cells can be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naïve T- cell responses. Dendritic cells can of course be engineered to express specific cell surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo and such modified dendritic cells are covered by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (termed exosomes) can be used in a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors 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, dendritic cells can be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow can be differentiated into dendritic cells by addition to the culture medium of combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/or other compound(s) which induces differentiation, maturation and proliferation of dendritic cells.

Dendritic cells are conveniently categorized as "immature" and "mature" cells, providing a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be used to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcy receptor and mannose receptor. The mature phenotype is typically characterized by lower expression of these markers, but a high expression of cell surface molecules responsible for T-cell activation such as class I and class II MHC, adhesion molecules (e.g. CD54 and CD11) and costimulatory molecules (e.g. eg CD40, CD80, CD86 and 4-1BB).

APC can generally be transfected with a polynucleotide according to the present invention (or part or other variant thereof) so that the coded polypeptide or an immunogenic part thereof is expressed on the cell surface. Such transfection can take place ex vivo and a pharmaceutical preparation comprising such transfected cells can then be used for therapeutic purposes, as described here. Alternatively, a gene delivery agent that targets a dendritic or other antigen-presenting cell can be administered to a patient, resulting in transfection occurring in vivo. For example, in vivo and ex vivo transfection of dendritic cells can generally be performed using any methods known in the art, such as those described in WO 97/24447 or the gene gun method described by Mahvi et al., Immunology and Cell Biology 75 :456-460, 1997. Antigen loading of dendritic cells can be achieved by incubating dendritic cells or progenitor cells with the tumor polypeptide, DNA (naked or in a plasmid vector) or RNA; or with antigen-expressing recombinant bacteria or viruses (eg vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T-cell help (eg, a carrier molecule). Alternatively, a dendritic cell can be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.

While any suitable carrier known to those skilled in the art may be used in the pharmaceutical compositions of the present invention, the type of carrier will typically vary depending on the method of administration. Preparations according to the present invention can be formulated for any suitable method of administration, including, for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use in such pharmaceutical preparations are biocompatible and may also be biodegradable. In certain embodiments, the preparation preferably provides a relatively constant level of active component release. In other embodiments, however, faster release rates immediately after administration may be desired. The composition of such preparations is well within the knowledge of ordinary professionals in the field using known techniques. Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative sustained-release carriers include supramolecular biovectors, which comprise a non-flowing hydrophilic core { e.g. a cross-linked polysaccharide or oligosaccharide) and, optionally, an outer layer comprising an amphiphilic compound, such as a phospholipid (see, e.g., U.S. Patent No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96 /06638). The amount of active compound contained in a sustained-release preparation depends on the site of implantation, the rate and expected duration of release, and the type of condition to be treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g. polylactate-polyglycolate) are used as carriers for the preparations according to the present invention. Suitable biodegradable microspheres are described, for example, in U.S. Pat. Patent No. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems. such as described in WO/99 40934 and references cited therein will also be applicable for many applications. Another illustrative carrier/delivery system utilizes a carrier comprising particulate-protein complexes, such as those described in U.S. Pat. patent No. 5,928,647, which can elicit class I-restricted cytotoxic T-lymphocyte responses in a host.

The pharmaceutical preparations according to the present invention will often further comprise one or more buffers (e.g. neutral-buffered saline or phosphate-buffered saline), carbohydrates (e.g. glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostatic agents, chelating agents such as EDTA or glutathione, adjuvants (e.g. aluminum hydroxide), soluble substances that make the preparation isotonic, hypotonic or slightly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives . Alternatively, preparations according to the present invention can be formulated as a lyophilisate.

The pharmaceutical preparations described herein may be presented in unit dose or multidose containers, such as sealed ampoules or vials. Such containers are typically sealed in such a way that they preserve the sterility and stability of the preparation until use. In general, preparations can be stored as suspensions, solutions or emulsions in oily or aqueous constituents. Alternatively, a pharmaceutical preparation can be stored in a freeze-dried state which only requires the addition of a sterile liquid carrier immediately prior to use.

Development of suitable dosage and treatment regimens for the use of the special preparations described here in a number of treatment regimens, including e.g. oral, parenteral, intravenous, intranasal and intramuscular administration and formulation, are well known in the art, some of which are briefly described below for general illustrative purposes.

In certain applications, the pharmaceutical compositions described herein can be delivered via oral administration to an animal. As such, these preparations may be formulated with an inert diluent or with an assimilable edible carrier or they may be enclosed in a hard or soft-shell gelatin capsule or they may be compressed into tablets or they may be introduced directly with food into the diet.

The active compounds can also be introduced with adjuvants and used in the form of edible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, biscuits and the like (see for example Matiowitz et al., Nature, 27 March 1997; 386( 6623):410-4; Hwang et al., Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U.S. Patent 5,641,515; U.S. Patent 5,580,579 and U.S. Patent 5,792,451). Tablets, troches, pills, capsules and the like may also contain any of a number of additional components, for example a binding agent, such as gum tragacanth, acacia, corn starch or gelatin; adjuvants, such as dicalcium phosphate; a disintegrant, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetener such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, wintergreen oil or cherry flavour. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills or capsules can be coated with shellac, sugar or both. Of course, any material used to make any dosage unit form must be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active compounds can be introduced into preparations and formulations with extended release.

Typically, these preparations will contain at least approx. 0.1% of the active compound or more, although the percentage of the active ingredient(s) can of course be varied and can suitably be between approx. 1 or 2% and approx. 60% or 70% or more of the weight or volume of the total preparation. Naturally, the amount of active compound(s) in each therapeutically useful preparation can be prepared in such a way that a suitable dose will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be understood by those skilled in the art of manufacturing such pharmaceutical preparations and as such, a variety of dosages and treatment regimens may be desirable.

For oral administration, the preparations according to the present invention can alternatively be introduced with one or more adjuvants in the form of a mouthwash, toothpaste, buccal tablet, oral spray or sublingual orally-administered preparation. Alternatively, the active ingredient may be introduced into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate or dispersed in a toothpaste or added in a therapeutically effective amount to a preparation which may include water, binders, abrasives, flavoring agents, foaming agents and humectants. funds. Alternatively, the preparations can be adapted in a tablet or solution form that can be placed under the tongue or otherwise dissolved in the mouth.

Under certain circumstances, it will be desirable to deliver the pharmaceutical preparations described here parenterally, intravenously, intramuscularly or also intraperitoneally. Such methods are well known to those skilled in the art, some of which are further described, for example, in U.S. Patent 5,543,158; U.S. patent 5,641,515 and U.S. patent 5,399,363. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water, suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under normal storage conditions and use, these preparations will generally contain a preservative to prevent the growth of microorganisms.

Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (see, for example, U.S. Patent 5,466,468). In all cases, the form must be sterile and must be liquid to the extent that easy sprayability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof and/or vegetable oils. Proper fluidity can be maintained, for example, by using a coating, such as lecithin, by maintaining the required particle size in the case of dispersion and/or by using surfactants. Prevention of the action of microorganisms can be remedied with various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferred to include isotonic agents, for example sugars or sodium chloride. Prolonged absorption of the injectable preparations can be produced by the use in the preparations of agents which delay absorption, for example aluminum monostearate and gelatin.

In one embodiment for parenteral administration in an aqueous solution, the solution must be appropriately buffered if necessary and the liquid diluent first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be used will be known to those skilled in the art in light of the present description. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences" 15th Ed., pages 1035-1038 and 1570-1580 ). Some variation in dose will necessarily occur depending on the condition of the individual being treated. Furthermore, for human administration, the preparations will of course preferably meet sterility, pyrogenicity and the general safety and purity standards required by the FDA Office of Biologics standards.

In another embodiment of the invention, the preparations described here can be formulated in neutral or salt form. Illustrative pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups in the protein) which are formed with inorganic acids such as for example hydrochloric or phosphoric acids or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron(III) hydroxides and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. After preparation, solutions will be administered in a manner compatible with the dose of preparation and in such an amount as is therapeutically effective.

The carriers can further include any and all solvents, dispersion media, constituents, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids and the like. Use of such media and agents for pharmaceutically active substances is well known in the field. Except where any conventional medium or agent is incompatible with the active ingredient, its use in the therapeutic preparations is encompassed. Supplementary active ingredients can also be introduced into the preparations. The term "pharmaceutically acceptable" denotes molecular entities and preparations which do not produce an allergic or similar adverse reaction when administered to a human.

In certain embodiments, the pharmaceutical preparations may be delivered by intranasal spray preparations, inhalation and/or other aerosol delivery agents. Methods for delivering genes, nucleic acids and peptide preparations directly to the lungs via nasal aerosol spray preparations are described in e.g. U.S. patent 5,756,353 and U.S. patent 5,804,212. Likewise, drug delivery using intranasal microparticle resins (Takenaga et al., J Controlled Release March 2, 1998; 52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U.S. patent 5,725,871) are also well known in the pharmaceutical area. Likewise, illustrative transmucosal drug delivery in the form of a polytetrafluoroethylene carrier matrix is described in U.S. Patent 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles and the like are used for introducing the preparations according to the present invention into suitable host cells/organisms. In particular, the preparations according to the present invention can be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere or a nanoparticle or the like. Alternatively, preparations according to the present invention can be bound, either covalently or non-covalently, to the surface of such carrier constituents.

The formation and use of liposome and liposome-like preparations as potential drug carriers is generally known to those skilled in the art (see, for example, Lasic, Trends Biotechnol, July 1998; 16(7):307-21; Takakura, Nippon Rinsho, March 1998 ; 56(3):691-5; Chandran et al., Indian J Exp Biol. Aug 1997; 35(8):801-9; Margalit, Crit RevTher Drug Carrier Syst. 1995;12(2-3): 233-61; U.S. Patent 5,567,434; U.S. Patent 5,552,157; U.S. Patent 5,565,213; U.S. Patent 5,738,868 and U.S. Patent 5,795,587, each specifically incorporated herein by reference in its entirety).

Liposomes have been used successfully with several cell types that are normally difficult to transfect by other procedures, including T-cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al., J Biol Chem. 25 Sep 1990; 265 (27):16337-42; Muller et al., DNA Cell Biol. Apr. 1990;9(3):221-9). In addition, liposomes are free of the DNA length restrictions typical of viral-based delivery systems. Liposomes have been effectively used to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors, and the like, into a variety of cultured cell lines and animals. Furthermore, the use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLV).

Alternatively, the invention provides, in other embodiments, pharmaceutically acceptable nanocapsule preparations of the preparations according to the present invention. Nanocapsules can generally encapsulate compounds in a stable and reproducible manner (see, for example, Quintanar-Guerrero et al., Drug Devlnd Pharm. Dec. 1998; 24(12): 1113-28). To avoid side effects due to intracellular polymer overload, such ultrafine particles (of size around 0.1 µm) can be designed using polymers that can be degraded in vivo. Such particles can be prepared as described, for example, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988; 5(1): 1 -20; zur Muhlen et al., Eur J Pharm Biopharm. March 1998; 45(2):149-55; Zambaux et al. J Controlled Release. Jan 2 1998; 50(1-3):31-40; and U.S. Patent 5,145,684.

Cancer therapeutic methods

In further aspects of the present invention, the pharmaceutical preparations described here can be used for the treatment of cancer, especially for immunotherapy of prostate cancer. By such methods, the pharmaceutical preparations described here are administered to a patient, typically a warm-blooded animal, preferably a human. A patient may, but need not, be affected by cancer. Accordingly, the above pharmaceutical preparations can be used to prevent the development of cancer or to treat a patient affected by cancer. Pharmaceutical preparations and vaccines can be administered either before or after surgical removal of primary tumors and/or treatment such as the administration of radiotherapy or conventional chemotherapeutic drugs. As described above, administration of the pharmaceutical preparations may be by any suitable method, including administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral routes.

In certain embodiments, immunotherapy may be active immunotherapy, where treatment is based on in vivo stimulation of the endogenous host immune system to respond to tumors by administration of immune response modifying agents (such as polypeptides and polynucleotides as set forth herein).

In other embodiments, immunotherapy can be passive immunotherapy, where treatment involves the delivery of agents with established tumor immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and do not necessarily depend on an intact host immune system. Examples of effector cells include T cells as described above, T lymphocytes (such as CD8<+>cytotoxic T lymphocytes and CD4<+>T helper tumor-infiltrating lymphocytes), killer cells (such as natural killer cells and lymphokine-activated killer cells ), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide set forth herein. T cell receptors and antibody receptors specific for the polypeptides disclosed herein can be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides provided herein can also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Patent No. 4,918,164) for passive immunotherapy.

Effector cells can generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Cultivation conditions for the expansion of simple antigen-specific effector cells to many billions in number while maintaining antigen recognition in vivo are well known in the field. Such in vitro culture conditions typically employ periodic stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As indicated above, immunoreactive polypeptides as provided herein can be used to rapidly expand antigen-specific T cell cultures to generate a sufficient number of cells for immunotherapy. In particular, antigen presenting cells, such as dendritic, macrophage, monocyte, fibroblast and/or B cells, can be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter suitable for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely and to survive for a long time in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long-term in significant numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al., Immunological Reviews 757:177, 1997).

Alternatively, a vector expressing a polypeptide set forth herein can be introduced into antigen-presenting cells taken from a patient and propagated by cloning ex vivo to be transplanted back into the same patient. Transfected cells can be reintroduced into the patient using any method known in the art, preferably in sterile form by intravenous, intracavital, intraperitoneal or intratumor administration.

Methods and frequency of administration of the therapeutic preparations described herein, as well as dose, will vary from individual to individual and can be readily established using standard techniques. In general, the pharmaceutical preparations and vaccines can be administered by injection { e.g. intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g. by aspiration) or orally. Preferably between 1 and 10 doses can be administered over a 52 week period. Preferably, 6 doses can be administered, at intervals of 1 month and booster vaccinations can be given periodically thereafter. Alternatively, protocols can be customized for individual patients. A suitable dose is an amount of a compound which, when administered as described above, can promote an anti-tumor immune response and is at least 10-50% above the basal (ie untreated) level. Such a response can be monitored by measuring anti-tumor antibodies in a patient or by vaccine-dependent formation of cytolytic effector cells that can kill the patient's tumor cells in vitro. Such vaccines must also be able to induce an immune response that leads to an improved clinical outcome (eg, more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients compared to non-vaccinated patients. In general, for pharmaceutical preparations and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 25 \ ±g to 5 mg per kg of patient. Suitable dose sizes will vary with the size of the patient, but will typically be in the range from approx. 0.1 ml to approx. 5 ml.

In general, an appropriate dose and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (eg, more frequent remissions, complete or partial or longer disease-free survival) in treated patients compared to untreated patients. Increases in pre-existing immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses can generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which can be performed using samples taken from a patient before and after treatment.

Cancer detection and diagnostic preparations, methods and kits

In general, cancer can be detected in a patient based on the presence of one or more prostate tumor proteins and/or polynucleotides encoding such proteins in a biological sample (eg blood, sera, sputum, urine and/or tumor biopsies) taken from the patient. In other words, such proteins can be used as markers to indicate the presence or absence of cancer such as prostate cancer. In addition, such proteins may be useful for the detection of other cancers. The binding agents provided herein generally allow detection of the level of antigen binding to the agent in the biological sample. Polynucleotide primers and probes can be used to detect the level of mRNA encoding a tumor protein, which is also indicative of the presence or absence of cancer. In general, a prostate tumor sequence should be present at a level that is at least three times higher in tumor tissue than in normal tissue.

There are a number of assay formats known to those skilled in the art for using a binding agent to detect polypeptide markers in a sample. See e.g. Harlowog Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of cancer in a patient can be determined by (a) contacting a biological sample taken from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide to a predetermined cut-off value.

In a preferred embodiment, the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the rest of the sample. The bound polypeptide can then be detected using a detection reagent that contains a reporter group and specifically binds to the binder/polypeptide complex. Such detection reagents can, for example, comprise a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive experiment can be used, where a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The degree to which components of the sample inhibit the binding of the labeled polypeptide to the binder is indicative of the reactivity of the sample with the immobilized binder. Suitable polypeptides for use in such experiments include full-length prostate tumor proteins and polypeptide portions thereof to which the binder binds, as described above.

The solid support can be any material known to those skilled in the art to which the tumor protein can bind. For example, the solid support can be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a ball or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinyl chloride. The carrier can also be a magnetic particle or a fiber optic sensor, such as those described in, for example, U.S. Pat. Patent No. 5,359,681. The binder can be immobilized on the solid support using a variety of techniques known to those skilled in the art, which are well described in the patent and scientific literature. In the context of the present invention, the term "immobilization" denotes both non-covalent association, such as adsorption and covalent binding (which may be a direct bond between the agent and functional groups on the support or may be a bond with a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption can be achieved by bringing the binder, in a suitable buffer, into contact with the solid support for a suitable period of time. The contact time varies with temperature, but is typically between approx. 1 hour and approx. 1 day. In general, contacting a well in a plastic microtiter plate (such as polystyrene or polyvinyl chloride) with an amount of binder in the range of approx. 10 ng to approx. 10 µg and preferably approx. 100 ng to approx. 1 µg, sufficient to immobilize a sufficient amount of binder.

Covalent bonding of binder to a solid support can generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binder. For example, the binding agent can be covalently bound to supports having a suitable polymer coating by using benzoquinone or by condensing an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnologi Catalog and Handbook, 1991, at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. This experiment can be carried out by first bringing an antibody that is immobilized on a solid support, usually the well of a microtiter plate, into contact with the sample, so that polypeptides in the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

More specifically, when the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those skilled in the art, such as bovine serum albumin or Tween 20™ (Sigma Chemical Co., St. Louis, MO). The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. The sample can be diluted with a suitable diluent such as phosphate-buffered saline (PBS) before incubation. In general, an appropriate contact time (ie, incubation time) is a period of time sufficient to detect the presence of polypeptide in a sample taken from a subject with prostate cancer. Preferably, the contact time is sufficient to achieve a level of bonding that is at least approx. 95% of that achieved by equilibrium between bound and unbound polypeptide. Those skilled in the art will appreciate that the time required to reach equilibrium can be easily determined by determining the level of binding that occurs over a period of time. At room temperature, an incubation time of approx. 30 minutes is generally sufficient.

Unbound sample can then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20™. The second antibody, which contains a reporter group, can then be added to the solid support. Preferred reporter groups include the groups listed above.

The detection reagent is then incubated with the immobilized antibody-polypeptide complex for a period of time sufficient to detect the bound polypeptide. An appropriate period of time can generally be determined by determining the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method used for detecting the reporter group depends on the type of reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods can be used to detect colorants, luminescent groups and fluorescent groups. Biotin can be detected using avidin, linked to another reporter group (usually a radioactive or fluorescent group or an enzyme). Enzyme reporter groups can generally be detected by addition of substrate (generally for a specific time period), followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of cancer, such as prostate cancer, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal corresponding to a predetermined cut-off value. In one preferred embodiment, the cut-off value for detection of cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without cancer. In general, a sample that generates a signal that is three standard deviations above the predetermined cutoff value is considered positive for cancer. In an alternative preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-7. Briefly, in this embodiment, the cut-off value can be determined from a plot of pairs of true positive values (ie, sensitivity) and false positive values (100% specificity) corresponding to each possible cut-off value for the diagnostic test result. The cutoff value for the plot closest to the upper left corner (ie the value that encompasses the largest area) is the most accurate cutoff value and a sample generating a signal higher than the cutoff value determined by this method can be considered positive. Alternatively, the cutoff value can be shifted to the left along the plot, to minimize the false positive value, or to the right, to minimize the false negative value. In general, a sample that generates a signal higher than the cutoff value determined by this method is considered positive for cancer.

In a related embodiment, the assay is performed in a flow-through or strip test format, where the binder is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides in the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binder then binds to the binder-polypeptide complex as a solution containing the second binder flows through the membrane. The detection of bound second binder can then be carried out as described above. In the strip test format, one end of the membrane to which binder is bound is immersed in a solution containing the sample. The sample migrates along the membrane through an area containing the second binder and into the area of immobilized binder. Concentration of the second binder in the area of immobilized antibody indicates the presence of cancer. Typically, the concentration of the second binder at that location forms a pattern, such as a line, that can be read visually. Absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is chosen to form a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to form a positive signal in the two-antibody sandwich assay, in the format described above. Preferred binders for use in such experiments are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane is in the range from approx. 25 ng to approx. 1 ^g and more preferably from approx. 50 ng to approx. 500 ng. Such tests can typically be performed with a very small amount of biological sample.

Of course, a number of other experimental protocols exist which are suitable for use with the tumor proteins or binders of the present invention. The above descriptions are only examples. For example, it will be clear to those skilled in the art that the above protocols can be easily modified to use tumor polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such tumor protein-specific antibodies can correlate with the presence of cancer.

Cancer can also or alternatively be detected based on the presence of T cells that specifically react with a tumor protein in a biological sample. In certain methods, a biological sample comprising CD4<+>and/or CD8<+>T cells isolated from a patient is incubated with a tumor polypeptide, a polynucleotide encoding such a polypeptide and/or APC expressing at least one immunogenic portion of such a polypeptide, and the presence or absence of specific activation of the T cells is detected. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells can be incubated in vitro for 2-9 days (typically 4 days) at 37°C with polypeptide (e.g. 5 - 25ng/ml). It may be desirable to incubate another aliquot of a T-cell sample in the absence of tumor polypeptide to serve as a control. For CD4<+>T cells, activation is preferably detected by assessing proliferation of the T cells. For CD8<+>T cells, activation is preferably detected by assessing cytolytic activity. A level of proliferation that is at least two times 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 indicated above, cancer can also or alternatively be detected based on the level of mRNA encoding a tumor protein in a biological sample. For example, at least two oligonucleotide primers can be used in a polymerase chain reaction (PCR)-based assay to amplify a portion of tumor cDNA derived from a biological sample, where at least one of the oligonucleotide primers is specific for (ie hybridizes to) a polynucleotide which codes for the tumor protein. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide that codes for a tumor protein can be used in a hybridization experiment to detect the presence of polynucleotide that codes for the tumor protein in a biological sample.

To allow hybridization under experimental conditions, oligonucleotide primers and probes must comprise an oligonucleotide sequence having at least approx. 60%, preferably at least approx. 75% and more preferably at least approx. 90%, identity to a part of a polynucleotide which codes for a tumor protein according to the present invention which is at least 10 nucleotides and preferably at least 20 nucleotides in length. Preferably, oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above. Oligonucleotide primers and/or probes that can be suitably used in the diagnostic methods described here are preferably at least 10-40 nucleotides long. In a preferred embodiment, the oligonucleotide primers comprise at least 10 consecutive nucleotides, more preferably at least 15 consecutive nucleotides, of a DNA molecule having a sequence as described here. Techniques for both PCR-based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 57:263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).

A preferred test uses RT-PCR, where PCR is used together with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and is reverse transcribed to produce cDNA molecules. PCR amplification using at least one specific primer generates a cDNA molecule, which can be separated and visualized using, for example, gel electrophoresis. Amplification can be performed on biological samples taken from a test patient and from an individual not affected by cancer. The amplification reaction can be performed on many dilutions of cDNA spanning orders of magnitude. A two-fold or greater increase in expression in multiple dilutions of the test patient sample compared to the same dilutions of a non-cancerous sample is typically considered positive.

