AU2003257511B2 - Compositions and methods for WT1 specific immunotherapy - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION DIVISIONAL APPLICATION

(ORIGINAL)

Name of Applicant: Actual Inventors: Address for Service: Invention Title: Corixa Corporation AND Alexander Gaiger GAIGER, Andrew; CHEEVER, Martin DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, Victoria, 3000, Australia "Compositions and methods for WT1 specific immunotherapy" Details of Associated Application, No: 64078/99 The following statement is a full description of this invention, including the best method of performing it known to us: ':\OI'IER\HPM\CORIXA CORPORATION\12362190\DIV APP FILED 23/10/03 COMPOSITIONS AND METHODS FOR WTI SPECIFIC IMMUNOTHERAPY TECHNICAL FIELD The present invention relates generally to the immunotherapy of malignant diseases such as leukemia and cancers. The invention is more specifically related to compositions for generating or enhancing an immune response to WTI, and to the use of such compositions for preventing and/or treating malignant diseases.

BACKGROUND OF THE INVENTION Cancer and leukemia are significant health problems in the United States and throughout the world.- Although advances have been made in detection and treatment of such diseases, no vaccine or other universally successful method for prevention or treatment of cancer and leukemia is currently available. Management of the diseases currently relies on a combination of early diagnosis and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. The course of treatment for a particular cancer is often selected based on a variety of prognostic parameters, including an analysis of specific tumor markers. However, the use of established markers often leads to a result that is difficult to interpret, and the high mortality continues to be observed in many cancer patients.

Immunotherapies have the potential to substantially improve cancer and leukemia treatment and survival. Recent data demonstrate that leukemia can be cured by immunotherapy in the context of bone marrow transplantation donor lymphocyte infusions). Such therapies may involve the generation or enhancement of an immune response to a tumor-associated antigen (TAA). However, to date, relatively few TAAs are known and the generation of an immune response against such antigens has, with rare exceptions, not been shown to be therapeutically beneficial.

Accordingly, there is a need in the art for improved methods for leukemia and cancer prevention and therapy. The present invention fulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION Briefly stated, this invention provides compositions and methods for the diagnosis and therapy of diseases such as leukemia and cancer. In one aspect, the present invention provides polypeptides comprising an immunogenic portion of a native WT1, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished. Within certain embodiments, the polypeptide comprises no more than 16 consecutive amino acid residues of a native WTI polypeptide. Within other.embodiments, the polypeptide comprises an immunogenic portion of amino acid residues 1 174 of a native WTI polypeptide or a variant thereof, wherein the polypeptide comprises no more than 16 consecutive amino acid residues present within amino acids 175 to 449 of the native WTI polypeptide. The immunogenic portion preferably binds to an MHC class I and/or class II molecule. Within certain embodiments, the polypeptide comprises a sequence selected from the group consisting of sequences recited in any one or more of Tables II XLVI, variants of the foregoing sequences that differ in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished and mimetics of the polypeptides recited above, such that the ability of the mimetic to react with antigen-specific antisera and/or T cell lines or clones is not substantially diminished.

Within other embodiments, the polypeptide comprises a sequence selected from the group consisting of ALLPAVPSL (SEQ ID NO:34), GATLKGVAA (SEQ ID NO:88), CMTWNQMNL (SEQ ID NOs: 49 and 258), SCLESQPTI (SEQ ID NOs: 199 and 296), SCLESQPAI (SEQ ID NO:198), NLYQMTSQL (SEQ ID NOs: 147 and 284), ALLPAVSSL (SEQ ID NOs: 35 and 255), RMFPNAPYL (SEQ ID NOs: 185 and 293), variants of the foregoing sequences that differ in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished and mimetics of the polypeptides recited above, such that the ability of the mimetic to react with antigen-specific antisera and/or T cell lines or clones is not substantially diminished. Mimetics may comprises amino acids in combination with one or more amino acid mimetics or may be entirely nonpeptide mimetics.

Within further aspects, the present invention provides polypeptides comprising a variant of an immunogenic portion of a WTI protein, wherein the variant differs from the immunogenic portion due to substitutions at between I and 3 amino acid positions within the immunogenic portion such that the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is enhanced relative to a native WTI protein, The present invention further provides WT1 polynucleotides that encode a WTI polypeptide as described above.

Within other aspects, the present invention provides pharmaceutical compositions and vaccines. Pharmaceutical compositions may comprise a polypeptide or mimetic as described above and/or one or more of a WT1 polynucleotide; (ii) an antibody or antigen-binding fragment thereof that specifically binds to a WTI polypeptide; (iii) a T cell that specifically reacts with a WT1 polypeptide or (iv) an antigen-presenting cell that expresses a WT1 polypeptide, in combination with a pharmaceutically acceptable carrier or excipient. Vaccines comprise a polypeptide as described above and/or one or more of a WTI polynucleotide, (ii) an antigenpresenting cell that expresses a WT1 polypeptide or (iii) an anti-idiotypic antibody, and a non-specific immune response enhancer. Within certain embodiments, less than 23 consecutive amino acid residues, preferably less than 17 amino acid residues, of a native WTI polypeptide are present within a WTI polypeptide employed within such pharmaceutical compositions and vaccines. The immune response enhancer may be an adjuvant. Preferably, an immune response enhancer enhances a T cell response.

The present invention further provides methods for enhancing or inducing an immune response in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as described above. In certain embodiments, the patient is a human.

The present invention further provides methods for inhibiting the development of a malignant disease in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as described above. Malignant diseases include, but are not limited to leukemias acute myeloid, acute lymphocytic and chronic myeloid) and cancers breast, lung, thyroid or gastrointestinal cancer or a melanoma). The patient may, but need not, be afflicted with the malignant disease, and the administration of the pharmaceutical composition or vaccine may inhibit the onset of such a disease, or may inhibit progression and/or metastasis of an existing disease.

The present invention, further provides, within other aspects, methods for removing cells expressing WT1 from bone marrow and/or peripheral blood or fractions thereof, comprising contacting bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood with T cells that specifically react with a WTI polypeptide, wherein the step of contacting is performed under conditions and fora time sufficient to permit the removal of WTI positive cells to less than 10%, preferably less than 5% and more preferably less than of the number of myeloid or lymphatic cells in the bone marrow, peripheral blood'or fraction Bone marrow, peripheral blood and fractions may be obtained from a patient afflicted with a disease associated with WTI expression, or may be obtained from a human or non-