In another embodiment, the preparations described here can be used as markers for the progression of cancer. In this embodiment, experiments as described above for the diagnosis of cancer can be performed over time and the change in the level of reactive polypeptide(s) or polynucleotide(s) evaluated. For example, the trials can be performed every 24-72 hours for a period of 6 months to 1 year and then performed as needed. In general, cancer develops in those patients where the level of polypeptide or polynucleotide detected increases over time. In contrast, cancer does not develop when the level of reactive polypeptide or polynucleotide either remains constant or decreases over time.

Certain in vivo diagnostic tests can be performed directly on a tumor. One such experiment involves bringing tumor cells into contact with a binding agent. The bound binder can then be detected directly or indirectly via a reporter group. Such binders can also be used in histological applications. Alternatively, polynucleotide probes can be used in such applications.

As indicated above, to improve sensitivity, multiple tumor protein markers can be examined in a given sample. It will be clear that binders specific for different proteins provided herein can be combined in a single experiment. Furthermore, multiple primers or probes can be used simultaneously. Selection of tumor protein markers can be based on routine trials to determine combinations that result in optimal sensitivity. In addition or alternatively, tests for tumor proteins given here can be combined with tests for other known tumor antigens.

The present invention further provides sets for use in any of the above diagnostic methods. Such sets typically comprise two or more components necessary for carrying out a diagnostic test. Components can be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a tumor protein. Such antibodies or fragments may be bound to a carrier material, as described above. One or more additional containers may include elements, such as reagents or buffers, to be used in the experiment. Such kits may also or alternatively contain a detection reagent as described above which contains a reporter group suitable for direct or indirect detection of antibody binding.

Alternatively, a kit may be designed to detect the level of mRNA encoding a tumor protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described above, which hybridizes to a polynucleotide encoding a tumor protein. Such an oligonucleotide can be used, for example, in PCR or hybridization experiments. Additional components that may be present in such a kit include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a tumor protein.

The following examples are given by way of illustration and not by way of limitation.

EXAMPLES

EXAMPLE 1

Isolation and characterization<g>of prostate-specific<p>ol<yp>e<p>times

This example describes the isolation of certain prostate-specific polypeptides from a prostate tumor cDNA library.

A human prostate tumor cDNA expression library was constructed from prostate tumor poly A<+>RNA using a Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning Kit (BRL Life Technologies, Gaithersburg, MD 20897) following the manufacturer's protocol. Specifically, prostate tumor tissue was homogenized with a polytron (Kinematica, Switzerland) and total RNA was extracted using Trizol reagent (BRL Life Technologies) as recommended by the manufacturer. Poly A<+>RNA was then purified using a Qiagen oligotex spin-column mRNA purification kit (Qiagen, Santa Clarita, CA 91355) according to the manufacturer's protocol. First strand cDNA was synthesized using the NotI/Oligo-dT18 primer. Double-stranded cDNA was synthesized, ligated with EcoRI/BAXI adapters (Invitrogen, San Diego, CA) and digested with Noti. After size fractionation with Chroma Spin-1000 columns (Clontech, Palo Alto, CA), cDNA was ligated into the EcoRI/NotI site of pCDNA3.1 (Invitrogen) and transformed into ElectroMax E. coli DH10B cells (BRL Life Technologies) by electroporation.

Using the same procedure, a normal human pancreas cDNA expression library was prepared from a pool of six tissue samples (Clontech). cDNA libraries were characterized by determining the number of independent colonies, the percentage of clones that had an insert, the average insert size, and by sequence analysis. Prostate tumor library contained 1.64 x 10<7> independent colonies, where 70% of clones have an insertion and the average insertion size is 1745 base pairs. The normal pancreatic cDNA library contained 3.3 x 10<6> independent colonies, with 69% of the clones having insertions and the average insertion size being 1120 base pairs. For both libraries, sequence analysis showed that the majority of clones had a full-length cDNA sequence and were synthesized from mRNA, with minimal rRNA and mitochondrial DNA contamination.

cDNA library subtraction was performed using the above prostate tumor and normal pancreas cDNA libraries, as described by Hara et al. ( Blood, 84:189-199, 1994 ) with some modifications. Specifically, the prostate tumor-specific subtracted cDNA library was generated as follows. Normal pancreas cDNA library (70 µg) was digested with EcoRI, Noti and Sful, followed by a fill-in reaction 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 (ag) of Photoprobe-biotin (Vector Laboratories, Burlingame, CA). As recommended by manufacturer, the resulting mixture was irradiated with a 270 W high mountain sun on ice for 20 min. Additional Photoprobe biotin (50 µl) was added and the biotinylation reaction was

repeated. After extraction with butanol five times, DNA was ethanol-precipitated and dissolved in 23 µl H 2 O to form driver DNA.

To generate tracer DNA, 10 µg prostate tumor cDNA library was digested with BamHI and XhoI, phenol chloroform extracted and passed through Chroma spinn-400 columns (Clontech). After ethanol precipitation, tracer DNA was dissolved in 5 µl H 2 O. Tracer DNA was mixed with 15 µl driver DNA and 20 µl of 2x hybridization buffer (1.5 M NaCl/10 mM EDTA/50 mM HEPES pH 7.5/0.2% sodium dodecyl sulfate), overlaid with mineral oil and completely heat-denatured. The sample was immediately transferred to a 68°C water bath and incubated for 20 hours (long hybridization [LH]). The reaction mixture was then subjected to streptavidin treatment followed by phenol/chloroform extraction. This process 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 of 2x hybridization buffer and subjected to hybridization at 68°C for 2 h (short hybridization [SH]). After removal of biotinylated double-stranded DNA, subtracted cDNA was ligated into the BamHI/XhoI site of chloramphenicol-resistant pBCSK<+>(Stratagene, La Jolla, CA 92037) and transformed into ElectroMax E. coli DH 10B cells by electroporation to generate a prostate tumor-specific subtracted cDNA library (referred to as "prostate subtraction 1").

To analyze the subtracted cDNA library, plasmid DNA was prepared from 100 independent clones, randomly selected from the subtracted prostate tumor-specific library and grouped based on insert size. Representative cDNA clones were further characterized by DNA sequencing with a Perkin Eimer/Applied Biosystems Division Automated Sequencer Model 373A (Foster City, CA). Six cDNA clones, hereafter referred to as F1-13, F1-12, F1-16, H1-1, H1-9 and H1-4, were shown to be abundant in the subtracted prostate-specific cDNA library. The determined 3' and 5' cDNA sequences for F1-12 are given in SEQ ID NO: 2 and 3, respectively, with determined 3' cDNA sequences for F1-13, F1-16, H1-1, H1-9 and H1 -4 are given respectively in SEQ ID NO: 1 and 4-7.

The cDNA sequences of the isolated clones were compared with known sequences in the gene bank using EMBL and GenBank databases (publication 96). Four of the prostate tumor cDNA clones, F1-13, F1-16, H1-1 and

H1-4, was determined to encode the following previously identified proteins: prostate-specific antigen (PSA), human glandular kallikrein, human tumor expression-enhanced gene, and mitochondrial cytochrome C oxidase subunit II. H1-9 was found to be identical to a previously identified human autonomously replicating sequence. No significant homologies to the cDNA sequence of F1-12 were found.

Subsequent investigations led to the isolation of a full-length cDNA sequence for F1-12 (also referred to as P504S). This sequence is given in SEQ ID NO: 107, with the corresponding putative amino acid sequence given in SEQ ID NO: 108. cDNA splice variants of P504S are given in SEQ ID NO: 600-605.

To clone less abundant prostate tumor-specific genes, cDNA library subtraction was performed by subtracting the prostate tumor cDNA library described above with the normal pancreas cDNA library and with the three most abundant genes in the previously subtracted prostate tumor-specific cDNA library : human glandular kallikrein, prostate-specific antigen (PSA) and mitochondrial cytochrome C oxidase subunit II. Specifically, 1 µg each of human glandular kallikrein, PSA, and mitochondrial cytochrome C oxidase subunit II cDNA in pCDNA3.1 was added to driver DNA and subtraction was performed as described above to yield a second subtracted cDNA library below referred to as "subtracted prostate tumor-specific cDNA library with tip potential ("spike")".

22 cDNA clones were isolated from the subtracted prostate tumor-specific cDNA library with peak potential. They determined 3' and 5' cDNA sequences for the 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 are respectively given in SEQ ID NO: 8-9, 10-11, 12-13, 14-15, 16-17, 18-19, 20-21, 22-23, 24- 25, 26-27 and 28-29. The determined 3'

cDNA sequences for the 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 is given respectively in SEQ ID NO: 30-40. Comparison of these sequences with those in the gene bank described above revealed no significant homologies with three of the five most abundant

DNA species, (J1-17, L1-12 and N1-1862; respectively SEQ ID NO: 8-9, 10-11 and 12-13). Of the two remaining most abundant species, one (J1-12; SEQ ID NO: 30) was found to be identical to the previously identified human pulmonary surfactant-associated protein and the other (K1-48; SEQ ID NO: 33) was found to have some homology with R. norvegicus mRNA in 2-arylpropionyl-CoA epimerase. Of the 17 less abundant cDNA clones isolated from the subtracted prostate tumor-specific cDNA library with peak potential, four (J1-16, K1-55, L1-6 and N1-1864; respectively SEQ ID NO:31, 34, 36 and 40) found to be identical to previously identified sequences, two (J1-21 and N1-1860; SEQ ID NO: 32 and 38, respectively) were found to show some homology to non-human sequences and two (L1-2 and N1 -1861; respectively SEQ ID NO: 35 and 39) were found to show some homology with known human sequences. No significant homologies were found with the 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, 18-19, 22-23, 24-25, 26-27, 28-29).

Subsequent investigations led to the isolation of full-length cDNA sequences for J1-17, L1-12 and N1-1862 (respectively SEQ ID NO: 109-111). The corresponding assumed amino acid sequences are given in SEQ ID NO: 112-114. L1-12 is also referred to as P501S. A cDNA splice variant of P501S is provided in SEQ ID NO: 606.

In a further experiment, four additional clones were identified by subtracting a prostate tumor cDNA library with normal prostate cDNA prepared from a pool of three normal prostate poly A+ RNAs (referred to as "prostate subtraction 2"). The determined cDNA sequences for these clones, hereinafter referred to as U1-3064, U1-3065, V1-3692 and 1A-3905, are provided in SEQ ID NOS: 69-72, respectively. Comparison of the determined sequences with those in the gene bank revealed no significant homologies with U1-3065.

A second spiking potential subtraction (referred to as "prostate subtraction spiking potential 2") was performed by subtracting a prostate tumor-specific cDNA library spiked with a normal pancreatic cDNA library and further spiked with PSA, J1-17, pulmonary surfactant-associated protein, mitochondrial DNA, cytochrome c oxidase subunit II, N1-1862, autonomous replicating sequence, L1-12 and tumor expression-enhanced gene. Four additional clones, hereafter referred to as V1-3686, R1-2330, 1B-3976 and V1-3679 were isolated. The determined cDNA sequences for these clones are given respectively in SEQ ID NO:73-76. Comparison of these sequences with those in the gene bank revealed no significant homologies with V1-3686 and R1-2330.

Further analysis of the three prostate subtractions described above (prostate subtraction 2, subtracted prostate tumor-specific peak potential cDNA library and prostate subtraction peak potential 2) resulted in the identification of 16 additional clones, referred to as 1G-4736, 1G-4738, 1G-4741, 1G-4744, 1G-4734, 1H-4774, 1H-4781, 1H-4785, 1H-4787, 1H-4796, 11- . 4810, 11-4811, 1J-4876, 1K-4884 and 1K-4896. The determined cDNA sequences for these clones are given in SEQ ID NO: 77-92, respectively. Comparison of these sequences with those in the gene bank as described above revealed no significant homologies with 1G-4741, 1G-4734, 11-4807, 1J-4876 and 1K-4896 (SEQ ID NO: 79, 81, 87, 90 and 92). Further analysis of the isolated clones led to the determination of extended cDNA sequences in 1G-4736, 1G-4738, 1G-4741, 1G-4744, 1H-4774, 1H-4781, 1H-4785, 1H-4787, 1H-4796 , 11-4807, 1J-4876, 1K-4884 and 1K-4896, given in SEQ ID NOS: 179-188 and 191-193, respectively, and determination of additional partial cDNA sequences in 11-4810 and 11-4811, given in SEKV ID NO: 189 and 190, respectively.

Further investigations with prostate subtraction tip potential 2 resulted in the isolation of three additional clones. Their sequences were determined as described above and compared to the latest GenBank. All three clones were found to have homology to known genes, which are cysteine-rich protein, KIAA0242 and KIAA0280 (SEQ ID NO: 317, 319 and 320, respectively). Further analysis of these clones with the Synteni microarray (Synteni, Palo Alto, CA) demonstrated that all three clones were overexpressed in most prostate tumors and prostatic BPH, as well as in the bulk of normal prostate tissue tested, but had low expression in all other normal loom.

A further subtraction was performed by subtracting a normal prostate cDNA library with normal pancreas cDNA (referred to as “prostate subtraction 3”). This led to the identification of six additional clones referred to as 1G-4761, 1G-4762, 1H-4766, 1H-4770, 1H-4771 and 1H-4772 (SEQ ID NOS: 93-98). Comparison of these sequences with those in the gene bank revealed no significant homologies with 1G-4761 and 1H-4771 (SEQ ID NO: 93 and 97, respectively). Further analysis of the isolated clones led to the determination of extended cDNA sequences for 1G-4761, 1G-4762, 1H-4766 and 1H-4772 given in SEQ ID NOS: 194-196 and 199, respectively, and to the determination of further partial cDNAs -sequences in 1H-4770 and 1H-4771, given in SEQ ID NO: 197 and 198, respectively.

Subtraction of a prostate tumor cDNA library, prepared from a pool of polyA+ RNA from three prostate cancer patients, with a normal pancreas cDNA library (prostate subtraction 4) led to the identification of eight clones, referred to as 1D-4297, 1D- 4309, 1D,1-4278, 1D-4288,1D-4283, 1D-4304, 1D-4296 and 1D-4280 (SEQ ID NO: 99-107). These sequences were compared with those in the gene bank as described above. No significant homologies were found with 1D-4283 and 1D-4304 (SEQ ID NO: 103 and 104, respectively). Further analysis of the isolated clones led to the determination of extended cDNA sequences in 1D-4309, 1D,1-4278,1D-4288, 1D-4283, 1D-4304, 1D-4296 and 1D-4280, given respectively in SEQ ID NO: NO: 200-206.

cDNA clones isolated in prostate subtraction 1 and prostate subtraction 2, described above, were colony-PCR amplified and their mRNA expression levels in prostate tumor, normal prostate and in various other normal tissues were determined using microarray technology (Synteni , Palo Alto, CA). Briefly, PCR amplification products were spotted onto slides in an array format, with each product occupying a unique location in the array. mRNA was extracted from the tissue sample to be tested, reverse transcribed and fluorescently labeled cDNA probes were generated. The microarrays were probed with the labeled cDNA probes, slides were scanned and fluorescence intensity was measured. This intensity correlates with the hybridization intensity. Two clones (referred to as P509S and P510S) were found to be overexpressed in prostate tumor and normal prostate and expressed at low levels in all other normal tissues tested (liver, pancreas, skin, bone marrow, brain, breast, adrenal gland, bladder, testis, salivary gland, colon, kidney, ovary, lung, spinal cord, skeletal muscle and colon). The determined cDNA sequences for P509S and P510S are provided in SEQ ID NO: 223 and 224, respectively. Comparison of these sequences with those in the gene bank as described above revealed some homology with previously identified ESTs.

Further investigations led to the isolation of the full-length cDNA sequence for P509S. This sequence is given in SEQ ID NO: 332, the corresponding putative amino acid sequence being given in SEQ ID NO: 339. Two variant full-length cDNA sequences for P510S are given in SEQ ID NO: 535 and 536, the corresponding putative amino acid sequences are given in SEQ ID NO: 537 and 538, respectively. Additional splice variants of P510S are given in SEQ ID NO: 598 and 599.

EXAMPLE 2

Determination of tissue-specificity of prostate-specific<p>ol<yp>e<p>times

Using gene-specific primers, mRNA expression levels were determined for 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) examined in a variety of normal and tumor tissues using RT-PCR.

Total RNA was extracted from a variety of normal and tumor tissues using Trizol reagent as described above. First strand synthesis was performed using 1-2 µg of total RNA with SuperScript II reverse transcriptase (BRL Life Technologies) at 42°C for one hour. The cDNA was then amplified by PCR with gene-specific primers. To ensure the semi-quantitative nature of RT-PCR, p-actin was used as an internal control for each of the tissues examined. First, serial dilutions of the first strand cDNA were prepared and RT-PCR experiments were performed using β-actin specific primers. A dilution was then chosen that incorporated linear range amplification of the β-actin template and that was sensitive enough to reflect the differences in initial copy numbers. Using these conditions, β-actin levels were determined for each reverse transcription reaction from each tissue. DNA contamination was minimized by DNase treatment and by ensuring a negative PCR result using first strand cDNA that was prepared without the use of reverse transcriptase.

mRNA expression levels were examined in four different types of tumor tissues (prostate tumor from 2 patients, breast tumor from 3 patients, colon tumor, lung tumor) and 16 different normal tissues, including prostate, colon, kidney, liver, lung, ovary, pancreas, skeletal muscle, skin , belly,

testicle, bone marrow and brain. F1-16 was found to be expressed at high levels in prostate tumor tissue, colon tumor and normal prostate and at lower levels in normal liver, skin and testes, expression being undetectable in the other tissues examined. H1-1 was found to be expressed at high levels in prostate tumor, lung tumor, breast tumor, normal prostate, normal colon and normal brain, at much lower levels in normal lung, pancreas, skeletal muscle, skin, small intestine, bone marrow and was not detected in the other tissues tested. J1-17 (P502S) and L1-12 (P501S) appear to be specifically overexpressed in the prostate, as both genes are expressed at high levels in prostate tumor and normal prostate, but at low to undetectable levels in all the other tissues examined. N1-1862 (P503S) was found to be overexpressed in 60% of prostate tumors and detectable in normal colon and kidney. RT-PCR results thus indicate that F1-16, H1-1, J1-17 (P502S), N1-1862 (P503S) and L1-12 (P501S) are either prostate-specific or are expressed at significantly elevated levels in the prostate .

Further RT-PCR studies showed that F1-12 (P504S) is overexpressed in 60% of prostate tumors, detectable in normal kidney, but not detectable in all other tissues tested. Similarly, R1-2330 was shown to be overexpressed in 40% of prostate tumors, detectable in normal kidney and liver, but not detectable in all other tissues tested. U1-3064 was found to be overexpressed in 60% of prostate tumors and also expressed in breast and colon tumors, but was not detectable in normal tissue.

RT-PCR characterization of R1-2330, U1-3064 and 1D-4279 showed that these three antigens are overexpressed in the prostate and/or prostate tumors.

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

The microarray technology described above was used to determine the expression levels of representative antigens described here in prostate tumor, breast tumor and the following normal tissues: prostate, liver, pancreas, skin, bone marrow, brain, breast, adrenal gland, bladder, testis, salivary gland, colon, kidney, ovary, lung, spinal cord, skeletal muscle and colon. L1-12 (P501S) was found to be overexpressed in normal prostate and prostate tumor, with some expression detected in normal skeletal muscle. Both J1-12 and F1-12 (P504S) were found to be overexpressed in prostate tumor, with expression being lower or undetectable in all other tissues tested. N1-1862 (P503S) was found to be expressed at high levels in prostate tumor and normal prostate and at low levels in normal colon and normal colon, expression being undetectable in all other tissues tested. R1-2330 was found to be overexpressed in prostate tumor and normal prostate and to be expressed at lower levels in all other tissues tested. 1D-4279 was found to be overexpressed in prostate tumor and normal prostate, expressed at lower levels in normal spinal cord and to be undetectable in all other tissues tested.

Further microarray analysis to specifically determine the extent to which P501S (SEQ ID NO: 110) was expressed in breast tumor revealed moderate overexpression not only in breast tumor but also in metastatic breast tumor (2/31), with negligible to low expression in normal tissue . These data indicate that P501S may be overexpressed in various breast tumors as well as in prostate tumors.

The expression levels of 32 ESTs (expressed sequence tags) described by Vasmatzis et al. ( Proe. Nati. Acad. Sei. USA 95:300-304, 1998 ) in a variety of tumor and normal tissues was examined by microarray technology as described above. Two of these clones (referred to as P1000C and P1001C) were found to be overexpressed in prostate tumor and normal prostate and expressed at low to undetectable levels in all other tissues tested (normal aorta, thymus, resting and activated PBMC, epithelial cells, spinal cord, adrenal , 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. The determined cDNA sequences for P1000C and P1001C are given in SEQ ID NO, respectively : 384 and 472. The sequence of P1001C was found to show some homology to the previously isolated human mRNA for JM27 protein. Subsequent comparison of the sequence in SEQ ID NO: 384 with sequences in public databases led to the identification of a full-length cDNA- sequence of P1000C (SEQ ID NO: 786), which encodes a 492 amino acid sequence. Analysis of the amino acid sequence using the PSORT II program led to the identification of a putative transmembrane domain from amino acids 84-100. c The DNA sequence of the open reading frame of P1000C, including the stop codon, is given in SEQ ID NO: 787, the open reading frame without the stop codon is given in SEQ ID NO: 788. The full-length amino acid sequence of P1000C is given in SEQ ID NO: 789. SEQ ID NO: 790 and 791 represent amino acids 1-100 and 100-492 of P1000C, respectively.

The expression of the polypeptide encoded by the full-length cDNA sequence for F1-12 (also referred to as P504S; SEQ ID NO: 108) was examined by immunohistochemical analysis. Rabbit anti-P504S polyclonal antibodies were raised against full-length P504S protein by standard techniques. Subsequent isolation and characterization of the polyclonal antibodies was also performed by techniques well known in the art. Immunohistochemical analysis showed that the P504S polypeptide was expressed in 100% of prostate carcinoma samples tested (n=5).

Rabbit anti-P504S polyclonal antibody did not appear to label benign prostate cells with the same cytoplasmic granular labeling, but rather with light nuclear labeling. Analysis of normal tissues showed that the encoded polypeptide was found to be expressed in some but not all normal human tissues. Positive cytoplasmic labeling with rabbit anti-P504S polyclonal antibody was found in normal human kidney, liver, brain, colon and lung-associated macrophages, while heart and bone marrow were negative.

These data indicate that P504S polypeptide is present in prostate cancer tissue and that there are qualitative and quantitative differences in the labeling between benign prostatic hyperplasia tissue and prostate cancer tissue, indicating that this polypeptide can be detected selectively in prostate tumors and therefore be applicable in diagnosis of prostate cancer.

EXAMPLE 3

Isolation and Characterization<g>of Prostate-Specific<p>ol<yp>e<p>times by PCR-BASED SUBTRACTION

A cDNA subtraction library, containing cDNA from normal prostate subtracted with ten other normal tissue cDNAs (brain, heart, kidney, liver, lung, ovary, placenta, skeletal muscle, spleen and thymus) and then subjected to a first round of PCR amplification, was acquired from Clontech. This library was subjected to a second round of PCR amplification, according to the manufacturer's protocol. The resulting cDNA fragments were subcloned into the pT7 Blue T vector (Novagen, Madison, WI) and transformed into XL-1 Blue MRF' E. coli (Stratagene). DNA was isolated from independent clones and sequenced using a Perkin Eimer/Applied Biosystems Division Automated Sequencer Model 373A: 59 positive clones were sequenced. Comparison of the DNA sequences from these clones with those in the gene bank, as described above, revealed no significant homologies with 25 of these clones, hereafter referred to as P5, P8, P9, P18, P20, P30, P34, P36, P38, P39 , P42, P49, P50, P53, P55, P60, P64, P65, P73, P75, P76, P79 and P84. The determined cDNA sequences for these clones are given respectively in SEQ ID NOS: 41-45, 47-52 and 54-65. P29, P47, P68, P80 and P82 (SEQ ID NOS: 46, 53 and 66-68, respectively) were found to show some degree of homology to previously identified DNA sequences. As far as the inventors know, none of these sequences have previously been shown to be present in the prostate.

Further investigation using the sequence of SEQ ID NO: 67 as a probe in standard full-length cloning methods resulted in the isolation of three cDNA sequences that appear to be splice variants of P80 (also known as P704P). These sequences are given in SEQ ID NO: 620-622.

Further investigations using the PCR-based methodology described above resulted in the isolation of more than 180 additional clones, of which 23 clones were found to show no significant homology to known sequences. The determined cDNA sequences for these clones are given in SEQ ID NO: 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) were found to show some homology to previously identified ESTs. An additional ten clones (SEQ ID NOS: 161-170) were found to have some degree of homology to known genes. Larger cDNA clones containing the P20 sequence represent splice variants of a gene referred to as P703P. The determined DNA sequence for the variants referred to as DE1, DE13 and DE14 are provided in SEQ ID NO:

ID NO: 171,175 and 177, the corresponding putative amino acid sequences being given in SEQ ID NO: 172, 176 and 178, respectively. The determined cDNA sequence for an extended spliced form of P703 is given in SEQ ID NO: 225. The DNA sequences for the splice variants referred to as DE2 and DE6 are given in SEQ ID NO: 173 and 174 respectively.

mRNA expression levels for representative clones in 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 activated and non-activated PBMC were determined by RT-PCR as described above. Expression was examined in one sample of each tissue type unless otherwise indicated.

P9 was found to be highly expressed in normal prostate and prostate tumor compared to all normal tissues tested, except normal colon which showed comparable expression. P20, part of the P703P gene, was found to be highly expressed in normal prostate and prostate tumor, compared to all twelve normal tissues tested. A modest increase in expression of P20 in breast tumor (n=2), colon tumor and lung tumor was seen compared to all normal tissues, except lung (1 of 2). Increased expression of P18 was found in normal prostate, prostate tumor and breast tumor compared to other normal tissues, except lung and stomach. A modest increase in expression of P5 was observed in normal prostate compared to most other normal tissues. However, slightly elevated expression was seen in normal lung and PBMC. Elevated expression of P5 was also observed in prostate tumors (2 of 5), breast tumor and one lung tumor sample. For P30, similar expression levels were seen in normal prostate and prostate tumor, compared to six of twelve other normal tissues tested. Increased expression was seen in breast tumors, one lung tumor sample and one colon tumor sample and also in normal PBMC. P29 was found to be overexpressed in prostate tumor (5 of 5) and normal prostate (5 of 5) compared to bulk normal tissue. However, significant expression of P29 was observed in normal colon and normal lung (2 of 2). P80 was found to be overexpressed in prostate tumor (5 of 5) and normal prostate (5 of 5) compared to all other normal tissues tested, with increased expression also seen in colon tumor.

Further investigations resulted in the isolation of twelve additional clones, hereafter referred to as 10-d8, 10-h10, 11-c8, 7-g6, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3, 8-h11, 9-f12 and 9-f3. The determined DNA sequences in 10-d8, 10-h10, 11-c8, 8-d4, 8-d9, 8-h11, 9-f12 and 9-f3 are given respectively in SEQ ID NO: 207, 208, 209 , 216, 217, 220, 221 and 222. The determined forward and reverse DNA sequences in 7-g6, 8-b5, 8-b6 and 8-g3 are given in SEQ ID NO: 210 and 211, respectively; 212 and 213; 214 and 215; and 218 and 219. Comparison of these sequences with those in the gene bank revealed no significant homologies with the sequence of 9-f3. Clones 10-d8, 11 -c8 and 8-h11 were found to show some homology to previously isolated ESTs, while 10-h10, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3 and 9- f12 was found to show some homology to previously identified genes. Further characterization of 7-G6 and 8-G3 showed identity with the known genes PAP and PSA, respectively.

mRNA expression levels for these clones were determined by application

of the micro-array technology described above. Clones 7-G6, 8-G3, 8-B5, 8-B6, 8-D4, 8-D9, 9-F3, 9-F12, 9-H3, 10-A2, 10-A4, 11-C9 and 11 -F2 was found to be overexpressed in prostate tumor and normal prostate, with expression in other tissues tested being low or undetectable. Increased expression of 8-F11 was seen in prostate tumor and normal prostate, bladder, skeletal muscle and colon. Increased expression of 10-H10 was seen in prostate tumor and normal prostate, bladder, lung, colon, brain and colon. Increased expression of 9-B1 was seen in prostate tumor, breast tumor and normal prostate, salivary gland, colon and skin, with increased expression of 11-C8 seen in prostate tumor and normal prostate and colon.

An additional cDNA fragment derived from the PCR-based normal prostate subtraction, described above, was found to be prostate-specific by both microarray technology and RT-PCR. The determined cDNA sequence of this clone (referred to as 9-A11) is provided in SEQ ID NO: 226. Comparison of this sequence with those in public databases revealed 99% identity with the known gene H0XB13.

Further investigations led to the isolation of clones 8-C6 and 8-H7. The determined cDNA sequences for these clones are provided in SEQ ID NO: 227 and 228, respectively. These sequences were found to show some homology to previously isolated ESTs.

PCR and hybridization-based methods were used to obtain longer cDNA sequences for clone P20 (also referred to as P703P), yielding three additional cDNA fragments that progressively extended the 5' end of the gene. These fragments, referred to as P703PDE5, P703P6.26 and P703PX-23 (SEQ ID NO: 326, 328 and 330, with the putative corresponding amino acid sequences given in SEQ ID NO: 327, 329 and 331, respectively) contain additional 5' sequence . P703PDE5 was recovered by screening a cDNA library (#141-26) with a portion of P703P as a probe. P703P6.26 was recovered from a mixture of three prostate tumor cDNAs and P703PX_23 was recovered from cDNA library (#438-48). Together, the additional sequences comprise the entire putative mature serine protease together with part of the putative signal sequence. The full-length cDNA sequence for P703P is provided in SEQ ID NO: 524, with the corresponding amino acid sequence provided in SEQ ID NO: 525.

Using computer algorithms, the following regions of P703P were predicted to represent potential HLA A2-binding CTL epitopes: amino acids 164-172 of SEQ ID NO: 525 (SEQ ID NO: 723); amino acids 160-168 of SEQ ID NO: 525 (SEQ ID NO: 724); amino acids 239-247 of SEQ ID NO: 525 (SEQ ID NO: 725); amino acids 118-126 of SEQ ID NO: 525 (SEQ ID NO: 726); amino acids 112-120 of SEQ ID NO: 525 (SEQ ID NO: 727); amino acids 155-164 of SEQ ID NO: 525 (SEQ ID NO: 728); amino acids 117-126 of SEQ ID NO: 525 (SEQ ID NO: 729); amino acids 164-173 of SEQ ID NO: 525 (SEQ ID NO: 730); amino acids 154-163 of SEQ ID NO: 525 (SEQ ID NO: 731); amino acids 163-172 of SEQ ID NO: 525 (SEQ ID NO: 732); amino acids 58-66 of SEQ ID NO: 525 (SEQ ID NO: 733); and amino acids 59-67 of SEQ ID NO: 525 (SEQ ID NO: 734).

P703P was found to show some homology to previously identified proteases, such as thrombin. The thrombin receptor has been shown to be preferentially expressed in highly metastatic breast carcinoma cells and breast carcinoma biopsy specimens. Introduction of thrombin receptor antisense cDNA has been shown to inhibit invasion of metastatic breast carcinoma cells in culture. Antibodies against thrombin receptor inhibit thrombin receptor activation and thrombin-induced platelet activation. Furthermore, peptides that resemble the receptor's bound ligand domain inhibit platelet aggregation by thrombin. P703P may play a role in prostate cancer through a protease-activated receptor on the cancer cell or on stromal cells. The potential trypsin-like protease activity of P703P can either activate a protease-activated receptor on the cancer cell membrane to promote tumorigenesis or activate a protease-activated receptor on adjacent cells (such as stromal cells) to secrete growth factors and/or proteases ( such as matrix metalloproteinases) that can promote tumor angiogenesis, invasion and metastasis. P703P can thus promote tumor progression and/or metastasis through activation of the protease-activated receptor. Polypeptides and antibodies that block P703P receptor interaction can therefore be suitably used in the treatment of prostate cancer.

To determine whether P703P expression increases with increased

severity of Gleason grade, an indicator of tumor stage, quantitative PCR analysis was performed on prostate tumor samples with a range of Gleason scores from 5 to > 8. The mean level of P703P expression increased with increasing Gleason score, indicating that P703P expression may correlate with increased disease severity.

Further investigations using a PCR-based subtraction library of a prostate tumor pool subtracted against a pool of normal tissue (referred to as JP: PCR subtraction) resulted in the isolation of 13 additional clones, seven of which had no significant homology to known GenBank sequences. The determined cDNA sequences for these seven clones (P711P, P712P, new 23, P774P, P775P, P710P and P768P) are given in SEQ ID NO: 307-311, 313 and 315, respectively. The remaining six clones (SEQ ID NO: 316 and 321-325) were shown to have some homology to known genes. By microarray analysis, all 13 clones showed three or more fold overexpression in prostate tissue, including prostate tumors, BPH and normal prostate compared to normal non-prostate tissue. Clones P711P, P712P, ny 23 and P768P showed overexpression in most prostate tumor and BPH tissues tested (n=29) and in the majority of normal prostate tissue (n=4), but background to low expression levels in all normal tissues. Clones P774P, P775P and P710P showed relatively lower expression, and expression in fewer prostate tumors and BPH samples, with negative to low expression in normal prostate.

Further investigations led to the isolation of an extended cDNA sequence for P712P (SEQ ID NO: 552). The amino acid sequences encoded by 16 predicted open reading frames present in the sequence in SEQ ID NO: 552 are given in SEQ ID NO: 553-568.

Full-length cDNA for P711P was obtained using the partial sequence of SEQ ID NO: 307 to screen a prostate cDNA library. Specifically, a directional cloned prostate cDNA library was prepared using standard techniques. One million colonies of this library were plated on LB/Amp plates. Nylon membrane filters were used to lift these colonies and the cDNA picked up by these filters was denatured and cross-linked to the filters by UV light. The P711P cDNA fragment of SEQ ID NO: 307 was radioactively labeled and used to hybridize with these filters. Positive clones were selected and cDNA was prepared and sequenced using an automatic Perkin Eimer/Applied Biosystems sequencer. The determined full-length sequence of P711P is provided in SEQ ID NO: 382, with the corresponding putative amino acid sequence provided in SEQ ID NO: 383.

Using PCR and hybridization-based methods, additional cDNA sequence information was derived for two clones described above, 11-C9 and 9-F3, hereafter referred to as P707P and P714P, respectively (SEQ ID NO: 333 and 334). After comparison with the latest GenBank, P707P was found to be a splice variant of the known gene HoxB13. In contrast, no significant homologies with P714P were found. Further investigation using the sequence of SEQ ID NO: 334 as a probe by standard full-length cloning methods resulted in an extended cDNA sequence for P714P. This sequence is provided in SEQ ID NO: 619. This sequence was found to show some homology to the gene encoding human ribosomal L23A protein.

Clones 8-B3, P89, P98, P130 and P201 (as described in U.S. Patent Application No. 09/020,956, filed February 9, 1998) were found to be contained in one contiguous sequence, referred to as P705P (SEQ ID NO: 335 , with the presumed amino acid sequence given in SEQ ID NO: 336), which was determined to be a splice variant of the known gene NKX 3.1.

Further investigation of P775P resulted in the isolation of four additional sequences (SEQ ID NO: 473-476) all of which are splice variants of the P775P gene. The sequence in SEQ ID NO: 474 was found to contain two open reading frames (ORFs). The putative amino acid sequences encoded by these ORFs are given in SEQ ID NO: 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: 479 ID NO: 473 was found to contain four ORFs. The putative amino acid sequences coded for by these ORFs are given in SEQ ID NO: 480-483. Additional splice variants of P775P are provided in SEQ ID NO: 593-597.

Subsequent investigations led to the identification of a genomic region on chromosome 22q11.2, known as the Cat's Eye syndrome region, which contains the five prostate genes P704P, P712P, P774P, P775P and B305D. The relative location of each of these five genes in the genomic region is shown in Fig. 10. This region may therefore be related to malignant tumors, and other potential tumor genes may be contained in this region. These investigations also led to the identification of a potential open reading frame (ORF) for P775P (given in SEQ ID NO: 533) which codes for the amino acid sequence in SEQ ID NO: 534.

Comparison of the clone with SEQ ID NO: 325 (referred to as P558S) with sequences in the GenBank and GeneSeq DNA databases showed that P558S is identical to the prostate-specific transglutaminase gene, which is known to have two forms. The full-length sequences for the two forms are provided in SEQ ID NO: 630 and 631, with the corresponding amino acid sequences provided in SEQ ID NO: 632 and 633, respectively. The cDNA sequence of SEQ ID NO: 631 has a 15 base pair insertion, resulting in a 5 amino acid insertion in the corresponding amino acid sequence (SEQ ID NO: 633). This insertion is not present in the sequence in SEQ ID NO: 630.

Further investigation of P768P (SEQ ID NO: 315) led to the identification of the putative full-length open reading frame (ORF). The cDNA sequence of the ORF with a stop codon is provided in SEQ ID NO: 764. The cDNA sequence of the ORF without a stop codon is provided in SEQ ID NO: 765, with the corresponding amino acid sequence provided in SEQ ID NO: 766. This sequence was found to show 86% identity with a rat calcium transporter protein, indicating that P768P may represent a human calcium transporter protein. The locations of transmembrane domains in P768P were predicted using the PSORT II computer algorithm. Six transmembrane domains were predicted at amino acid positions 118-134, 172-188, 211-227, 230-246, 282-298 and 348-364. The amino acid sequences in SEQ ID NO: 767-772 respectively represent amino acids 1-134, 135-188, 189-227, 228-246, 247-298 and 299-511 of P768P.

EXAMPLE 4

Synthesis of<p>ol<yp>e<p>times

Polypeptides can be synthesized on a Perkin Eimer/Applied Biosystems 430A peptide synthesizer using FMOC chemistry with HPTU (O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) activation. A Gly-Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method for conjugation, attachment to an immobilized surface or labeling of the peptide. Cleavage of the peptides from the solid support can be performed using the following cleavage mixture: trifluoroacetic acid: ethanedithiol: thioanisole: water: phenol (40:1:2:2:3). After cleavage for 2 hours, the peptides can be precipitated in cold methyl-t-butyl ether. The peptide pellet can then be dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized before purification by C18 reverse phase HPLC. A gradient of 0%-60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) can be used to elute the peptides. After lyophilization of the pure fractions, the peptides can be characterized by using electrospray or other types of mass spectrometry and by amino acid analysis.

EXAMPLE 5

Further isolation and characterization of prostate-specific <p>ol<yp>e<p>times by PCR-based subtraction

A cDNA library generated from prostate primary tumor mRNA as described above was subtracted with cDNA from normal prostate. The subtraction was performed using a PCR-based protocol (Clontech), which was modified to generate larger fragments. In this protocol, tester and driver double-stranded cDNAs were separately digested with five restriction enzymes that recognize six-nucleotide restriction sites (Mlul, Mscl, Pvull, Sali, and Stul). This digestion resulted in an average cDNA size of 600 bp, instead of the average size of 300 bp resulting from digestion with RsaI according to the Clontech protocol. This modification did not affect the subtraction efficiency. Two tester populations were then formed with different adapters and the driver library remained without adapters.

Tester and driver libraries were then hybridized using excess driver cDNA. In the first hybridization step, drivers were separately hybridized with each of the two tester cDNA populations. This resulted in populations of (a) unhybridized tester cDNA, (b) tester cDNA hybridized to other tester cDNAs, (c) tester cDNA hybridized to driver cDNA, and (d) unhybridized driver cDNA. The two separate hybridization reactions were then combined and rehybridized in the presence of additional denatured driver cDNA. After this second hybridization, in addition to populations (a) to (d), a fifth population (e) was formed, where the tester cDNA with one adapter hybridized to the tester cDNA with the other adapter. Consequently, the second hybridization step resulted in the enrichment of 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 performed using adapter-specific primers. Only population(s), containing tester cDNA that does not hybridize to driver cDNA, was amplified exponentially. A second PCR amplification step was then performed, to reduce background and further enrich differentially expressed sequences.

This PCR-based subtraction technique normalizes differentially expressed cDNA so that rare transcripts overexpressed in prostate tumor tissue can be recovered. Such transcripts would be difficult to recover by traditional subtraction methods.

In addition to genes known to be overexpressed in prostate tumor, 77 additional clones were identified. Sequences of these partial cDNAs are provided in SEQ ID NO: 29 to 305. Most of these clones had no significant homology to database sequences. Exceptions were JPTPN23 (SEQ ID NO: 231; similarity to pig valosin-containing protein), JPTPN30 (SEQ ID NO: 234; similarity to rat proteasome subunit mRNA), JPTPN45 (SEQ ID NO: 243; similarity to rat norvegicus cytosolic NADP-dependent isocitrate dehydrogenase), JPTPN46 (SEQ ID NO: 244; similarity to human subclone H8 4 d4 DNA sequence), JP1D6 (SEQ ID NO: 265; similarity to 6th gallus dynein light chain-A), JP8D6 ( SEQ ID NO: 288; similarity to human BAC clone RG016J04), JP8F5 (SEQ ID NO: 289; similarity to human subclone H8 3 b5 DNA sequence) and JP8E9 (SEQ ID NO: 299; similarity to human Alu sequence) .

Further studies using the PCR-based subtraction library consisting of a prostate tumor pool subtracted against a normal prostate pool (referred to as PT-PN PCR subtraction) yielded three additional clones. Comparison of the cDNA sequences of these clones with the latest publication from GenBank revealed no significant homologies with the two clones referred to as P715P and P767P (SEQ ID NOS: 312 and 314). The remaining clone was found to have some homology to the known gene KIAA0056 (SEQ ID NO: 318). Using microarray analysis to measure mRNA expression levels in different tissues, all 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 a factor of three or greater, with elevated expression seen in the majority of normal prostate samples and in fetal tissue, but negative to low expression in all other normal tissues. Clone P767P was overexpressed in many prostate tumors and BPH tissues, with moderate expression levels in half of the normal prostate samples and background to low expression in all other normal tissues tested.

Further microarray analysis as described above of the PT-PN PCR subtraction library and of a DNA subtraction library containing cDNA from prostate tumor subtracted with a pool of normal tissue cDNA led to the isolation of 27 additional clones (SEQ ID NOS: 340-365 and 381) that were found to be overexpressed in prostate tumor. The clones of SEQ ID NO: 341, 342, 345, 347, 348, 349, 351, 355-359, 361, 362 and 364 were also found to be expressed in normal prostate. Expression of all 26 clones in a variety of normal tissues was found to be low or undetectable, with the exception of P544S (SEQ ID NO: 356) which was found to be expressed in the small intestine. Of the 26 clones, 11 (SEQ ID NOS: 340-349 and 362) were found to show some homology with previously identified sequences. No significant homologies were found with the clones of SEQ ID NO: 350, 351, 353-361 and 363-365.

Comparison of the sequence in SEQ ID NO: 362 with sequences in GenBank and GeneSeq DNA databases showed that this clone (referred to as P788P) is identical to GeneSeq accession no. X27262, which codes for a protein found in the GeneSeq protein accession no. Y00931. The full-length cDNA sequence of P788P is provided in SEQ ID NO: 634, with the corresponding predicted amino acid provided in SEQ ID NO: 635. Subsequently, a full-length cDNA sequence for P788P containing polymorphisms not found in the sequence in SEQ ID NO: 635 ID NO: 634, cloned multiple times by PCR amplification from cDNA prepared from many RNA templates from three individuals. This particular cDNA sequence of this polymorphic variant of P788P is provided in SEQ ID NO: 636, with the corresponding amino acid sequence provided in SEQ ID NO: 637. The sequence in SEQ ID NO: 637 differs from that in SEQ ID NO: 635 by six amino acid residues. The P788P protein has 7 potential transmembrane domains at the C-terminal part and is predicted to be a plasma membrane protein with an extracellular N-terminal region.

Further investigation of the clone of SEQ ID NO: 352 (referred to as P790P) led to the isolation of the full-length cDNA sequence of SEQ ID NO: 526. The corresponding predicted amino acid is given in SEQ ID NO: 527. Data from two quantitative PCR experiments showed that P790P is overexpressed in 11/15 prostate tumor samples tested and is expressed at low levels in spinal cord, with no expression seen in all other normal samples tested. Data from additional PCR experiments and microarray experiments showed overexpression in normal prostate and prostate tumor with little or no expression in other tissues tested. P790P was subsequently found to show significant homology to a previously identified G protein-coupled prostate tissue receptor.

Further investigation of the clone of SEQ ID NO: 354 (referred to as P776P) led to the isolation of an extended cDNA sequence, provided in SEQ ID NO: 569. The determined cDNA sequences of three additional splice variants of P776P are provided in SEQ ID NO: NO: 570-572. The amino acid sequences encoded by two putative open reading frames (ORFs) contained in SEQ ID NO: 570, one putative ORF contained in SEQ ID NO: 571 and 11 putative ORFs contained in SEQ ID NO: 569 are given in SEQ ID NO: 573, respectively -586. Further investigations led to the isolation of the full-length sequence of the clone of SEQ ID NO: 570 (given in SEQ ID NO: 737). Full-length cloning efforts for the clone of SEQ ID NO: 571 led to the isolation of two sequences (provided in SEQ ID NO: 738 and 739), representing a single clone, which is identical except for a polymorphic insertion/deletion at position 1293 Specifically, the clone with SEQ ID NO: 739 (referred to as clone F1) has a C at position 1293. The clone with SEQ ID NO: 738 (referred to as clone F2) has a single base pair deletion at position 1293. The putative amino acid sequences encoded for of 5 open reading frames located in SEQ ID NO: 737 are given in SEQ ID NO: 740-744, with the putative amino acid sequences encoded by the clone of SEQ ID NO: 738 and 739 given in SEQ ID NO: 745-750.

Comparison of the cDNA sequences of clones P767P (SEQ ID NO: 314) and P777P (SEQ ID NO: 350) with sequences in the GenBank human EST database showed that the two clones shared many EST sequences, indicating that P767P and P777P may represent the same gene. A DNA consensus sequence derived from a DNA sequence alignment of P767P, P777P and multiple EST clones is provided in SEQ ID NO: 587. The amino acid sequences encoded by three putative ORFs located in SEQ ID NO: 587 are provided in SEQ ID NO: 588-590.

The clone of SEQ ID NO: 342 (referred to as P789P) was found to show homology to a previously identified gene. The full-length cDNA sequence for P789P and the corresponding amino acid sequence are provided in SEQ ID NO: 735 and 736, respectively.

EXAMPLE 6

Pe<p>tid<p>rimin<g>of mice and<p>ro<p>a<g>erin<g>of CTL lines

6.1. This example illustrates the generation of a CTL cell line specific for cells expressing the P502S gene.

Mice expressing the human HLA A2Kb transgene (provided by Dr L. Sherman, The Scripps Research Institute, La Jolla, CA) were immunized with P2S#12 peptide (VLGWVAEL; SEQ ID NO: 306), which is derived from P502S- gene (also referred to herein as J1-17, SEQ ID NO: 8), as described by Theobald et al., Proe. Nati. Acad. Pollock. USA 92:11993-11997, 1995 with the following modifications. Mice were immunized with 100 µg of P2S#12 and 120 µg of an I-Ab binding peptide derived from hepatitis B virus protein emulsified in incomplete Freund's adjuvant. Three weeks later, these mice were euthanized and, using a nylon mesh, simple cell suspensions were prepared. Cells were then resuspended at 6 x 10<6> cells/ml in complete medium (RPMI-1640; Gibco BRL, Gaithersburg, MD) containing 10% FCS, 2 mM Glutamine (Gibco BRL), sodium pyruvate (Gibco BRL), non-essential amino acids (Gibco BRL), 2 x 10'<5>M 2-mercaptoethanol, 50 U/ml penicillin and streptomycin and cultured in the presence of the irradiated (3000 rad) P2S#12 pulse (5 mg/ml P2S#12 and 10 mg/ml β2-microglobulin) LPS blasts (A2 transgenic spleen cells cultured in the presence of 7 ug/ml dextran sulfate and 25 ug/ml LPS for 3 days). Six days later the cells (5 x 105/ml) were restimulated with 2.5 x 10<6>/ml peptide pulsed irradiated (20,000 rad) EL4A2KB cells (Sherman et al, Science 258:815-818, 1992) and 3 x 10<6>/ml A2 transgenic spleen feeder cells. Cells were cultured in the presence of 20 U/ml IL-2. Cells were still restimulated on a weekly basis as described, in preparation for cloning the line.

P2S#12 line was cloned by limiting dilution assay with peptide-pulsed EL4 A2Kb tumor cells (1 x 10<4>cells/well) as stimulators and A2 transgenic spleen cells as feeders (5 x 10<5>cells/well) grown in the presence of 30 U/ml IL-2. On day 14, the cells were restimulated as before. On day 21, clones that grew were isolated and maintained in culture. Many of these clones demonstrated significantly higher reactivity (lyse) against human fibroblasts (HLA A2Kb-expressing) transduced with P502S than against control fibroblasts. An example is presented in Figure 1.

These data indicate that P2S #12 represents a naturally processed epitope of the P502S protein that is expressed in association with the human HLA A2Kb molecule.

6.2. This example illustrates the generation of murine CTL lines and CTL clones specific for cells expressing the P501S gene.

This series of experiments was performed similarly to that described above. Mice were immunized with the P1S#10 peptide (SEQ ID NO: 337), which is derived from the P501S gene (also referred to herein as L1-12, SEQ ID NO: 110). The P1S#10 peptide was deduced by analysis of the putative polypeptide sequence of P501S for potential HLA-A2 binding sequences as defined by published HLA-A2 binding motifs (Parker, KC, et al, J. Immunol., 152:163, 1994) . P1S#10 peptide was synthesized as described in Example 4 and empirically tested for HLA-A2 binding using a T cell-based competitive assay. Predicted A2-binding peptides were tested for their ability to compete with HLA-A2-specific peptide presentation to an HLA-A2-restricted CTL clone (D150M58), which is specific for the HLA-A2-binding influenza matrix peptide fluM58. D150M58 CTL secrete TNF in response to self-presentation of peptide fluM58. In this competitive assay, test peptides at 100-200 µg/ml were added to cultures of D150M58 CTL to bind HLA-A2 on CTL. After 30 min, CTL cultured with test peptides or control peptides were tested for their antigen dose response to the fluM58 peptide in a standard TNF bioassay. As shown in Figure 3, peptide P1S#10 competes with HLA-A2-restricted presentation of fluM58, demonstrating that peptide P1S#10 binds HLA-A2.

Mice expressing the human HLA A2Kb transgene were immunized as described by Theobald et al. ( Proe. Nati. Acad. Sei. USA 92:11993-11997, 1995 ) with the following modifications. Mice were immunized with 62.5 µg of P1S #10 and 120 µg of an I-Ab binding peptide derived from Hepatitis B Virus protein emulsified in incomplete Freund's adjuvant. Three weeks later, these mice were euthanized and single cell suspensions were prepared using a nylon mesh. The cells were then resuspended at 6 x 10<6>cells/ml in complete medium (as described above) and cultured in the presence of irradiated (3000 rad) P1S#10-pulsed (2ug/ml P1S#10 and 10 mg/ml p2 -microglobulin) LPS blasts (A2 transgenic spleen cells cultured in the presence of 7 ug/ml dextran sulfate and 25 ug/ml LPS for 3 days). Six days later, cells (5 x 10<5>/ml) were restimulated with 2.5 x 10<6>/ml peptide-pulsed irradiated (20,000 rad) EL4A2KB cells, as described above and 3 x 10<6>/ ml A2 transgenic spleen feeder cells. The cells were cultured in the presence of 20 U/ml IL-2. The cells were restimulated on a weekly basis in preparation for cloning. After three rounds of in vitro stimulations, one line was formed that recognized P1S#10-pulsed Jurkat A2Kb targets and P501S-transduced Jurkat targets as shown in Figure 4.

A P1S#10-specific CTL line was cloned by limiting dilution assay with peptide-pulsed EL4 A2Kb tumor cells (1 x 10<4>cells/well) as stimulators and A2 transgenic spleen cells as feeders (5 x 10<5>cells/well well) cultured in the presence of 30U/ml IL-2. On day 14, the cells were restimulated as before. On day 21, viable clones were isolated and maintained in culture. As shown in Figure 5, five of these clones demonstrated specific cytolytic reactivity against P501S-transduced Jurkat A2Kb targets. These data indicate that P1S#10 represents a naturally processed epitope of the P501S protein that is expressed in association with the human HLA-A2.1 molecule.

EXAMPLE 7

Priming<g>of CTL in vivo using naked DNA immuniserin<g>

WITH A PROSTATE ANTIGEN

Prostate-specific antigen L1-12, as described above, is also referred to as P501S. HLA A2Kb Tg mice (provided by Dr L. Sherman, The Scripps Research Institute, La Jolla, CA) were immunized with 100 µg P501S in the VR1012 vector either intramuscularly or intradermally. The mice were immunized three times, with a two-week interval between immunizations. Two weeks after the last immunization, immune spleen cells were cultured with Jurkat A2Kb-P501S-transduced stimulator cells. CTL lines were stimulated weekly. After two weeks of in vitro stimulation, CTL activity was assessed against P501S-transduced targets. Two of 8 mice developed strong anti-P501S CTL responses. These results demonstrate that P501S contains at least one naturally processed HLA-A2-restricted CTL epitope.

EXAMPLE 8

Ability of human T cells to<g>recognize prostate-specific<p>ol<yp>e<p>times

This example illustrates the ability of T cells specific for a prostate tumor polypeptide to recognize human tumor.

Human CD8<+>T cells were primed in vitro to the P2S-12 peptide (SEQ ID NO: 306) derived from P502S (also referred to as J1-17) using dendritic cells according to the protocol of Van Tsai et eel. { Critical Reviews in Immunology 78:65-75,1998). The resulting CD8<+>T cell microcultures were tested for their ability to recognize the P2S-12 peptide presented by autologous fibroblasts or fibroblasts transduced to express the P502S gene in a γ-interferon ELISPOT assay (see Lalvani et al., J. Exp. Med. 786:859-865, 1997). Briefly, titration numbers for T cells were examined in duplicate on 10<4> fibroblasts in the presence of 3 (ag/ml human p2-microglobulin and 1 ug/ml P2S-12 peptide or control E75 peptide. In addition, T- cells simultaneously examined autologous fibroblasts transduced with the P502S gene or, as a control, fibroblasts transduced with HER-2/neu Before the experiment, the fibroblasts were treated with 10 ng/ml γ-interferon for 48 hours to upregulate class I MHC expression. One of the microcultures (#5) demonstrated strong recognition of both peptide-pulsed fibroblasts as well as transduced fibroblasts in a γ-interferon ELISPOT assay.Figure 2A demonstrates that there was a strong increase in the number of γ-interferon spots with increasing numbers of T cells on fibroblasts pulsed with the P2S-12 peptide (filled bars) but not with control E75 peptide (open bars). This demonstrates the ability of these T cells to specifically recognize the P2S-12 peptide. As shown in Figure 2B, this microculture also demonstrated an increase in the number of γ-interferon stains with increasing numbers of T cells on fibroblasts transduced to express the P502S gene but not the HER-2/neu gene. These results provide further confirmatory evidence that the P2S-12 peptide is a naturally processed epitope of the P502S protein. Furthermore, this also demonstrates that there exist in the human T cell repertoire, high affinity T cells that can recognize this epitope. These T cells will also be able to recognize human tumors that express the P502S gene.

EXAMPLE 9

Elicitation of prostate antigen-specific CTL responses

in human blood

This example illustrates the ability of a prostate-specific antigen to elicit a CTL response in blood from normal humans.

Autologous dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC from normal donors by growth for five days in RPMI medium containing 10% human serum, 50 ng/ml GMCSF and 30 ng/ml IL-4. After culture, DC were infected overnight with recombinant P501S-expressing vaccinia virus at an M.O.I. of 5 and matured for 8 hours by the addition of 2 micrograms/ml CD40 ligand. Viruses were inactivated by UV irradiation, CD8<+> cells were isolated by positive selection using magnetic beads and priming cultures were initiated in 24-well plates. After five cycles of stimulation using autologous fibroblasts retrovirally transduced to express P501S and CD80, CD8+ lines were identified as specifically producing interferon-gamma when stimulated with autologous P501S-transduced fibroblasts. The P501S-specific activity of cell line 3A-1 could be maintained after further stimulation cycles on autologous B-LCL transduced with P501S. Line 3A-1 was shown to specifically recognize autologous B-LCL transduced to express P501S, but not EGFP-transduced autologous B-LCL, as measured by cytotoxicity assays (<51>Cr release) and interferon-gamma production (Interferon-gamma Elispot; see supra and Lalvani et al., J. Exp. Med. 186:859-865,1997). The results of these experiments are presented in Figures 6A and 6B.

EXAMPLE 10

Identification of a naturally processed CTL epitope contained in the prostate-specific antibody P703P

The 9-mer peptide p5 (SEQ ID NO: 338) was derived from the P703P antigen (also referred to as P20). The p5 peptide is immunogenic in human HLA-A2 donors and is a naturally processed epitope. Antigen-specific human CD8+ T cells can be primed after repeated in vitro stimulations with monocytes pulsed with p5 peptide. These CTL specifically recognize p5-pulsed and P703P-transduced target cells in both ELISPOT (as described above) and chromium release assays. In addition, immunization of HLA-A2Kb transgenic mice with p5 leads to generation of CTL lines that recognize a variety of HLA-A2Kb- or HLA-A2-transduced target cells expressing P703P.

Initial studies demonstrating that p5 is a naturally processed epitope were performed using HLA-A2Kb transgenic mice. HLA-A2Kb transgenic mice were immunized subcutaneously in the foot pad with 100 µg of p5 peptide together with 140 µg of hepatitis B virus core peptide (a Th peptide) in Freund's incomplete adjuvant. Three weeks after immunization, spleen cells from immunized mice were stimulated in vitro with peptide-pulsed LPS blasts. CTL activity was assessed by chromium release assay five days after primary in vitro stimulation. Retrovirally transduced cells expressing the control antigen P703P and HLA-A2Kb were used as targets. CTL lines that specifically recognize both p5-pulsed targets as well as P703P-expressing targets were identified.

Human in vitro priming experiments demonstrated that the p5 peptide is immunogenic in humans. Dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC from normal human donors by culture for five days in RPMI medium containing 10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml human IL-4 . After culture, DC were pulsed with 1 µg/ml p5 peptide and cultured with CD8 + T cell-enriched PBMC. CTL lines were restimulated on a weekly basis with p5-pulsed monocytes. Five to six weeks after initiation of the CTL cultures, CTL recognition of p5-pulsed target cells was demonstrated. CTL were additionally shown to recognize human cells transduced to express P703P, demonstrating that p5 is a naturally processed epitope.

Studies identifying an additional peptide epitope (referred to as peptide 4) derived from prostate tumor-specific antigen P703P that can be recognized by CD4 T cells on the surface of cells in association with HLA class II molecules were performed as follows. The amino acid sequence of peptide 4 is provided in SEQ ID NO: 638, with the corresponding cDNA sequence provided in SEQ ID NO: 639. 20 15-mer peptides overlapping by 10 amino acids and derived from the carboxy-terminal fragment of P703P were generated using of standard procedures. Dendritic cells (DC) were derived from PBMC from a normal female donor using GM-CSF and IL-4 by standard protocols. CD4 T cells were generated from the same donor as DC using MACS beads and negative selection. DC were pulsed overnight with pools of 15-mer peptides, with each peptide at a final concentration of 0.25 micrograms/ml. The pulsed DC were washed and plated at 1 x 10<4> cells/well of 96-well V-bottomed plates and purified CD4 T cells were added at 1 x 10<5>/well. Cultures were supplemented with 60 ng/ml IL-6 and 10 ng/ml IL-12 and incubated at 37°C. Cultures were restimulated as above on a weekly basis using DC generated and pulsed as above, as antigen presenting cells, supplemented with 5 ng/ml IL-7 and 10 u/ml IL-2. After 4 in vitro stimulation cycles, 96 lines (each line corresponding to one well) were tested for specific proliferation and cytokine production in response to the stimulation pools, with an irrelevant pool of peptides derived from mammaglobin used as a control. One line (referred to as 1-F9) was identified from pool #1 that demonstrated specific proliferation (as measured by 3H proliferation assay) and cytokine production (as measured by interferon-gamma ELISA assay) in response to pool #1 of P703P peptides. This line was further tested for specific recognition of the peptide pool, specific recognition of individual peptides in the pool and in HLA mismatch assays to identify the relevant restriction allele. Line 1-F9 was found to specifically proliferate and produce interferon-gamma in response to peptide pool #1 and also peptide 4 (SEQ ID NO: 638). Peptide 4 corresponds to amino acids 126-140 of SEQ ID NO: 327. Peptide titration experiments were performed to assess the sensitivity of lane 1-F9 to the specific peptide. The line was found to respond specifically to peptide 4 at concentrations as low as 0.25 ng/ml, indicating that the T cells are highly sensitive and therefore likely to have a high affinity for the epitope.

To determine the HLA restriction of the P703P response, a panel of antigen-presenting cells (APC) was generated that was partially matched to the donor used to generate the T cells. APC were pulsed with the peptide and used in proliferation and cytokine assays together with line 1-F9. APC matched to the donor by HLA-DRB0701 and HLA-DQB02 alleles were able to present the peptide to the T cells, indicating that the P703P-specific response is restricted to one of these alleles.

Antibody blocking experiments were used to determine whether the restriction allele was HLA-DR0701 or HLA-DQ02. Anti-HLA-DR blocking antibody L243 or an irrelevant isotype matched lgG2a was added to T cells and APC cultures pulsed with the peptide RMPTVLQCVNVSWS (SEQ ID NO: 638) at 250 ng/ml. Standard interferon-gamma and proliferation assays were performed. While the control antibody had no effect on the ability of the T cells to recognize the peptide-pulsed APC, in both experiments anti-HLA-DR antibody completely blocked the ability of the T cells to specifically recognize the peptide-pulsed APC.

To determine whether the peptide epitope RMPTVLQCVNVSWS (SEQ ID NO: 638) was naturally processed, the ability of line 1-F9 to recognize APC pulsed with recombinant P703P protein was examined. For these experiments, several recombinant P703P sources were used; E. co//-derived P703P, Pichia-derived P703P and baculovirus-derived P703P. Irrelevant protein controls used were E. coli -derived L3E, a lung-specific antigen) and baculovirus-derived mammaglobin. In interferon-gamma ELISA assays, line 1-F9 was able to efficiently recognize both E. coli forms of P703P as well as Pichia-derived recombinant P703P, while baculovirus-derived P703P was recognized less efficiently. Subsequent Western blot analysis showed that E coli and Pichia P703P protein preparations were intact, while the baculovirus P703P preparation was approximately 75% degraded. Thus, peptide RMPTVLQCVNVSWS (SEQ ID NO: 638) from P703P is a naturally processed peptide epitope derived from P703P and presented to T cells in the context of HLA-DRB-0701.

In further investigations, 24 15-mer peptides overlapping by 10 amino acids and derived from the N-terminal fragment of P703P (corresponding to amino acids 27-154 of SEQ ID NO: 525), were generated by standard procedures and their ability to be recognized by CD4 cells were determined essentially as described above. DC were pulsed overnight with pools of the peptides with each peptide at a final concentration of 10 micrograms/ml. A large number of individual CD4 T cell lines (65/480) demonstrated significant proliferation and cytokine release (IFN-gamma) in response to the P703P peptide pools, but not to a control peptide pool. CD4 T cell lines demonstrating specific activity were restimulated on the appropriate pool of P703P peptides and again tested on the individual peptides of each pool as well as a peptide dose titration of the pool of peptides in an IFN-gamma release assay and in a proliferation assay.

16 immunogenic peptides were recognized by the T cells from the entire set of peptide antigens tested. The amino acid sequences of these peptides are given in SEQ ID NO: 656-671, with the corresponding cDNA sequences given in SEQ ID NO: 640-655, respectively. In some cases, the peptide reactivity of the T cell line could be mapped to a single peptide, however, some could be mapped to more than one peptide in each collection. Those CD4 T cell lines that showed a representative pattern of recognition from each peptide pool with a reasonable affinity for peptide were selected for further analysis (1-1 A, -6A; II-4C, -5E; III-6E, IV- 4B, -3F, -9B, -10F, V-5B, -4D and -10F). These CD4 T-cell lines were restimulated on the appropriate individual peptide and again examined on autologous DC pulsed with a shortened form of recombinant P703P protein produced in E. coli (amino acids 96 - 254 in SEQ ID NO: 525), full-length P703P produced in the baculovirus expression system and a fusion between influenza virus NS1 and P703P produced in E. coli. Of the T cell lines tested, line 1-1A specifically recognized the truncated form of P703P (E. coli), but no other recombinant form of P703P. This line also recognized the peptide used to elicit the T cells. Lines 2-4C recognized the truncated form of P703P (E. coli) and the full-length form of P703P produced in baculovirus, as well as peptide. The remaining T cell lines tested were either peptide-specific only (II-5E, II-6F, IV-4B, IV-3F, IV-9B, IV-10F, V-5B and V-4D) or were non-responsive for any antigen tested (V-10F). These results demonstrate that the peptide sequence RPLLANDLMLIKLDE (SEQ ID NO: 671; corresponding to amino acids 110-124 of SEQ ID NO: 525) recognized by the T-cell line 1-1A and the peptide sequences SVSESDTIRSISIAS (SEQ ID NO: 668; corresponding to amino acids 125-139 in SEQ ID NO: 525) and ISIASQCPTAGNSCL (SEQ ID NO: 667; corresponding to amino acid residues 35-149 in SEQ ID NO: 525) recognized by the T-cell line II-4C, may be naturally processed epitopes of the P703P protein.

EXAMPLE 11

Expression of a breast tumor-derived antigen in the prostate

Isolation of the antigen B305D from breast tumor by differential display is described in US Patent Application No. 08/700,014, filed August 20, 1996. Many different splice forms of this antigen were isolated. The determined cDNA sequences for these splice forms are given in SEQ ID NO: 366-375, with the putative amino acid sequences corresponding to the sequences in SEQ ID NO: 292, 298 and 301-303 given respectively in SEQ ID NO: 299-306. In further investigations, a splice variant of the cDNA sequence of SEQ ID NO: 366 was isolated, which was found to contain an additional guanine residue at position 884 (SEQ ID NO: 530), leading to a frameshift in the open reading frame. The determined DNA sequence of this ORF is given in SEQ ID NO: 531. This frameshift generates a protein sequence (given in SEQ ID NO: 532) of 293 amino acids that contains the C-terminal domain common to the other isoforms of B305D, but which differ in the N-terminal region.

The expression levels of B305D in a variety of tumor tissues and normal tissues were examined by real-time PCR and by Northern analysis. The results showed that B305D is strongly expressed in breast tumor, prostate tumor, normal prostate and normal testis, with expression being low or undetectable in all other tissues examined (colon tumor, lung tumor, ovarian tumor and normal bone marrow, colon, kidney,

liver, lung, ovary, skin, small intestine, stomach). Using real-time PCR on a panel of prostate tumors, expression of B305D in prostate tumors was shown

to increase with increasing Gleason grade, demonstrating that expression of B305D increases as prostate cancer progresses.

EXAMPLE 12

Generation of human CTL in vitro using whole-gen<p>rimin<g>and stimulation<g>techniques with the prostate-specific antibody P501S

Using in vitro whole-gene priming with P501S vaccinia-infected DC (see, for example, Yee et al, The Journal of Immunology, 157(9):4079-86, 1996), human CTL lines were derived that specifically recognize autologous fibroblasts transduced with P501S (also known as L1-12), as determined by interferon-γ ELISPOT assay as described above. Using a panel of HLA-mismatched B-LCL lines transduced with P501S, these CTL lines were shown to be likely to be restricted to the HLAB class I allele. Specifically, dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC from normal human donors, by culture for five days in RPMI medium containing 10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml human IL -4. After culture, DC were infected overnight with recombinant P501S vaccinia virus at a multiplicity of infection (M.O.I) of five and matured overnight by the addition of 3 ng/ml CD40 ligand. Viruses were inactivated by UV irradiation. CD8+ T cells were isolated using a magnetic bead system and priming cultures were initiated using standard culture techniques. Cultures were restimulated every 7-10 days using autologous primary fibroblasts retrovirally transduced with P501S and CD80. After four cycles of stimulation, CD8+ T cell lines were identified that specifically produced interferon-γ when stimulated with P501S and CD80-transduced autologous fibroblasts. A panel of HLA-mismatched B-LCL lines transduced with P501S was generated to define the restriction allele of the response. By measuring interferon-γ in an ELISPOT assay, the P501S-specific response was shown to be likely limited by HLA B alleles. These results demonstrate that a CD8+ CTL response to P501S can be elicited.

To identify the epitope(s) recognized, the cDNA encoding P501S was fragmented by various restriction digests and sub-cloned into the retroviral expression vector pBIB-KS. Retroviral supernatants were generated by transfection of the helper packaging line Phoenix-Ampho. Supernatants were then used to transduce Jurkat/A2KB cells for CTL screening. CTL were screened in IFN-gamma ELISPOT assays against these A2Kb targets transduced with the "library" of P501S fragments. Initial positive fragments P501S/H3 and P501S/F2 were sequenced and found to encode amino acids 106-553 and amino acids 136-547, respectively, of SEQ ID NO: 113. A truncation of H3 was performed to encode amino acid residues 106-351 of SEQ ID NO: 113, which was unable to stimulate CTL, thus localizing the epitope to amino acid residues 351-547. Additional fragments encoding amino acids 1-472 (Fragment A) and amino acids 1-351 (Fragment B) were also constructed. Fragment A, but not Fragment B, stimulated CTL and thus localized the epitope to amino acid residues 351-472. Overlapping 20-mer and 18-mer peptides representing this region were tested by pulsing Jurkat/A2KB cells against CTL in an IFN-gamma assay. Only peptides P501S-369(20) and P501S-369(18) stimulated CTL. 9-mer and 10-mer peptides representing this region were synthesized and correspondingly tested. Peptide P501S-370 (SEQ ID NO: 539) was the minimal 9-mer that gave a strong response. Peptide P501S-376 (SEQ ID NO: 540) also gave a weak response, indicating that it could represent a cross-reactive epitope.

In subsequent studies, the ability of primary human B cells transduced with P501S to prime MHC class I-restricted, P501S-specific, autologous CD8 T cells was examined. Primary B cells were derived from PBMC from a homozygous HLA-A2 donor by culture in CD40 ligand and IL-4, transduced at high frequency with recombinant P501S in the vector pBIB and selected with blastocidin-S. 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 examined for specificity. After this initial screening, microcultures with significant signal above background cloned on autologous EBV-transformed B cells (BLCL) were also transduced with P501S. Using IFN-gamma ELISPOT for detection, many of these CD8 T-cell clones were found to be specific for P501S, as demonstrated by reactivity to BLCL/P501S but not BLCL transduced with control antigen. It was further demonstrated that anti-P501S CD8 T cell specificity is HLA-A2 restricted. First, antibody blocking experiments with anti-HLA-A,B,C monoclonal antibody (W6.32), anti-HLA-B,C monoclonal antibody (B1.23.2) and a control monoclonal antibody showed that only anti-HLA -A,B,C antibody blocked 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.

A natively processed, CD8, class I-restricted peptide epitope of P501S was identified as follows. Dendritic cells (DC) were isolated by Percol gradient followed by differential adherence and cultured for 5 days in the presence of RPMI medium containing 1% human serum, 50ng/ml GM-CSF and 30ng/ml IL-4. After culture, DC were infected for 24 h with P501S-expressing adenovirus at an MOI of 10 and matured for an additional 24 h with the addition of 2 µg/ml CD40 ligand. CD8 cells were enriched by subtraction of CD4 + , CD14 + and CD16 + populations from PBMC with magnetic beads. Priming cultures containing 10,000 P501S-expressing DC and 100,000 CD8+ T cells per well was set up in 96-well V-bottom plates with RPMI containing 10% human serum, 5ng/ml IL-12 and 10ng/ml IL-6. Cultures were stimulated every 7 days using autologous fibroblasts retrovirally transduced to express P501S and CD80 and were treated with IFN-gamma for 48-72 hours to upregulate MHC Class I expression. 10 u/ml IL-2 was added at the time of stimulation and on days 2 and 5 after stimulation. After 4 cycles of stimulation, one P501S-specific CD8+ T cell line (referred to as 2A2) was identified that produced IFN-gamma in response to IFN-gamma-treated P501S/CD80-expressing autologous fibroblasts, but not in response to IFN- gamma-treated P703P/CD80-expressing autologous fibroblasts in a γ-IFN Elispot assay. Line 2A2 was cloned in 96-well plates at 0.5 cells/well or 2 cells/well in the presence of 75,000 PBMC/well, 10,000 B-LCL/well, 30ng/ml OKT3 and 50u/ml IL-2. Twelve clones were isolated that showed strong P501S specificity in response to transduced fibroblasts.

Fluorescence-activated cell sorting (FACS) analysis was performed on P501S-specific clones using CD3-, CD4-, and CD8-specific antibodies conjugated to PercP, FITC, and PE, respectively. Consistent with the use of CD8-enriched T cells in the priming cultures, P5401S-specific clones were found to be CD3+, CD8+ and CD4-.

To identify the relevant P501S epitope recognized by P501S-specific CTL, pools of 18-20 mer or 30-mer peptides spanning the bulk of the amino acid sequence of P501S were loaded onto autologous B-LCL and tested in γ-IFN Elispot assays for the ability to stimulate two P501S-specific CTL clones, referred to as 4E5 and 4E7. One pool, composed of five 18-20 more peptides spanning amino acids 411-486 of P501S (SEQ ID NO: 113), was found to be recognized by both P501S-specific clones. To identify the specific 18-20mer peptide recognized by the clones, each of the 18-20mer peptides comprising the positive pool was tested individually in γ-IFN Elispot assays for the ability to stimulate the two P501S-specific CTL clones , 4E5 and 4E7. Both 4E5 and 4E7 specifically recognized a 20-mer peptide (SEQ ID NO: 710; cDNA sequence provided in SEQ ID NO: 711) spanning amino acids 453-472 of P501S. Since the minimal epitope recognized by CD8+ T cells is almost always either a 9- or 10-mer peptide sequence, 10-mer peptides spanning the entire sequence of SEQ ID NO: 710 were synthesized that differed by 1 amino acid. Each of these 10-mer peptides was tested for the ability to stimulate two P501S-specific clones, (referred to as 1D5 and 1E12). One 10-mer peptide (SEQ ID NO: 712; cDNA sequence provided in SEQ ID NO: 713) was identified as specifically stimulating P501S-specific clones. This epitope spans amino acids 463-472 of P501S. This sequence defines a minimal 10-mer epitope from P501S that can be naturally processed and to which CTL responses can be identified in normal PBMC. Thus, this epitope is a candidate for use as a vaccine entity and as a therapeutic and/or diagnostic reagent against prostate cancer.

To identify the class I restriction element for the P501S-derived sequence of SEQ ID NO: 712, HLA blocking and mismatch analyzes were performed. In γ-IFN Elispot assays, the specific response of clones 4A7 and 4E5 to P501S-transduced autologous fibroblasts was blocked by preincubation with 25 µg/ml W6/32 (pan-Class I blocking antibody) and B1.23.2 (HLA-B/ C blocking antibody). These results demonstrate that SEQ ID NO: 712-

specific response is limited to an HLA-B or HLA-C allele.

For HLA mismatch analysis, autologous B-LCL (HLA-A1,A2,B8,B51, Cw1, Cw7) and heterologous B-LCL (HLA-A2,A3,B18,B51,Cw5,Cw14) which have a common HLAB51 allele, pulsed for one hour with 20ug/ml peptide of SEQ ID NO: 712, washed and tested in γ-IFN Elispot assay for the ability to stimulate clones 4A7 and 4E5. Antibody blocking experiments with B1.23.2 (HLA-B/C blocking antibody) were also performed. SEQ ID NO: 712-specific response was detected using both autologous (D326) and heterologous (D107) B-LCL and further the responses were blocked by preincubation with 25ug/ml B1.23.2 HLA-B/C blocking antibody. Together, these results demonstrate that the P501S-specific response to the peptide of SEQ ID NO: 712 is restricted to the HLA-B51 class 1 allele. Molecular cloning and sequence analysis of the HLA-B51 allele from D3326 showed that the HLA-B51 subtype of D326 is HLA-B51011.

Based on the 10-mer P501S-derived epitope with SEQ ID NO: 712, two 9-mers with the sequences SEQ ID NO: 714 and 715 were synthesized and tested in Elispot assays for the ability to stimulate two P501S-specific CTL clones derived from line 2A2. The 10-mer peptide of SEQ ID NO: 712, as well as the 9-mer peptide of SEQ ID NO: 715, but not the 9-mer peptide of SEQ ID NO: 714, were able to stimulate P501S-specific CTL to produce IFN-gamma. These results demonstrate that the peptide of SEQ ID NO: 715 is a 9-mer P501S-derived epitope recognized by P501S-specific CTL. The DNA sequence encoding the epitope of SEQ ID NO: 715 is provided in SEQ ID NO: 716.

To identify the class I-restricting allele for the P501S-derived peptide of SEQ ID NO: 712 and 715 specific response, each of the HLA B and C alleles cloned from the donor was used in the in vitro priming experiment. Sequence analysis showed that the relevant alleles were HLA-B8, HLA-B51, HLA-Cw01 and HLA-Cw07. Each of these alleles was subcloned into an expression vector and cotransfected together with the P501S gene into VA-13 cells. Transfected VA-13 cells were then tested for the ability to specifically stimulate P501S-specific CTL in ELISPOT assays. VA-13 cells transfected with P501S and HLA-B51 were able to stimulate P501S-specific CTL to secrete IFN-gamma. VA-13 cells transfected with HLA-B51 alone or P501S + the other HLA alleles were unable to stimulate P501S-specific CTL. These results demonstrate that the limiting allele for the P501S-specific response is the HLAB51 allele. Sequence analysis showed that the subtype of the relevant restriction allele is HLA-B51011.

To determine whether P501S-specific CTL could recognize prostate tumor cells expressing P501S, the P501S-positive lines LnCAP and CRL2422 (which both express "moderate" amounts of P501S mRNA and protein) and PC-3 (which express low amounts of P501S mRNA and protein), plus the P501S-negative cell line DU-145, retrovirally transduced with the HLA-B51011 allele cloned from the donor, were used to generate P501S-specific CTL. HLA-B51011- or EGFP-transduced and selected tumor cells were treated with gamma-interferon and androgen (to upregulate stimulatory functions and P501S, respectively) and used in gamma-interferon Elispot experiments with the P501S-specific CTL clones 4E5 and 4E7. Untreated cells were used as control.

Both 4E5 and 4E7 efficiently and specifically recognized LnCAP and CRL2422 cells transduced with the HLA-B51011 allele, but not the same cell lines transduced with EGFP. In addition, both CTL clones specifically recognized PC-3 cells transduced with HLA-B51011, but not the P501S-negative tumor cell line DU-145. Treatment with gamma interferon or androgen did not improve the ability of CTL to recognize tumor cells. These results demonstrate that P501 S-specific CTL, generated by in vitro whole-gene priming, specifically and efficiently recognize prostate tumor cell lines expressing P501S.

A naturally processed CD4 epitope of P501S was identified as follows.

CD4 cells specific for P501S were prepared as described above. A series of 16 overlapping peptides were synthesized spanning approximately 50% of the amino-terminal portion of the P501S gene (amino acids 1-325 of SEQ ID NO: 113). For priming, peptides were collected in pools of 4 peptides, pulsed at 4 µg/ml on dendritic cells (DC) for 24 hours, with TNF-alpha. DC were then washed and mixed with negatively selected CD4+ T cells in 96 well U-bottom plates. The cultures were restimulated weekly on fresh DC loaded with peptide pools. After a total of 4 stimulation cycles, cells were rested for an additional week and tested for specificity to APC pulsed with peptide pools using γ-IFN ELISA and proliferation assays. For these experiments, adherent monocytes were loaded with either the relevant peptide pool at 4 µg/ml or an irrelevant peptide at µg/ml, used as APC. T cell lines demonstrating either specific cytokine secretion or proliferation were then tested for recognition of individual peptides present in the pool. T-cell lines could be identified from pools A and B that recognized individual peptides from these pools.

From collection A, lines AD9 and AE10 specifically recognized peptide 1 (SEQ ID NO: 719) and line AF5 recognized peptide 39 (SEQ ID NO: 718). From collection B, line BC6 could be identified as recognizing peptide 58 (SEQ ID NO: 717). Each of these lines was stimulated on the specific peptide and tested for specific recognition of the peptide in a titration experiment as well as cell lysates generated by infection of HEK 293 cells with adenovirus expressing either P501S or an irrelevant antigen. For these experiments, APC-adherent monocytes were pulsed with either 10.1 or 0.1 µg/ml individual P501S peptides and DC were pulsed overnight with a 1:5 dilution of adenovirally infected cell lysates. Lines AD9, AE10 and AF5 retained significant recognition of the relevant P501S-derived peptides even at 0.1 mg/ml. Furthermore, line AD9 demonstrated significant (8.1-fold stimulation index) specific activity for lysates from adenovirus-P501S-infected cells. These results demonstrate that high affinity CD4 T cell lines can be generated against P501S-derived epitopes and that at least one subset of these T cells specific for the P501S-derived sequence of SEQ ID NO: 719 is specific for an epitope that is naturally processed by human cells. The DNA sequences encoding the amino acid sequences with SEQ ID NO: 717-719 are given in SEQ ID NO: 720-722, respectively.

To further characterize the P501S-specific activity of AD9, the line was cloned using anti-CD3. Three clones, referred to as 1A1, 1A9 and 1F5, were identified which were specific for the P501S-1 peptide (SEQ ID NO: 719). To determine the HLA restriction allele for the P501 S-specific response, each of these clones was tested in class II antibody blocking and HLA mismatch assays using proliferation and gamma interferon assays. In antibody blocking assays and measurement of gamma interferon production using ELISA assays, the ability of all three clones to recognize peptide-pulsed APC was specifically blocked by coincubation with either a pan-class II blocking antibody or an HLA-DR blocking antibody, but not with HLA-DQ or an irrelevant antibody. Proliferation experiments carried out simultaneously with the same cells confirmed these results. These data indicate that the P501S-specific response of the clones is limited by an HLA-DR allele. Further investigations demonstrated that the restriction allele for the P501S-specific response is HLA-DRB1501.

EXAMPLE 13

Identification of prostate-specific antigens by microarray analysis

This example describes the isolation of certain prostate-specific polypeptides from a prostate tumor cDNA library.

A human prostate tumor cDNA expression library as described above was screened using microarray analysis to identify clones showing at least a three-fold overexpression in prostate tumor and/or normal prostate tissue, compared to non-prostate normal tissue (not including testis). 372 clones were identified and 319 were successfully sequenced. Table I presents a summary of these clones, which are shown in SEQ ID NO:385-400. Of these sequences, SEQ ID NOs: 386, 389, 390 and 392 correspond to new genes and SEQ ID NOs: 393 and 396 correspond to previously identified sequences. The others (SEQ ID NOS: 385, 387, 388, 391, 394, 395 and 397-400) correspond to known sequences, as shown in Table I.

CGI-82 showed 4.06-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 43% of prostate tumors, 25% normal prostate, not detected in other normal tissues tested. L-iditol-2-dehydrogenase showed 4.94-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 90% of prostate tumors, 100% of normal prostate and not detected in other normal tissues tested. Ets transcription factor PDEF showed 5.55-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 47% prostate tumors, 25% normal prostate and not detected in other normal tissues tested. hTGR1 showed 9.11-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 63% of prostate tumors and was not detected in 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, and was low to undetectable in normal tissues tested including normal prostate tissue. Prostatic acid phosphatase showed 9.14-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 67% of prostate tumors, 50% of normal prostate and not detected in other normal tissues tested. Transglutaminase showed 14.84 times overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 30% of prostate tumors, 50% of normal prostate and 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, 75% of normal prostate and is undetectable in all other normal tissues tested. CGI-69 showed 3.56-fold overexpression in prostate tissue compared to other normal tissues tested. It is a rare gene, detected in more than 90% of prostate tumors and in 75% of normal prostate tissue. The expression of this gene in normal tissue was very low. KIAA0122 showed 4.24-fold overexpression in prostate tissue compared to other normal tissues tested. It was overexpressed in 57% of prostate tumors, it was undetectable in all normal tissues tested including normal prostate tissue. 19142.2 bangur showed 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 undetectable 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, 75% of normal prostate and was also overexpressed in normal bone marrow, pancreas and activated PBMC. New clone 23379 (also referred to as P553S) showed 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 prostate and was 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 and was undetectable in all normal tissues tested, including normal prostate tissue. New clone 23320 showed 3.15-fold overexpression in prostate tissue compared to other normal tissues tested. It was detectable in all prostate tumors and 50% of normal prostate tissue. It was also expressed in normal colon and trachea. Other normal tissues do not express this gene at a high level.

Subsequent full-length cloning studies of P553S, using standard techniques, showed that this clone is an incompletely spliced form of P501S. The determined cDNA sequences for four splice variants of P553S are provided in SEQ ID NO: 623-626. An amino acid sequence encoded by SEQ ID NO: 626 is provided in SEQ ID NO: 627. The cDNA sequence of SEQ ID NO: 623 was found to contain two open reading frames (ORFs). The amino acid sequences encoded by these two ORFs are given in SEQ ID NO: 628 and 629.

EXAMPLE 14

Identification of prostate-specific antigens by electronic subtraction

This example describes the use of an electronic subtraction technique to identify prostate-specific antigens.

Potential prostate-specific genes present in the GenBank human EST database were identified by electronic subtraction (similar to that described by

Vasmatizis et al., Proe. Nati. Acad. Pollock. USA 95:300-304,1998). The sequences of EST clones (43,482) derived from different prostate libraries were obtained from the GenBank public human EST database. Each prostate EST sequence was used as a query sequence in a BLASTN (National Center for Biotechnology Information) search against the human EST database. All matches considered identical (length of matching sequence >100 base pairs, density of identical matches over this region >70%) were grouped (aligned) together in a duster. Clusters containing more than 200 ESTs were discarded as they likely represented repetitive elements or highly expressed genes such as those for ribosomal proteins. If two or more clusters shared ESTs, these clusters were grouped together in a "superduster," resulting in 4,345 prostate superclusters.

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

II.

Each superduster was analyzed expressed as EST in the supercluster. The tissue source of each EST clone was noted and used to classify the superdusters into four groups: Type 1 - EST clones found only in Plus group libraries; no expression detected in Minus or Other group libraries; Type 2- EST clones derived only from Plus and Other group libraries; no expression detected in the Minus group; Type 3- EST clones derived from Pluss, Minus and Other group libraries, but the number of ESTs derived from Pluss group is higher than in Minus or Other groups; and Type 4 EST clones derived from Pluss, Minus and Other group libraries, but the number derived from the Pluss group is higher than the number derived from the Minus group. This analysis identified 4345 breast clusters (see Table III). From these dusters, 3172 EST clones were ordered from Research Genetics, Inc. and received as frozen glycerol stocks in 96-well plates.

The EST clone inserts were PCR amplified using amino-linked PCR primers for Synteni microarray analysis. When more than one PCR product was obtained for a particular clone, that PCR product was not used for expression analysis. A total of 2528 clones from the electronic subtraction method were analyzed by microarray analysis to identify breast clones from electronic subtraction that had high levels of tumor vs. normal tissue mRNA. Such screenings were performed using a Synteni (Palo Alto, CA) microarray, according to the manufacturer's instructions (and essentially as described by Schena et al., Proe. Nati. Acad. Sei. USA 93:10614-10619, 1996 and Heller et al., Proe. Nat. Acad. Sei. USA 94:2150-2155, 1997). In these assays, the clones were arrayed on the chip, which was then probed with fluorescent probes formed from normal and tumor prostate cDNA, as well as various other normal tissues. Slides were scanned and fluorescence intensity was measured.

Clones with an expression ratio greater than 3 (ie, the level in prostate tumor and normal prostate mRNA was at least three times the level in 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 certain new sequences shown in SEQ ID NOS: 407, 413, 416-419, 422, 426, 427 and 450.

Further examination of the clone of SEQ ID NO: 407 (also referred to as P1020C) led to the isolation of an extended cDNA sequence provided in SEQ ID NO: 591. This extended cDNA sequence was found to contain an open reading frame encoding the putative amino acid sequence in SEQ ID NO: 592. The P1020C cDNA and amino acid sequences were found to show some similarity to the human endogenous retroviral HERV-K pol gene and protein.

EXAMPLE 15

Further identification of prostate-specific antigens by microarray analysis

This example describes the isolation of additional prostate-specific polypeptides from a prostate tumor cDNA library.

A human prostate tumor cDNA expression library as described above was screened using microarray analysis to identify clones that showed at least threefold overexpression in prostate tumor and/or normal prostate tissue, compared to non-prostate normal tissue (not including testis). 142 clones were identified and sequenced. Certain of these clones are shown in SEQ ID NO: 454-467. Of these sequences, SEQ ID NO: 459-460 represent new genes. The others (SEQ ID NOS: 454-458 and 461-467) correspond to known sequences. Comparison of the determined cDNA sequence of SEQ ID NO: 461 with sequences in the Genbank database using the BLAST program revealed homology to the previously identified transmembrane protease serine 2 (TMPRSS2). The full-length cDNA sequence for this clone is provided in SEQ ID NO: 751, with the corresponding amino acid sequence provided in SEQ ID NO: 752. The cDNA sequence encoding the first 209 amino acids of TMPRSS2 is provided in SEQ ID NO: 753 , the first 209 amino acids being given in SEQ ID NO: 754.

The sequence in SEQ ID NO: 462 (referred to as P835P) was found to correspond to the previously identified clone FLJ13518 (Accession AK023643; SEQ ID NO: 774), which had no associated open reading frame (ORF). This clone was used for searching the Geneseq DNA database and matched a clone previously identified as a G protein-coupled receptor protein (DNA Geneseq Accession A09351; amino acid Geneseq Accession Y92365), which is characterized by the presence of seven transmembrane domains. The sequences of fragments between these domains are given in SEQ ID NO: 778-785, where SEQ ID NO: 778, 780, 782 and 784 represent extracellular domains and SEQ ID NO: 779, 781, 783 and 785 represent intracellular domains. SEQ ID NO: 778-785 represent respectively amino acids 1-28, 53-61, 83-103, 124-143, 165-201, 226-238, 263-272 and 297-381 of P835P. The full-length cDNA sequence for P835P is provided in SEQ ID NO: 773. The cDNA sequence of the open reading frame for P835P, including the stop codon, is provided in SEQ ID NO: 775, with the open reading frame without the stop codon provided in SEQ ID NO : 776 and the corresponding amino acid sequence given in SEQ ID NO: 777.

EXAMPLE 16

Further characterization of prostate-specific antigen P710P

This example describes full-length cloning of P710P.

The prostate cDNA library described above was screened with the P710P fragment described above. One million colonies were plated on LB/Ampicillin plates. Nylon membrane filters were used to lift these colonies and the cDNA captured by these filters was then denatured and cross-linked to the filters with UV light. The p71 OP fragment was radiolabeled and used to hybridize with the filters. Positive cDNA clones were selected and their cDNA recovered and sequenced with an automatic Perkin Eimer/Applied Biosystems Division sequencer. Four sequences were obtained and are presented in SEQ ID NO: 468-471. These sequences appear to represent different splice variants of the P710P gene. Subsequent comparison of the cDNA sequences of P710P with those in Genbank revealed homology to the DD3 gene (Genbank accession numbers AF103907 & AF103908). The cDNA sequence of DD3 is given in SEQ ID NO: 618.

EXAMPLE 17

Protein expression of prostate-specific antigens

This example describes the expression and purification of prostate-specific antigens in E. coli, baculovirus, mammalian and yeast cells.

a) Expression of P501S in E . coli

Expression of the full-length form of P501S was attempted by first cloning

P501S without the leader sequence (amino acids 36-553 of SEQ ID NO: 113) downstream of the first 30 amino acids of M. tuberculosis antigen Ra 12 (SEQ ID NO: 484) in pET17b. Specifically, P501S DNA was used to perform PCR using primers AW025 (SEQ ID NO: 485) and AW003 (SEQ ID NO: 486). AW025 is a sense cloning primer containing a HindIII site. AW003 is an antisense cloning primer containing an EcoRI site. DNA amplification was performed using 5 µl 10X Pfu buffer, 1 µl 20 mM dNTP, 1 µl each of the PCR primers at 10 µM concentration, 40 µl water, 1 µl Pfu DNA polymerase (Stratagene, La Jolla, CA) and 1 µl DNA at 100 ng/µl Denaturation at 95°C for 30 sec followed by 10 cycles of 95°C for 30 sec, 60°C for 1 min and at 72°C for 3 min 20 cycles of 95°C for 30 sec, 65°C for 1 min and at 72°C for 3 min and finally with 1 cycle of 72°C for 10 min PCR product was cloned into Ra12m/pET17b using Hindi11 and EcoRI The sequence of the resulting fusion construct (referred to as Ra12-P501S-F) was confirmed by DNA sequencing.

The fusion construct was transformed into BL21(DE3)pLysE, pLysS and CodonPlus E. coli (Stratagene) and grown overnight in LB medium with kanamycin. The resulting culture was developed with IPTG. Protein was transferred to PVDF membrane and blocked with 5% non-fat milk (in PBS-Tween buffer), washed three times and incubated with mouse anti-His tag antibody (Clontech) for 1 hour. The membrane was washed 3 times and probed with HRP-Protein A (Zymed) for 30 min. Finally, the membrane was washed 3 times and developed with ECL (Amersham). No expression was detected by Western blot. Similarly, no expression was detected by Western blot when the Ra12-P501S-F fusion was used for expression in BL21 CodonPlus with CE6 phage (Invitrogen).

An N-terminal fragment of P501S (amino acids 36-325 of SEQ ID NO: 113) was cloned downstream of the first 30 amino acids of the M. tuberculosis antigen Ra12 in pET17b as follows. P501S DNA was used to perform PCR using 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 Ra 12 in pET17b at the Hindi 11 and EcoRI sites. The fusion construct (referred to as 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. Using Western blot analysis, protein bands were observed at the expected molecular weight of 36 kDa. Some high molecular weight bands were also observed, which were probably due to aggregation of the recombinant protein. No expression was detected by Western blot when the Ra12-P501S-F fusion was used for expression in BL21 CodonPlus of CE6 phage.

A fusion construct comprising a C-terminal part of P501S (amino acids 257-553 of SEQ ID NO: 113) located downstream of the first 30 amino acids of the M. tuberculosis antigen Ra12 (SEQ ID NO: 484) was prepared as follows. P501S DNA was used to perform PCR using 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 Ra12 in pET17b at the HindIII and EcoRI sites. 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 by Western blot, with some molecular weight aggregates also observed. Expression was also detected by Western blot when the Ra12-P501S-C fusion was used for expression in BL21 CodonPlus elicited by CE6 phage.

A fusion construct comprising a fragment of P501S (amino acids 36-298 in SEQ ID NO: 113) located downstream of M. tuberculosis antigen Ra12 (SEQ ID NO: 705) was prepared as follows. P501S DNA was used to perform PCR using primers AW042 (SEQ ID NO: 706) and AW053 (SEQ ID NO: 707). AW042 is a sense cloning primer containing an EcoRI site. AW053 is an antisense primer with stop and Xho I sites. DNA amplification was performed essentially as described above. The resulting PCR product was cloned into Ra12 in pET17b at EcoRI and Xho I sites. The resulting fusion construct (referred to as Ra12-P501S-E2) was expressed in B834 (DE3) pLys S E. coli host cells in TB media for 2 h at room temperature. Expressed protein was purified by washing the inclusions and running on a Ni-NTA column. The purified protein remained soluble in buffer containing 20 mM Tris-HCl (pH 8), 100 mM NaCl, 10 mM B-Me and 5% glycerol. The determined cDNA and amino acid sequences for the expressed fusion protein are given in SEQ ID NO: 708 and 709, respectively.

b) Expression of P501S in Baculovirus

Bac-to-Bac baculovirus expression system (BRL Life Technologies, Inc.)

was used to express P501S protein in insect cells. Full-length P501S (SEQ ID NO: 113) was amplified by PCR and cloned into the XbaI site of the donor plasmid pFastBacl. The recombinant bacmid and baculovirus were prepared according to the manufacturer's instructions. The recombinant baculoviruses were amplified in Sf9 cells and high titer viral vectors were used to infect High Five cells (Invitrogen) to produce the recombinant protein. The identity of the full-length protein was confirmed by N-terminal sequencing of the recombinant protein and by Western blot analysis (Figure 7). Specifically, 0.6 million High Five cells in 6-well plates were infected with either unrelated control virus BV/ECD_PD (lane 2), with recombinant baculovirus for P501S at various amounts or MOIs (lanes 4-8) or non- infected (lane 3). Cell lysates were run on SDS-PAGE under reducing conditions and analyzed by Western blot with anti-P501S monoclonal antibody P501S-10E3-G4D3 (prepared as described below). Lane 1 is the biotinylated protein molecular weight marker (BioLabs).

The localization of recombinant P501S in the insect cells was investigated as follows. The insect cells overexpressing P501S were fractionated into nucleus, mitochondria, membrane and cytosol fractions. Equal amounts of protein from each fraction were analyzed by Western blot with a monoclonal antibody against P501S. Because of the fractionation scheme, both nuclear and mitochondrial fractions contain some plasma membrane components. However, the membrane fraction is basically free of mitochondria and nucleus. P501S was found to be present in all fractions containing the membrane component, indicating that P501S may be associated with the plasma membrane of the insect cells expressing the recombinant protein.

c) Expression of P501S in mammalian cells

Full-length P501S (553 amino acids; SEQ ID NO: 113) was cloned into

various mammalian expression vectors, including pCEP4 (Invitrogen), pVR1012 (Vical, San Diego, CA) and a modified form of the retroviral vector pBMN, referred to as pBIB. Transfection of P501S/pCEP4 and P501S/pVR1012 into HEK293 fibroblasts 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 min. This mixture was added to 1 µg of P501S plasmid DNA, mixed briefly and incubated for 30 min at room temperature. The Fugene/DNA mixture was added to cells and incubated for 24-48 hours. Expression of recombinant P501S in transfected HEK293 fibroblasts was detected by Western blot using a monoclonal antibody to P501S.

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

FACS analysis of P501S transiently infected CHO-K cells demonstrated surface expression of P501S. Expression was detected using rabbit polyclonal antisera against a P501S peptide, as described below. Flow cytometric analysis was performed using a FaCScan

(Becton Dickinson) and the data were analyzed using the Cell-Quest program.

d) Expression of P501S in S. cerevisiae

P501S was expressed in yeast, targeting membranes using a yeast prepro signal sequence. The natural signal sequence and first lumenal domain of P501S were deleted to preserve the natural positioning of the expressed P501S protein.

Specifically, the α-prepro signal sequence of S. cerevisiae linked to amino acids 55-553 of SEQ ID NO: 113 with a His tag tail was cloned into plasmid pRIT15068 with CUP1 promoter and transfected into S. cerevisiae strain Y1790. The Y1790 strain is Leu+ and His-. Expression of protein was induced by the addition of either 500 µM or 250 µM CuSO 4 at 30°C in minimal medium supplemented with histidine. Cells were harvested 24 hours after induction. Extracts were prepared by growing cells to a concentration of OD600 5.0 in 50 mM citrate phosphate buffer (pH 4.0) plus 130 mM NaCl supplemented with protease inhibitors. Cells were disrupted using glass beads and centrifuged for 20 min. at 15,000 g. The recombinant protein was found to be 100% pellet-associated.

Expression of the recombinant protein (molecular weight 63 kD) was demonstrated by Western blot analysis, using the anti-P501S monoclonal antibody 10E-D4-G3 described below. The amino acid sequence of the expressed protein is given in SEQ ID NO: 792.

Fermentation processes for the production of a prepro-P501S-His-tag recombinant protein in S. cerevisiae (strain Y1790 - CUP1 inducible promoter) were evaluated as follows. 100 µl of master seed containing 2.5 x 10<8> cells/ml transformed S. cerevisiae Y1790 was spread on FSC004AA solid medium. The composition of FSC004AA medium is as follows: glucose 10 g/l; Na 2 Mo 0 4 , 2 H 2 O 0.0002 g/l; folic acid 0.000064 g/l; KH2P041 g/l; MnSO 4 .H 2 O 0.0004 g/l; Inositol 0.064 g/l; MgSO 4 , 7H 2 O 0.5 g/l; H3B030.0005 g/l; Pyridoxine 0.008 g/l; CaCl 2 , 2 H 2 O 0.1 g/l; At 0.0001 g/l; Thiamine 0.008 g/l; NaCl 0.1 g/l; CoCl2.6H2O 0.00009 g/l; Niacin 0.000032 g/l; FeCl3.6H2O 0.0002 g/l; Riboflavin 0.000016 g/l; Panthotenate Ca 0.008 g/l; CuS04.5H20 0.00004 g/l; Biotin 0.000064 g/l; para-aminobenzoic acid 0.000016 g/l; ZnSO 4 , 7H 2 O 0.0004 g/l; (NH 4 ) 2 SO 45 g/l; agar 18 g/l; Histidine 0.1 g/l.

Two plates were incubated for 26 hours at 30°C. These solid pre-cultures were harvested in 5 ml liquid medium FSC007AA and 0.5 ml (or 9.3 x 10<7> cells) of this suspension was used to inoculate 2 liquid pre-cultures.

The composition of FSC007AA medium is as follows: Glucose 10 g/l; Na2Mo04.2H2O 0.0002 g/l; folic acid 0.000064 g/l; KH2P041 g/l; MnSO 4 .H 2 O 0.0004 g/l; Inositol 0.064 g/l; MgSO 4 .7H 2 O 0.5 g/l; H3B030.0005 g/l; Pyridoxine 0.008 g/l; CaCl2.2H20 0.1 g/l; At 0.0001 g/l; Thiamine 0.008 g/l; NaCl 0.1 g/l; CoCl2.6H2O 0.00009 g/l; Niacin 0.000032 g/l; FeCl3.6H20 0.0002 g/l; Riboflavin 0.000016 g/l; Panthotenate Ca 0.008 g/l; CuS04.5H20 0.00004 g/l; Biotin 0.000064 g/l; para-aminobenzoic acid 0.000016 g/l; ZnSO 4 .7H 2 O 0.0004 g/l; (NH 4 ) 2 SO 45 g/l; Histidine 0.1 g/l.

These pre-cultures were run for 20 hours in 2 L flasks containing 400 ml medium FSC007AA to achieve an OD of 1.8. The other characteristics of these pre-cultures are as follows: pH 2.8; glucose 2.3 g/l; ethanol 3.4 g/l.

The best timing for liquid pre-cultures for strain Y1790 was determined in preliminary experiments. Liquid pre-cultures containing 400 ml of medium and inoculated with different volumes of Master germ (0.25, 0.5, 1 or 2 ml) were monitored to identify the best inoculum size and timing. Glucose, ethanol, pH, OD and cell numbers (determined by flow cytometry) were monitored between 16 and 23 hours of culture. Glucose depletion and maximum biomass were achieved after 20 h of incubation with 0.5 inoculum. These conditions were used for transfer of the pre-culture to fermentation.

A total of 800 ml of pre-culture was used to inoculate a 20 L fermentor containing 5 L of medium FSC002AA. Three ml of irradiated antifoam was added before inoculation. The composition of FSC002AA medium is as follows: (NH 4 ) 2 SO 46.4 g/l; Na2Mo04.2H2O 2.05 mg/l; folic acid 0.54 mg/l; KH2P048.25 g/l; MnSO 4 .H 2 O 4.1 mg/l; inositol 540 mg/; MgSO 4 , 7H 2 O 4.69 g/l; H3B035.17 m/l; pyridoxine 68 mg/l; CaCl2.2H2O 0.92 g/l; At 1.03 mg/l; thiamine 68 mg/l; NaCl 0.06 g/l; CoCl2.6H20 0.92 mg/l; Niacin 0.27 mg/l; HC11 ml/l; FeCl3.6H20 9.92 mg/l; Riboflavin 0.13 mg/l; CuS04.5H20 0.41 mg/l; Glucose 0.14 g/l; Panthotenate About 68 mg/l; ZnSO4.7H2O 4.1 mg/l; Biotin

0.54 mg/l; para-aminobenzoic acid 0.13 mg/l; Histidine 0.3 g/l.

The carbon source (glucose) was supplemented by continuous feeding of FFB004AA medium. The composition of the FFB004AA medium is as follows: glucose 350 g/l; Na2Mo04.2H2O 5.15 mg/l; folic acid 1.36 mg/l; KH2P0420.6 g/l; MnSO 4 .H 2 O 10.3 mg/l; inositol 1350 mg/l; MgSO 4 .7H 2 O 11.7 g/l; H3B0312.9 m/l; pyridoxine 170 mg/l; CaCl2.2H20 2.35 g/l; At 2.6 mg/l; thiamine 170 g/l; NaCl 0.15 g/l; CoCl2.6H2O 2.3 mg/l; niacin 0.67 mg/l; HCl 2.5 ml/l; FeCl3.6H20 24.8 mg/l; riboflavin; 0.33 mg/l; CuS04.5H20 1.03 mg/l; biotin 1.36 mg/l; panthotenate About 170 mg/l; ZnSO4.7H2O 10.3 mg/l; para-aminobenzoic acid: 0.33 mg/l; histidine 5.35 g/l.

The remaining glucose concentration was kept very low (>0 mg/l) to minimize ethanol production during the fermentation. This was achieved by limiting the growth of the microorganism using a limited glucose feed rate. Standard biomass content (OD 80-90) was reached by fermentation after a 44-hour growth phase.

CUP1 promoter was then induced by addition of 500 µM CuSO 4 to produce P501S antigen. CuSO 4 addition was followed by ethanol accumulation (up to 6 g/l) and the glucose feed rate was then reduced to consume the ethanol. The copper available to the microorganism was monitored by testing Cu ion concentration in the medium supernatant using a spectrophotometric copper assay (DETC method). The fermentation was then supplemented with CuSO 4 throughout the induction phase to maintain the concentration between 150 and 250 uM in the supernatant. The biomass reached an OD of 100 at the end of induction. Cells were harvested after 8 hours of induction.

Cell homogenate was prepared and analyzed by SDS-PAGE and Western Blot using standard protocols. A major protein band with the expected molecular weight of 62KD was detected by Western blot using anti-P501S monoclonal antibodies. Western blot analysis also showed that the significant 62KD band was progressively produced from 30 minutes of induction and reached a maximum after 3 hours. No more antigen appeared to be produced between 3 and 12 hours of induction.

The number of passages through a French Press required to extract all the antigen from the cells was evaluated. One, three and five passages were tested and total cell lysates, supernatants and pellets of cell lysates were analyzed by Western blot. Three passages through a French Press were sufficient to complete extraction of the antigen. The antigen was present in the insoluble fraction.

e) Expression of P703P in Baculovirus

cDNA for full-length P703P-DE5 (SEQ ID NO: 326), together with

many flanking restriction sites, was obtained by digestion of the plasmid pCDNA703 with restriction endonucleases Xba I and Hind III. The resulting restriction fragment (about 800 base pairs) was ligated into the transfer plasmid pFastBacI which was digested with the same restriction enzymes. The sequence of the insertion was confirmed by DNA sequencing. The recombinant transfer plasmid pFBP703 was used to produce recombinant bacmid DNA and baculovirus using the Bac-To-Bac Baculovirus expression system (BRL Life Technologies). High Five cells were infected with the recombinant virus BVP703, as described above, to obtain recombinant P703P protein.

e) Expression of P788P in E . Coli

A truncated, N-terminal part, of P788P (residues 1-644 of SEQ ID NO: 777; referred to as P788P-N) fused with a C-terminal 6xHis tag was expressed in E. coli as follows. P788P cDNA was amplified using primers AW080 and AW081 (SEQ ID NO: 672 and 673). AW080 is a sense cloning primer with an Ndel seat. AW081 is an antisense cloning primer with an Xhol site. The PCR-amplified P788P, as well as the vector pCRX1, were digested with NdeI and XhoI. Vector and insert were ligated and transformed into NovaBlue cells. Colonies were randomly screened for insertion and then sequenced. P788P-N clone #6 was confirmed to be identical to the designed construct. The expression construct P788P-N #6/pCRX1 was transformed into E. coli BL21 CodonPlus-RIL competent cells. After induction, most of the cells grew well, reaching OD600 of more than 2.0 after 3 hours. Coomassie labeled SDS-PAGE showed an overexpressed band at ca. 75 kD. Western blot analysis using a 6xHis-tagged antibody confirmed that the band was P788P-N. The determined cDNA sequence for P788P-N is provided in SEQ ID NO: 674, with the corresponding amino acid sequence provided in SEQ ID NO: 675.

f) Expression of P510S in E . Coli

The P510S protein has 9 potential transmembrane domains and is

predicted to be located at the plasma membrane. The C-terminal protein of this protein as well as the putative third extracellular domain of P510S were expressed in E. coli as follows.

The expression construct referred to as Ra12-P501S-C was designed to have a 6 His tag at the N-terminal end, followed by the M. tuberculosis antigen Ra12 (SEQ ID NO: 676) and then the C-terminal portion of P510S ( amino residues 1176-1261 of SEQ ID NO: 538). Full-length P510S was used to amplify the P510S-C fragment by PCR using primers AW056 and AW057 (SEQ ID NO: 677 and 678, respectively). AW056 is a sense cloning primer with an EcoRI site. AW057 is an antisense primer with stops and Xhol seats. The amplified P501S fragment and Ra12/pCRX1 were digested with EcoRI and XhoI and then purified. The insert and vector were ligated together and transformed into NovaBlue. Colonies were randomly screened for insertion and sequences. For protein expression, the expression construct was transformed into E. coli BL21 (DE3) CodonPlus-RIL competent cells. A mini-induction screen was performed to optimize the expression conditions. After the induction, the cells grew well, reaching an OD 600 nm greater than 2.0 after 3 hours. Coomassie labeled SDS-PAGE showed a highly overexpressed band at ca.

30 kD. Although this is higher than the expected molecular weight, Western blot analysis was positive, showing that this band was His-tag-containing protein. The optimized culture conditions are as follows. Dilute overnight culture/day culture (LB + kanamycin + chloramphenicol) in 2xYT (with kanamycin and chloramphenicol) at a ratio of 25 ml of culture to 1 liter of 2xYT. Let grow at 37°C until OD600 = 0.6. Take an aliquot as TO sample. Add 1 mM IPTG and incubate at 30°C for 3 h. Take out a T3 sample, spin down cells and store at -80°C. The determined cDNA and amino acid sequences for the Ra12-P510S-C construct are provided in SEQ ID NO: 679 and 682, respectively.

The expression construct P510S-C was designed to have a 5' added start codon and a glycine (GGA) codon and then the P510S C terminal fragment followed by the in-frame 6x histidine tag and stop codon from the pET28b vector. The cloning strategy is similar to that used for Ra12-P510S-C, except that the PCR primers used were those shown in SEQ ID NO: 685 and 686, respectively, and the NcoI/XhoI cut in pET28b was used. The primer of SEQ ID NO: 685 formed a 5' NcoI site and added a start codon. Antisense primer with SEQ ID NO: 686 creates an Xhol site on the P510S C terminal fragment. Clones were confirmed by sequencing. For protein expression, the expression construct was transformed into E. coli BL21 (DE3) CodonPlus-RIL competent cells. An OD600 greater than 2.0 was achieved 30 hours after induction. Coomassie-labeled SDS-PAGE showed an overexpressed band at ca. 11 kD. Western blot analysis confirmed that the band was P510S-C, as did N-terminal protein sequencing. The optimized culture conditions are as follows: dilute overnight culture/day culture (LB + kanamycin + chloramphenicol) in 2x YT (+ kanamycin and chloramphenicol) at a ratio of 25 ml culture to 1 liter 2x YT and grow at 37°C up to an OD 600 of approx. 0.5 is achieved. Take an aliquot as TO sample. Add 1 mM IPTG and incubate at 30°C for 3 h. Spin down the cells and store at -80 °C until purification. The determined cDNA and amino acid sequences for the P510S-C construct are shown in SEQ ID NO: 680 and 683, respectively.

The putative third extracellular domain of P510S (P510S-E3; residues 328-676 in SEQ ID NO: 538) was expressed in E. coli as follows. The p510S fragment was amplified by PCR using the primers shown in SEQ ID NO: 687 and 688. The primer of SEQ ID NO: 687 is a sense primer with an Ndel site for use in ligation to pPDM. The primer with SEQ ID NO: 688 is an antisense primer with an added XhoI site for use in ligation to pPDM. The resulting fragment was cloned into pPDM at the NdeI and XhoI sites. Clones were confirmed by sequencing. For protein expression, the clone was transformed into E. coli BL21 (DE3) CodonPlus-RIL competent cells. After induction, an OD600 greater than 2.0 was achieved after 3 hours. Coomassie-labeled SDS-PAGE showed an overexpressed band at ca. 39 kD and N-terminal sequencing confirmed that the N-terminus was that of P510S-E3. Optimized culture conditions are as follows: dilute overnight culture/daytime culture (LB + kanamycin + chloramphenicol) in 2x YT (kanamycin and chloramphenicol) at a ratio of 25 ml culture to 1 liter 2x YT. Grow at 37°C until OD 600 is 0.6. Take an aliquot as a T0 sample. Add 1 mM IPTG and incubate at 30°C for 3 h. Take out a T3 sample, spin down the cells and store at -80 °C until purification. The determined cDNA and amino acid sequences for the P501S-E3 construct are provided in SEQ ID NO: 681 and 684, respectively.

g) Expression of P775S in E . Coli

The antigen P775P contains multiple open reading frames (ORFs). It

third ORF, which codes for the protein in SEQ ID NO: 483, has the best emotive score. An expression fusion construct containing M. tuberculosis antigen Ra12 (SEQ ID NO: 676) and P775P-ORF3 with an N-terminal 6x His tag was prepared as follows. P775P-ORF3 was amplified using sense PCR primers with SEQ ID NO: 689 and anti-sense PCR primer with SEQ ID NO: 690. The PCR-amplified fragment of P775P and Ra12/pCRX1 was digested with the restriction enzymes EcoRI and Xhol . Vector and insert were ligated and then transformed into NovaBlue cells. Colonies were randomly screened for insertion and then sequenced. A clone having the desired sequence was transformed into E. coli BL21 (DE3) CodonPlus-RIL competent cells. Two hours after induction, cell density peaked at OD600 of approximately 1.8. Coomassie-labeled SDS-PAGE showed an overexpressed band at ca. 31 kD. Western blot using 6x His-tag antibody confirmed that the band was Ra12-P775P-ORF3. The determined cDNA and amino acid sequences for the fusion construct are given in SEQ ID NO: 691 and 692, respectively.

H) Expression of a P703P His-tag fusion protein in E. coli

cDNA for the coding region of P703P was prepared by PCR using the primers of SEQ ID NO: 693 and 694. The PCR product was digested with EcoRI restriction enzyme, gel purified and cloned into a modified pET28 vector with a His tag in frame, which was digested with Eco72l and EcoRI restriction enzymes. Correct construction was confirmed by DNA sequence analysis and then transformed into E. coli BL21 (DE3) pLys S expression host cells. The determined amino acid and cDNA sequences of the expressed recombinant P703P are provided in SEQ ID NO: 695 and 696, respectively.

I) Expression of a P705P His-tag fusion protein in E. coli

cDNA for the coding region of P705P was prepared by PCR using the primers of SEQ ID NO: 697 and 698. The PCR product was digested with EcoRI restriction enzyme, gel purified and cloned into a modified pET28 vector with a His tag in frame, which was digested with Eco72l and EcoRI restriction enzymes. Correct construction was confirmed by DNA sequence analysis and then transformed into E. coli BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells. The determined amino acid and cDNA sequences of the expressed recombinant P705P are provided in SEQ ID NO: 699 and 700, respectively.

J) Expression of a P711P His-tag fusion protein in E. coli

cDNA for the coding region of P711P was prepared by PCR using the primers of SEQ ID NO: 701 and 702. The PCR product was digested with EcoRI restriction enzyme, gel purified and cloned into a modified pET28 vector with a His tag in frame, which was digested with Eco72l and EcoRI restriction enzymes. Correct construction was confirmed by DNA sequence analysis and then transformed into E. coli BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells. The determined amino acid and cDNA sequences for the expressed recombinant P711P are provided in SEQ ID NO: 703 and 704, respectively.

EXAMPLE 18

Production and characterization of antibodies against prostate-specific <p>ol<yp>e<p>times a) Production and characterization of polyclonal antibodies against P703P.

P504S and P509S

Polyclonal antibodies against P703P, P504S and P509S were prepared as follows.

Each prostate tumor antigen expressed in an E. coli recombinant expression system was grown overnight in LB medium with appropriate antibiotics at 37°C in a shaking incubator. The next morning, 10 ml of the overnight culture was added to 500 ml of 2x YT plus appropriate antibiotics in a 21 whipped Erlenmeyer flask. When the optical density (at 560 nm) of the culture reached 0.4-0.6, the cells were developed with IPTG (1 mM). Four hours after induction with IPTG, the cells were harvested by centrifugation. The cells were then washed with phosphate-buffered saline and centrifuged again. The supernatant was discarded and the cells were either frozen for future use or immediately processed.

20 ml of lysis buffer was added to the cell pellet and vortexed. To break up the E. coli cells, this mixture was then passed through the French Press at a pressure of 1120 kg/cm<2>. The cells were then centrifuged again and the supernatant and pellet were checked by SDS-PAGE for distribution of the recombinant protein. For proteins located in the cell pellet, the pellet was resuspended in 10 mM Tris pH 8.0, 1% CHAPS and the inclusion material pellet was washed and centrifuged again. This procedure was repeated two more times. The washed inclusion material pellet was solubilized with either 8 M urea or 6 M guanidine-HCl containing 10 mM Tris pH 8.0 plus 10 mM imidazole. The solubilized protein was added to 5 ml of nickel-chelate resin (Qiagen) and incubated for 45 min. to 1 hour at room temperature with continuous agitation. After incubation, the resin and protein mixture was poured through a disposable column and the flow-through was collected. The column was then washed with 10-20 column volumes of the solubilization buffer. The antigen was then eluted from the column using 8M urea, 10 mM Tris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. An SDS-PAGE gel was run to determine which fractions should be collected for further purification.

As a final purification step, a strong anion exchange resin such as HiPrepQ (Biorad) was equilibrated with the appropriate buffer and the pooled fractions from above were loaded onto the column. Each antigen was eluted from the column with an increasing salt gradient. Fractions were collected while the column was running and another SDS-PAGE gel was run to determine which fractions from the column should be collected. The combined fractions were dialyzed against 10 mM Tris pH 8.0. The proteins were then loaded onto glass after filtration through a 0.22 micron filter and the antigens were frozen until needed for immunization.

400 micrograms of each prostate antigen was combined with 100 micrograms of muramyl dipeptide (MDP). Every four weeks, rabbits received a booster dose of 100 micrograms mixed with an equal volume of Incomplete Freund's Adjuvant (IFA). Seven days after each boost, the animals were bled. Sera were made by incubating the blood at 4°C for 12-4 hours followed by centrifugation.

Ninety-six-well plates were coated with antigen by incubation with 50 microliters (typically 1 microgram) of recombinant protein at 4°C for 20 hours. 250 microliters of BSA blocking buffer was added to the wells and incubated at room temperature for 2 hours. Plates were washed 6 times with PBS/0.01% Tween. Rabbit sera were diluted in PBS. 50 microliters of diluted serum was added to each well and incubated at room temperature for 30 min. Plates were washed as described above before 50 microliters of goat anti-rabbit horseradish peroxidase (HRP) at a 1:10,000 dilution was added and incubated at room temperature for 30 min. The plates were again washed as described above and 100 microliters of TMB microwell peroxidase substrate was added to each well. After 15 min. incubation in the dark at room temperature, the colorimetric reaction was stopped with 100 microliters of 1N H 2 SO 4 and read immediately at 450 nm. All polyclonal antibodies showed immunoreactivity to the relevant antigen.

b) Preparation and characterization of antibodies against P501S

A murine monoclonal antibody directed against the carboxy terminus of it

prostate-specific antigen P501S was prepared as follows.

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

Mice were immunized with truncated P501S protein. Serum aliquots from mice potentially containing anti-P501S polyclonal sera were tested for P501S-specific reactivity using ELISA assay with purified P501S and trx-P501S proteins. Serum samples that appeared to react specifically with P501S were then screened for P501S reactivity by Western analysis. Mice harboring a P501 S-specific antibody component were sacrificed and spleen cells were used to generate anti-P501S antibody-producing hybridomas using standard techniques. Hybridoma supernatants were tested for P501 S-specific reactivity initially by ELISA and then by FACS analysis of reactivity with P501S-transduced cells. Based on these results, the monoclonal hybridoma referred to as 10E3 was selected for further subcloning. Several subclones were generated, tested for specific reactivity for P501S using ELISA and typed for IgG isotype. The results of this analysis are shown below in Table V. Of the 16 subclones tested, the monoclonal antibody 10E3-G4-D3 was selected for further investigation.

The specificity of 10E3-G4-D3 for P501S was examined by FACS analysis. Specifically, cells were fixed (2% formaldehyde, 10 min), permeabilized (0.1% saponin, 10 min) and labeled with 10E3-G4-D3 at 0.5-1 µg/ml, followed by incubation with a secondary, FITC -conjugated goat anti-mouse lg antibody (Pharmingen, San Diego, CA). Cells were then analyzed for FITC fluorescence using an Excalibur fluorescence-activated cell sorter. For FACS analysis of transduced cells, B-LCL were retrovirally transduced with P501S. For analysis of infected cells, B-LCL were infected with a vaccinia vector expressing P501S. To demonstrate specificity in these experiments, B-LCL were transduced with a different antigen (P703P) and uninfected B-LCL vectors were used. 10E3-G4-D3 was shown to bind with P501S-transduced B-LCL and also with P501S-infected B-LCL, but not with either uninfected cells or P703P-transduced cells.

To determine whether the epitope recognized by 10E3-G4-D3 was found on the surface or in an intracellular compartment of cells, B-LCL were transduced with P501S or HLA-B8 as a control antigen and either fixed and permeabilized as described above or directly labeled with 10E3-G4-D3 and analyzed as above. Specific recognition of P501S by 10E3-G4-D3 was found to require permeabilization, indicating that the epitope recognized by this antibody is intracellular.

The reactivity of 10E3-G4-D3 with the three prostate tumor cell lines Lncap, PC-3, and DU-145, which are known to express high, medium, and very low levels of P501S, respectively, was investigated by permeabilizing the cells and treating them as described above. Higher reactivity for 10E3-G4-D3 was seen with Lncap than with PC-3, which in turn showed higher reactivity than DU-145. These results are consistent with real-time PCR and demonstrate that the antibody specifically recognizes P501S in these tumor cell lines and that the epitope recognized in prostate tumor cell lines is also intracellular.

Specificity of 10E3-G4-D3 for P501S was also demonstrated by Western blot analysis. Lysates from prostate tumor cell lines Lncap, DU-145 and PC-3, from P501S transiently transfected HEK293 cells and from non-transfected HEK293 cells were prepared. Western blot analysis of these lysates with 10E3-G4-D3 revealed a 46 kDa immunoreactive band in Lncap, PC-3 and P501S-transfected HEK cells, but not in DU-145 cells or non-transfected HEK293 cells. P501S mRNA expression is consistent with these results since semi-quantitative PCR analysis showed that P501S mRNA is expressed in Lncap, at a lower but detectable level in PC-3 and not at all in DU-145 cells. Bacterial expressed and purified recombinant P501S (referred to as P501SStr2) was recognized by 10E3-G4-D3 (24 kDa), which was full-length P501S transiently expressed in HEK293 cells using either the expression vector VR1012 or pCEP4. Although the predicted molecular weight of P501S is 60.5 kDa, both transfected and "native" P501S had a somewhat lower mobility due to its hydrophobic nature.

Immunohistochemical analysis was performed on prostate tumor and a panel of normal tissue sections (prostate, adrenal gland, breast, cervix, colon, duodenum, gallbladder, ileum, kidney, ovary, pancreas, parotid gland, skeletal muscle, spleen and testis). Tissue samples were fixed in formalin solution for 24 hours and embedded in paraffin before being cut into 10 micron sections. Tissue sections were permeabilized and incubated with 10E3-G4-D3 antibody for 1 hour. HRP-labeled anti-mouse followed by incubation with DAB chromogen was used to visualize P501S immunoreactivity. P501S was found to be highly expressed in both normal prostate and prostate tumor tissues, but was not detected in any of the other tissues tested.

To identify the epitope recognized by 10E3-G4-D3, an epitope mapping was performed. A series of 13 overlapping 20-21 mers (5 amino acid overlap; SEQ ID NO: 489-501) were synthesized spanning the fragment of P501S used to generate 10E3-G4-D3. Flat-bottomed 96-well microtiter plates were coated with either the peptides or the P501S fragment used to immunize mice, at 1 microgram/ml for 2 hours at 37°C. Wells were then aspirated and blocked with phosphate-buffered saline containing 1% (w/v) BSA for 2 h at room temperature and then washed in PBS containing 0.1% Tween 20 (PBST). Purified antibody 10E3-G4-D3 was added at 2-fold dilutions (1000 ng -16 ng) in PBST and incubated for 30 minutes at room temperature. This was followed by washing 6 times with PBST and then incubation with HRP-conjugated donkey anti-mouse IgG (H+L)Affinipure F(ab') fragment (Jackson Immunoresearch, West Grove, PA) at 1:20000 for 30 minutes. The plates were then washed and incubated for 15 minutes in tetramethylbenzidine. Reactions were stopped by the addition of 1N sulfuric acid and the plates were read at 450 nm using an ELISA plate reader. As shown in Fig. 8, reactivity was seen with the peptide of SEQ ID NO: 496 (corresponding to amino acids 439-459 of P501S) and with the P501S fragment, but not with the remaining peptides, demonstrating that the epitope recognized by 10E3-G4- D3 is located at amino acids 439-459 in SEQ ID NO: 113.

To further evaluate the tissue specificity of P501S, multi-array immunohistochemical analysis was performed on approximately 4700 different human tissues including all major normal organs as well as neoplasias derived from these tissues. 65 of these human tissue samples were of prostate origin. Tissue sections 0.6 mm in diameter were formalin-fixed and paraffin-embedded. Samples were pretreated with HIER using 10 mM citrate buffer pH 6.0 and boiling for 10 min. The sections were labeled with 10E3-G4-D3 and P501S immunoreactivity was visualized with HRP. All 65 prostate tissue samples (5 normal, 55 untreated prostate tumors, 5 hormone-resistant prostate tumors) were positive, showing distinct perinuclear labeling. All other tissues examined were negative for P501S expression.

c) Preparation and characterization of antibodies against P503S

A fragment of P503S (amino acids 113-241 of SEQ ID NO: 114) was

expressed and purified from bacteria essentially as described above for P501S and used to immunize both rabbits and mice. Mouse monoclonal antibodies were isolated using standard hybridoma technology as described above. Rabbit monoclonal antibodies were isolated using Selected Lymphocyte Antibody Method (SLAM) technology at Immgenics Pharmaceuticals (Vancouver, BC, Canada). Table VI below lists the monoclonal antibodies that were developed against P503S.

The DNA sequences encoding the complementarity determining regions (CDR) of the rabbit monoclonal antibodies 20D4 and JA1 were determined and are given in SEQ ID NO: 502 and 503, respectively.

To better define the epitope-binding region in each of the antibodies, a series of overlapping peptides was generated spanning amino acids 109-213 of SEQ ID NO: 114. These peptides were used to epitope-map anti-P503S monoclonal antibodies by ELISA as follows. The recombinant fragment of P503S used as the immunogen was used as a positive control. 96 well microtiter plates were coated with either peptide or recombinant antigen at 20 ng/well overnight at 4°C. Plates were aspirated and blocked with phosphate-buffered saline containing 1% (w/v) BSA for 2 h at room temperature and then washed in PBS containing 0.1% Tween 20 (PBST). Purified rabbit monoclonal antibodies diluted in PBST were added to the wells and incubated for 30 min. at room temperature. This was followed by washing 6 times with PBST and incubation with Protein-A HRP conjugate at a 1:2000 dilution for a further 30 min. Plates were washed six times in PBST and incubated with tetramethylbenzidine (TMB) substrate for an additional 15 min. The reaction was stopped by the addition of 1N sulfuric acid and plates were read at 450 nm using an ELISA plate reader. ELISA with mouse monoclonal antibodies was performed with tissue culture supernatants run undiluted in the experiment.

All antibodies bound to the recombinant P503S fragment, with the exception of the negative control SP2 supernatant. 20D4, JA1 and 1D12 bound tightly to peptide #2101 (SEQ ID NO: 504), which corresponds to amino acids 151-169 of SEQ ID NO: 114. 1C3 bound to peptide #2102 (SEQ ID NO: 505), which corresponds to amino acids 165-184 of SEQ ID NO: 114. 9C12 bound to peptide #2099 (SEQ ID NO: 522), which corresponds to amino acids 120-139 of SEQ ID NO: 114. The other antibodies bound to regions not examined in these studies.

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

To determine which tissues express P503S, immunohistochemical analysis was performed, essentially as described above, on a panel of normal tissues (prostate, adrenal gland, breast, cervix, colon, duodenum, gallbladder, ileum, kidney, ovary, pancreas, parotid gland , skeletal muscle, spleen and testis). HRP-labeled anti-mouse or anti-rabbit antibody followed by incubation with TMB was used to visualize P503S immunoreactivity. P503S was found to be highly expressed in prostate tissue, with lower levels of expression observed in cervix, colon, ileum and kidney and no expression observed in adrenal, breast, duodenum, gallbladder, ovary, pancreas, parotid gland, skeletal muscle, spleen and testis.

Western blot analysis was used to characterize anti-P503S monoclonal antibody specificity. SDS-PAGE was performed on recombinant (rec) P503S expressed in and purified from bacteria and on lysates from HEK293 cells transfected with full-length P503S. Protein was transferred to nitrocellulose and then Western blot analyzed with each of the anti-P503S monoclonal antibodies (20D4, JA1, 1D12, 6D12 and 9C12) at an antibody concentration of 1 µg/ml. Protein was detected using horseradish peroxidase (HRP) conjugated to either a goat anti-mouse monoclonal antibody or to protein A-sepharose. The monoclonal antibody 20D4 detected the relevant molecular weight 14 kDa recombinant P503S (amino acids 113-241) and 23.5 kDa species in HEK293 cell lysates transfected with full-length P503S. Other anti-P503S monoclonal antibodies showed similar specificity by Western blot.

d) Preparation and characterization of antibodies against P703P

Rabbits were immunized with either the truncated (P703Ptr1; SEQ ID NO: 172) or full-length mature form (P703Pfl; SEQ ID NO: 523) of recombinant P703P protein expressed in and purified from bacteria as described above. Affinity-purified polyclonal antibody was generated using immunogen P703Pfl or P703Ptr1 bound to a solid support. Rabbit monoclonal antibodies were isolated using SLAM technology at Immgenics Pharmaceuticals. Table VII below lists both the polyclonal and monoclonal antibodies raised against P703P.

The DNA sequences encoding the complementarity determining regions (CDR) of rabbit monoclonal antibodies 8H2, 7H8 and 2D4 were determined and are given 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 the peptides in SEQ ID NO: 509 (corresponding to amino acids 145-159 in SEQ ID NO: 172) and SEQ ID NO: 510 (corresponding to amino acids 11-25 in SEQ ID NO: 172) respectively. . The polyclonal antibody 2594 was found to bind to the peptides in SEQ ID NO: 511-514, the polyclonal antibody 9427 binding to the peptides in SEQ ID NO: 515-517.

The specificity of the anti-P703P antibodies was determined by Western blot analysis as follows. SDS-PAGE was performed on (1) bacterially expressed recombinant antigen; (2) lysates of HEK293 cells and Ltk-/- cells either untransfected or transfected with a plasmid expressing full-length P703P; and (3) supernatant isolated from these cell cultures. Protein was transferred to nitrocellulose and then Western blot analyzed using the anti-P703P polyclonal antibody #2594 at an antibody concentration of 1 µg/ml. Protein was detected using horseradish peroxidase (HRP) conjugated to an anti-rabbit antibody. A 35 kDa immunoreactive band could be observed with recombinant P703P. Recombinant P703P runs at a slightly higher molecular weight since it is epitope-tagged. In lysates and supernatants from cells transfected with full-length P703P, a 30 kDa band corresponding to P703P was observed. To ensure specificity, lysates from HEK293 cells stably transfected with a control plasmid were also tested and were negative for P703P expression. Other anti-P703P antibodies showed similar results.

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

e) Preparation and characterization of antibodies against P504S

Full-length P504S (SEQ ID NO: 108) was expressed and purified from bacteria

essentially as described above for P501S and used to induce

rabbit monoclonal antibodies using Selected Lymphocyte Antibody Method (SLAM) technology at Immgenics Pharmaceuticals (Vancouver, BC, Canada). The anti-P504S monoclonal antibody 13H4 was shown by Western blot to bind to both expressed recombinant P504S and to native P504S in tumor cells.

Immunohistochemical studies using 13H4 to assess P504S expression in various prostate tissues were performed as described above. A total of 104 cases, comprising 65 cases of radical prostatectomies with prostate cancer (PC), 26 cases of prostate biopsies and 13 cases of benign prostatic hyperplasia (BPH), were labeled with the anti-P504S monoclonal antibody 13H4. P504S showed strong cytoplasmic granular labeling in 64/65 (98.5%) of PC in prostatectomies and 26/26 (100%) of PC in prostatic biopsies. P504S was labeled strongly and diffusely in carcinomas (4+ in 91.2% of cases of PC; 3+ in 5.5%; 2+ in 2.2% and 1+ in 1.1%) and high-grade prostatic intraepithelial neoplasia (4+ in all cases). The expression of P504S did not vary with Gleason score. Only 17/91 (18.7%) of cases of NP/BPH around PC and 2/13 (15.4%) of BPH cases were focal (1+, none 2+ to 4+ in all cases) and weak positive for P504S in large glands. Expression of P504S was not found in small atrophic glands, postatrophic hyperplasia, basal cell hyperplasia and transition cell metaplasia in either biopsies or prostatectomies. Thus, P504S was found to be overexpressed in all Gleason scores of prostate cancer (98.5 to 100% sensitivity) and only showed focal positivity in large normal glands in 19/104 cases (82.3% specificity). These findings indicate that P504S can be appropriately used for the diagnosis of prostate cancer.

EXAMPLE 19

Characterization<g>of cell surface ex<p>resion and

chromosome localization of the prostate-specific anti<g>en P501S

This example describes studies demonstrating that prostate-specific antigen P501S is expressed on the surface of cells, along with studies to determine the probable chromosomal location of P501S.

The protein P501S (SEQ ID NO: 113) is predicted to have 11 transmembrane domains. Based on the discovery that the epitope recognized by the anti-P501 S monoclonal antibody 10E3-G4-D3 (described above in Example 17) is intracellular, it was hypothesized that the following transmembrane determinants would allow the prediction of extracellular domains of P501S. Fig. 9 is a schematic representation of the P501S protein showing the putative location of the transmembrane domains and the intracellular epitope described in Example 17. Underlined sequence represents the putative transmembrane domains, bold sequence represents the putative extracellular domains and italic sequence represents the putative intracellular domains. Sequence that is both highlighted and underlined represents a sequence used to generate polyclonal rabbit serum. The location of the transmembrane domains was predicted using HHMTOP as described by Tusnady and Simon (Principles Governing Amino Acid Composition of Integral Membrane Proteins: Applications to Topology Prediction, J. Mol. Biol. 283:489-506, 1998).

Based on Fig. 9, the P501 S domain flanked by the transmembrane domains corresponding to amino acids 274-295 and 323-342 is believed to be extracellular. The peptide in SEQ ID NO: 518 corresponds to amino acids 306-320 in P501S and is located in the presumed extracellular domain. The peptide of SEQ ID NO: 519, which is identical to the peptide of SEQ ID NO: 518 except for the substitution of histidine with asparginine, was synthesized as described above. Cys-Gly was added to the C-terminus of the peptide to facilitate conjugation to the carrier protein. Cleavage of the peptide from the solid support was performed using the following cleavage mixture: trifluoroacetic acid:ethanediol:thioanisole:water:phenol (40:1:2:2:3). After cleavage for two hours, the peptide was precipitated in cold ether. The peptide pellet was then dissolved in 10% vol/vol acetic acid and lyophilized before purification by C18 reverse phase hplc. A gradient of 5-60% acetonitrile (containing 0.05% TFA) in water (containing 0.05% TFA) was used to elute the peptide. The purity of the peptide was verified by hplc and mass spectrometry and was found to be

>95%. The purified peptide was used to generate rabbit polyclonal antisera as described above.

Surface expression of P501S was examined by FACS analysis. Cells were labeled with polyclonal anti-P501S peptide serum at 10 µg/ml, washed, incubated with secondary FITC-conjugated goat anti-rabbit Ig antibody (ICN), washed and analyzed for FITC fluorescence using an Excalibur fluorescence-activated cell sorter . For FACS analysis of transduced cells, B-LCL were retrovirally transduced with P501S. To demonstrate specificity in these experiments, B-LCL were transduced with an irrelevant antigen (P703P) or non-transduced labeled in parallel. For FACS analysis of prostate tumor cell lines, Lncap, PC-3 and DU-145 were used. Prostate tumor cell lines were dissociated from tissue culture plates using cell dissociation medium and labeled as above. All samples were treated with propidium iodide (Pl) before FACS analysis and data were obtained from Pl-exclusive (ie, intact and non-permeabilized) cells. Rabbit polyclonal serum raised against the peptide of SEQ ID NO: 519 was shown to specifically recognize the surface of cells transduced to express P501S, demonstrating that the epitope recognized by the polyclonal serum is extracellular.

To determine biochemically whether P501S is expressed on the cell surface, peripheral membranes from Lncap cells were isolated and subjected to Western blot analysis. Specifically, Lncap cells were lysed using a dounce homogenizer in 5 ml of homogenization buffer (250 mM sucrose, 10 mM HEPES, 1 mM EDTA, pH 8.0, 1 complete protease inhibitor tablet (Boehringer Mannheim)). Lysate samples were spun at 1000 g for 5 min. at 4°C. The supernatant was then spun at 8000 g for 10 min. at 4°C. Supernatant from the 8000 g centrifugation was recovered and subjected to 100,000 g spinning for 30 min. at 4°C to recover peripheral membrane. Samples were then separated by SDS-PAGE and Western blot analyzed with the mouse monoclonal antibody 10E3-G4-D3 (described above in Example 17) using the conditions described above. Recombinant purified P501S as well as HEK293 cells transfected with and overexpressing P501S were included as positive controls for P501S detection. LCL cell lysate was included as a negative control. P501S could be detected in Lncap total cell lysate, the 8000 g (inner membrane) fraction and also in the 100,000 g (plasma membrane) fraction. These results indicate that P501S is expressed at, and localizes to, the peripheral membrane.

To show that rabbit polyclonal antiserum raised against the peptide of SEQ ID NO: 519 specifically recognizes this peptide as well as the corresponding native peptide of SEQ ID NO: 518, ELISA assays were performed. For these assays, flat-bottomed 96-well microtiter plates were coated with either the peptide of SEQ ID NO: 519, the longer peptide of SEQ ID NO: 520 that spans the entire predicted extracellular domain, the peptide of SEQ ID NO: 521 that represents the epitope recognized by the P501S-specific antibody 10E3-G4-D3 or a P501S fragment (corresponding to amino acids 355-526 of SEQ ID NO: 113) which does not include the immunizing peptide sequence, at 1 µg/ml for 2 hours at 37°C . The wells were aspirated, blocked with phosphate buffered saline containing 1% (w/v) BSA for 2 h at room temperature and then washed in PBS containing 0.1% Tween 20 (PBST). Purified anti-P501S rabbit polyclonal serum was added at 2-fold dilutions (1000 ng - 125 ng) in PBST and incubated for 30 min. at room temperature. This was followed by washing 6 times with PBST and incubation with HRP-conjugated goat anti-rabbit IgG (H+L) Affinipure F(ab') fragment at 1:20000 for 30 min. The plates were then washed and incubated for 15 min. in tetramethyl-benzidine. Reactions were stopped by the addition of 1N sulfuric acid and the plates were read at 450 nm using an ELISA plate reader. As shown in Fig. 11, the anti-P501S polyclonal rabbit serum specifically recognized the peptide of SEQ ID NO: 519 used for the immunization as well as the longer peptide of SEQ ID NO: 520, but did not recognize the irrelevant P501S-derived peptides and fragments.

In further investigations, rabbits were immunized with peptides derived from P501S sequence and assumed to be either extracellular or intracellular, as shown in Fig. 9. Polyclonal rabbit sera were isolated and polyclonal antibodies in the serum were purified, as described above. To determine specific reactivity with P501S, FACS analysis was employed, using either B-LCL transduced with P501S or the irrelevant antigen P703P from B-LCL infected with vaccinia virus expressing P501S. For surface expression, dead and non-intact cells were excluded from the analysis as described above. For intracellular labeling, cells were fixed and permeabilized as described above. Rabbit polyclonal serum raised against the peptide of SEQ ID NO: 548, which corresponds to amino acids 181-198 of P501S, was found to recognize a surface epitope of P501S. Rabbit polyclonal serum raised against the peptide of SEQ ID NO: 551, which corresponds to amino acids 543-553 of P501S, was found to recognize an epitope that was either potentially extracellular or intracellular, as in various experiments intact or permeabilized cells were recognized by the polyclonal sera. Based on similar deductive reasoning, the sequences in SEQ ID NO: 541-547, 549 and 550, which correspond to amino acids 109-122, 539-553, 509-520, 37-54, 342-359, 295-323, 217 -274, 143-160 and 75-88 of P501S, are considered to be potential surface epitopes of P501S recognized by antibodies.

In further investigations, mouse monoclonal antibodies were raised against amino acids 296 to 322 of P501S, which are believed to be in an extracellular domain. A/J mice were immunized with P501S/adenovirus, followed by subsequent boosting with an E. coli recombinant protein, referred to as P501N, containing amino acids 296 to 322 of P501S and with peptide 296-322 (SEQ ID NO: 755) linked with KLH. The mice were then used for splenic B-cell fusions to form anti-peptide hybridomas. The resulting 3 clones, referred to as 4F4 (lgG1 .kappa), 4 G5 (lgG2a,kappa) and 9B9 (lgG1,kappa), were cultured for antibody production. 4 The G5 mAb was purified by passing the supernatant over a Protein A-sepharose column, followed by antibody elution using 0.2M glycine, pH 2.3. Purified antibody was neutralized by adding 1M Tris, pH 8 and buffer changed to PBS.

For ELISA analysis, 96-well plates were coated with P501S peptide 296-322 (referred to as P501-long), an irrelevant P775 peptide, P501S-N, P501TR2, P501S-long-KLH, P501S peptide 306-319 (referred to as P501-short)-KLH or the irrelevant peptide 2073-KLH, all at a concentration of 2 µg/ml and allowed to incubate for 60 minutes at 37°C. After labeling, the plates were washed 5X with PBS + 0.1% Tween and then blocked with PBS, 0.5% BSA, 0.4% Tween20 for 2 hours at room temperature. After addition of supernatants or purified mAb, the plates were incubated for 60 min at room temperature. Plates were washed as above and donkey anti-mouse IgHRP-linked secondary antibody was added and incubated for 30 min at room temperature, followed by a final wash as above. TMB-peroxidase substrate was added and incubated for 15 minutes at room temperature in the dark. The reaction was stopped by the addition of 1N H 2 SO 4 and the OD was read at 450 nM. All three hybrid clones secreted mAbs that recognized peptide 296-322 and the recombinant protein P501N.

For FACS analysis, HEK293 cells were transiently transfected with P501SA/R1012 expression constructs using Fugene 6 reagent. After 2 days of culture, cells were harvested and washed, then incubated with purified 4 G5 mAb for 30 min on ice. After numerous washes in PBS, 0.5% BSA, 0.01% azide, goat anti-mouse Ig-FITC was added to the cells and incubated for 30 min on ice. The cells were washed and resuspended in wash buffer containing 1% propidium iodide and subjected to FACS analysis. The FACS analysis confirmed that amino acids 296-322 of P501S are in an extracellular domain and are cell surface expressed.

The chromosomal location of P501S was determined using the GeneBridge 4 Radiation Hybrid panel (Research Genetics). PCR primers with SEQ ID NO: 528 and 529 were used in PCR with DNA collections from the hybrid panel according to the manufacturer's guidelines. After 38 cycles of amplification, the reaction products were separated on a 1.2% agarose gel and the results were analyzed through the Whitehead Institute/M IT Center for Genome Research web server (http://www-genom.wi.mit.edu/cgi-bin /contig/rhmapper.pl) to determine the likely chromosomal location. Using this method, P501S was mapped to the long arm of chromosome 1 at W1-9641 between q32 and q42. This region of chromosome 1 is linked to prostate cancer susceptibility in 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 indicate that P501S may play a role in malignant prostate cancer.

EXAMPLE 20

Regulation of expression of the prostate-specific antibody P501S

Steroid (androgen) hormone modulation is a common treatment modality for prostate cancer. Expression of several prostate tissue-specific antigens has previously been demonstrated to respond to androgen. The responsiveness of prostate-specific antigen P501S to androgen treatment was investigated in a tissue culture system as follows.

Cells from the prostate tumor cell line LNCaP were plated at 1.5 x 106 cells/T75 flask (for RNA isolation) or 3 x 105 cells/well of a 6-well plate (for FACS analysis) and cultured overnight in RPMI 1640 media containing 10% charcoal-stripped fetal calf serum (BRL Life Technologies, Gaithersburg, MD). Cell culture was continued for an additional 72 h in RPMI 1640 media containing 10% charcoal-stripped fetal calf serum, with 1 nM of the synthetic androgen Methyltrienolone (R1881; New England Nuclear) added at various times. Cells were then harvested for RNA isolation and FACS analysis at 0, 1, 2, 4, 8, 16, 24, 28 and 72 h after androgen addition. FACS analysis was performed using anti-P501S antibody 10E3-G4-D3 and permeabilized cells.

For Northern analysis, 5-10 micrograms of total RNA was run on a

formaldehyde denaturing gel, transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ), cross-linked and labeled with methylene blue. The filter was then prehybridized with Church's Buffer (250 mM Na 2 HPO 4 , 70 mM H 3 PO 4 , 1 mM EDTA, 1% SDS, 1% BSA in pH 7.2) at 65°C for 1 hour. P501S DNA was labeled with 32P using the High Prime random-primed DNA labeling kit (Boehringer Mannheim). Unincorporated labeling was removed using MicroSpin S300-HR columns (Amersham Pharmacia Biotech). The RNA filters were then hybridized with fresh Churcl<Y>s Buffer containing labeled cDNA overnight, washed with 1X SCP (0.1 M NaCl, 0.03 M Na2HP04.7H2O, 0.001 M Na2EDTA), 1% Sarkosyl (n- lauroylsarcosine) and exposed to X-ray film.

Using both FACS and Northern analysis, P501S message and protein levels were found to increase in response to androgen treatment.

EXAMPLE 20

Production of fusion proteins of prostate-specific antigens

The example describes the production of a fusion protein of prostate-specific antigen P703P and a shortened form of the known prostate antigen PSA. The shortened form of PSA has a 21 amino acid deletion around the active serine site. The expression construct for the fusion protein also has a restriction site at the 3' end, immediately before the termination codon, to aid in the addition of cDNA for additional antigens.

Full-length cDNA for PSA was obtained by RT-PCR from a pool of RNA from human prostate tumor tissues using the primers of SEQ ID NO: 607 and 608 and cloned into the vector pCR-Blunt 11-TOPO. The resulting cDNA was used as a template to prepare two different fragments of PSA by PCR with two sets of primers (SEQ ID NO: 609 and 610; and SEQ ID NO: 611 and 612). The PCR products having the expected size were used as templates to produce shortened forms of PSA by PCR with the primers of SEQ ID NO: 611 and 613, which formed PSA (delta 208-218 in amino acids). The cDNA for the mature form of P703P with a 6X histidine tag at the 5' end was prepared by PCR with P703P and the primers of SEQ ID NO: 614 and 615. The cDNA for the fusion of P703P with the shortened form of PSA (refer to as FOPP) was then obtained by PCR using the modified P703P cDNA and the shortened form of PSA cDNA as templates and the primers with SEQ ID NO: 614 and 615. The FOPP cDNA was cloned into the NdeI site and the XhoI site of the expression vector pCRX1 and confirmed by DNA sequencing. The determined cDNA sequence for the FOPP fusion construct is provided in SEQ ID NO: 616, with the amino acid sequence provided in SEQ ID NO: 617.

The fusion FOPP was expressed as a single recombinant protein in E. coli as follows. The expression plasmid pCRX1 FOPP was transformed into E. coli strain BL21 -CodonPlus RIL. The transformant was shown to express FOPP protein after induction with 1 mM IPTG. The culture with the corresponding expression clone was inoculated into 25 ml LB medium containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, grown at 37°C to OD 600 of approx. 1 and stored at 4°C overnight. The culture was diluted in 1 liter TB LB containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol and grown at 37°C to OD600 of 0.4. IPTG was added to a final concentration of 1 mM and the culture was incubated at 30°C for 3 h. The cells were pelleted by centrifugation at 5000 rpm for 8 min. To purify the protein, the cell pellet was suspended in 25 ml of 10 mM Tris-Cl pH 8.0, 2 mM PMSF, complete protease inhibitor and 15 µg of lysozyme. The cells were lysed at 4°C for 30 min, sonicated several times and the lysate was centrifuged for 30 min at 10,000 x g. The pellet, which contained the inclusion material, was washed twice with 10 mM Tris-Cl pH 8.0 and 1% CHAPS . The inclusion material was dissolved in 40 ml of 10 mM Tris-Cl pH 8.0, 100 mM sodium phosphate and 8 M urea. The solution was bound to 8 ml of Ni-NTA (Qiagen) for one hour at room temperature. The mixture was poured into a 25 ml column and washed with 50 ml of 10 mM Tris-Cl pH 6.3, 100 mM sodium phosphate, 0.5% DOC and 8M urea. The bound protein was eluted with 350 mM imidazole, 10 mM Tris-Cl pH 8.0, 100 mM sodium phosphate and 8 M urea. The fractions containing FOPP proteins were collected and extensively dialyzed against 10 mM Tris-Cl pH 4.6, aliquoted and stored at -70°C.

EXAMPLE 21

Real-time PCR characterization<g>of prostate-specific antibody<g>en P501S in<p>erified blood from<p>prostate cancer patients

Circulating epithelial cells were isolated from fresh blood of normal individuals and metastatic prostate cancer patients, mRNA isolated and cDNA prepared using real-time PCR procedures. Real-time PCR was performed by the Taqman™ procedure using both gene-specific primers and probes to determine the levels of gene expression.

Epithelial cells were enriched from blood samples using an immunomagnetic bead separation method (Dynal A.S., Oslo, Norway). Isolated cells, the light and the magnetic beads were removed. The lysate was then processed for poly A+ mRNA isolation using magnetic beads coated with Oligo(dT)25. After washing the beads in buffer, bead/poly A+ RNA samples were suspended in 10 mM Tris-HCl, pH 8.0 and subjected to reverse transcription. The resulting cDNA was subjected to real-time PCR using gene-specific primers. Beta-actin content was also determined and used for normalization. Samples with P501S copies greater than the mean of the normal samples + 3 standard deviations were considered positive. Real-time PCR on blood samples was performed using the Taqman™ procedure, but extended to 50 cycles using forward and reverse primers and probes specific for P501S. Of the eight samples tested, 6 were positive for P501S and B-actin signal. The remaining 2 samples did not have detectable B-actin or P501S. No P501 S signal was observed in the four normal blood samples tested.

EXAMPLE 22

Expression of prostate-specific antigens P703P and P501S in

SCID MICE-PASSED PROSTATE TUMORS

When assessing the effectiveness of antigens in the treatment of prostate cancer, the continued presence of the antigens in tumors during androgen ablation therapy is important. The presence of prostate-specific antigens P703P and P501S in prostate tumor samples grown in SCID mice in the presence of testosterone was evaluated as follows.

Two prostate tumors that had metastasized to bone were removed from patients, implanted into SCID mice, and cultured in the presence of testosterone. Tumors were evaluated for mRNA expression of P703P, P501S and PSA using quantitative real-time PCR with the SYBR-green assay method. Expression of P703P and P501S in a prostate tumor was used as a positive control and absence in normal intestine and normal heart as negative controls. In both cases, specific mRNA was present in late-passage tumors. Since the bone metastases were grown in the presence of testosterone, this implies that the presence of these genes would not be lost during androgen ablation therapy.

EXAMPLE 23

ANTI-P503S MONOCLONAL antibody inhibits tumor growth in vivo

The ability of the anti-P503S monoclonal antibody 20D4 to suppress tumor formation in mice was examined as follows.

Ten SCID mice were injected subcutaneously with HEK293 cells expressing P503S. Five mice received 150 micrograms of 20D4 intravenously on day 0 (time of tumor cell injection), day 5 and day 9. Tumor size was measured for 50 days. Of the five animals that did not receive 20D4, three developed detectable tumors after approx. 2 weeks which continued to grow throughout the survey. In contrast, none of the five mice that received 20D4 developed tumors. These results demonstrate that anti-P503S Mab 20D4 exhibits potent anti-tumor activity in vivo.

EXAMPLE 24

Characterization<g>of a T-cell receptor clone from a

<p>501 s-specific t-cell clone

T cells have a limited lifespan. However, cloning of T-cell receptor (TCR) chains and subsequent transfer enables essentially infinite propagation of T-cell specificity. Cloning of tumor-antigen TCR chains allows transfer of specificity to T cells isolated from patients that share the TCR MHC restriction allele. Such T cells could then be expanded and used in adoptive transfer settings to introduce tumor-antigen specificity to patients bearing tumors expressing the antigen. T cell receptor alpha and beta chains from a CD8 T cell clone specific for prostate specific antigen P501S were isolated and sequenced as follows.

Total mRNA from 2 x 10<6> cells of CTL clone 4E5 (described above in Example 12) was isolated using Trizol reagent and cDNA was synthesized. To determine Va and Vb sequences in this clone, a panel of Va and Vb subtype-specific primers was synthesized and used in RT-PCR reactions with cDNA generated from each of the clones. The RT-PCR reactions demonstrated that each of the clones expressed a common Vb sequence corresponding to the Vb7 subfamily. Furthermore, using cDNA generated from the clone, the expressed Vα sequence was determined to be Vα6. To clone the full TCR alpha and beta chains from clone 4E5, primers were designed to span the initiator and terminator encoding TCR nucleotides. The primers were as follows: TCR Valfa-6 5' (sense): GGATCC—GCCGCCACC— ATGTCACTTTCTAGCCTGCT (SEQ ID NO: 756) BamHI site Kozak TCR alpha sequence TCR alpha 3' (antisense): GTCGAC—TCAGCTGGACCACAGCCGCAG (SEQ ID NO: 757 ) Sali sete TCR alpha constant sequence TCR Vbeta-7.

5'(sense): GGATCC—GCCGCCACC--ATGGGCTGCAGGCTGCTCT (SEQ ID NO

NO: 758) BamHI site Kozak TCR alpha sequence TCR beta 3' (antisense): GTCGAC—TCAGAAATCCTTTCCTTGAC (SEQ ID NO: 759) Sali site TCR beta constant sequence. Standard 35 cycle RT-PCR reactions were established using cDNA synthesized from the CTL clone and the above primers using the proofreading thermostable polymerase PWO (Roche, Nutley, NJ).

The resulting specific bands (about 850 bp for alpha and about 950 for beta) were ligated into PCR blunt vector (Invitrogen) and transformed into E. coli. E. coli transformed with plasmids containing full-length alpha and beta chains were identified and large-scale preparations of the corresponding plasmids were made. Plasmids containing full-length TCR alpha and beta chains were subjected to sequencing. The sequencing reactions demonstrated cloning of full-length TCR alpha and beta chains with the determined cDNA sequences for the Vb and Va chains shown in SEQ ID NO: 760 and 761, respectively. The corresponding amino acid sequences are shown in SEQ ID NO: 762, respectively and 763. The Va sequence was shown by nucleotide sequence alignment to be 99% identical (347/348) to Va6,2 and Vb to be 99% identical to Vb7 (336/338).

From the foregoing, it will be understood that although specific embodiments of the present invention are described herein for illustrative purposes, various modifications may be made without departing from the scope of the present invention. Accordingly, the invention is limited only by the following claims.

Claims (18)

1. Isolated polynucleotide comprising a sequence selected from the group consisting of: (a) sequences given in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; (b) complements of the sequences given in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; (c) sequences consisting of at least 20 consecutive residues of a sequence given in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340 -375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636 , 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; (d) sequences that hybridize to a sequence given in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328,330, 332-335, 340-375, 381 , 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655 , 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788 under moderately stringent conditions; (e) sequences that have at least 75% identity with a sequence in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; (f) sequences that have at least 90% identity with a sequence in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788; and (g) degenerate variants of a sequence given in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639- 655, 674, 680, 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 and 786-788. 2. Isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) sequences set forth in SEQ ID NO: 112-114, 172, 176, 178,327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 627-629, 632, 633, 635, 637, 638, 656-671, 675, 683, 684, 710, 712, 714, 715, 717-719, 723-734, 736, 740-750, 752, 754, 755, 766-772, 777-785 and 789-791; (b) sequences that have at least 70% identity with a sequence in SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383,477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 627-629, 632, 633, 635, 637, 638, 656- 671, 675, 683, 684, 710, 712, 714, 715, 717-719, 723-734, 736, 740-750, 752, 754, 755, 766-772, 777-785 and 789-791; (c) sequences that have at least 90% identity with a sequence in SEQ ID NO: 112-114, 172, 176, 178, 327,329, 331,336,339,376-380,383,477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 627-629, 632, 633, 635, 637, 638, 656-671, 675, 683, 684, 710, 712, 714, 715, 717-719, 723-734, 736, 740-750, 752, 754, 755, 766-772, 777-785 and 789-791; (d) sequences encoded by a polynucleotide according to claim 1; (e) sequences having at least 70% identity with a sequence encoded by a polynucleotide according to claim 1; and (f) sequences which have at least 90% identity with a sequence encoded by a polynucleotide according to claim 1. 3. Expression vector comprising a polynucleotide according to claim 1 operably linked to an expression control sequence. 4. Host cell transformed or transfected with an expression vector according to claim 3. 5. Isolated antibody or antigen-binding fragment thereof, which specifically binds to a polypeptide according to claim 2. 6. Method for detecting the presence of cancer in a patient, comprising the steps: (a) obtaining a biological sample from the patient; (b) contacting the biological sample with a binding agent that binds to a polypeptide according to claim 2; (c) detecting in the sample an amount of polypeptide that binds to the binding agent; and (d) comparing the amount of polypeptide to a predetermined cut-off value and thereby determining the presence of cancer in the patient. 7. Fusion protein comprising at least one polypeptide according to claim 2. 8. Fusion protein according to claim 7, where the fusion protein comprises a sequence selected from the group consisting of: (a) sequences given in SEQ ID NO: 682, 692, 695, 699, 703 and 709; and (b) sequences encoded by SEQ ID NO: 679, 691, 696, 700, 704 and 708. 9. Oligonucleotide that hybridizes to a sequence indicated in SEQ ID NO: 1-111,115-171,173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384 -476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-626, 630, 631, 634, 636, 639-655, 674, 680 , 681, 711, 713, 716, 720-722, 735, 737-739, 751, 753, 764, 765, 773-776 or 786-788 under moderately stringent conditions. 10. Method for stimulating and/or expanding T cells specific for a tumor protein, comprising contacting T cells with at least one component selected from the group consisting of: (a) polypeptides according to claim 2; (b) polynucleotides according to claim 1; and (c) antigen presenting cells expressing a polypeptide according to claim 1, under conditions and for a time sufficient to allow stimulation and/or expansion of T cells. 11. Isolated T cell population, comprising T cells produced by methods according to claim 10. 12. Preparation comprising a first component selected from the group consisting of physiologically acceptable carriers and immunostimulants and a second component selected from the group consisting of: (a) polypeptides according to claim 2; (b) polynucleotides according to claim 1; (c) antibodies according to claim 5; (d) fusion proteins according to claim 7; (e) T cell populations according to claim 11; and (f) antigen-presenting cells expressing a polypeptide according to claim 2. 13. Method for stimulating an immune response in a patient, comprising administering to the patient a preparation according to claim 12. 14. Method for treating cancer in a patient, comprising administering to the patient a preparation according to claim 12. 15. Method for determining the presence of cancer in a patient, comprising the steps of: (a) obtaining a biological sample from the patient; (b) contacting the biological sample with an oligonucleotide according to claim 9; (c) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; and (d) comparing the amount of polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value and thereby determining the presence of cancer in the patient. 16. Diagnostic kit comprising at least one oligonucleotide according to claim 9. 17. Diagnostic kit comprising at least one antibody according to claim 5 and a detection reagent, where the detection reagent comprises a reporter group. 18. Method for inhibiting the development of cancer in a patient, comprising the steps: (a) incubating CD4+ and/or CD8+ T cells isolated from a patient with at least one component selected from the group consisting of: (i) polypeptides according to claim 2; (ii) polynucleotides according to claim 1; and (iii) antigen-presenting cells expressing a polypeptide according to claim 2, so that the T cell proliferates; and (b) administering to the patient an effective amount of the proliferated T cells; thereby inhibiting the development of cancer in the patient.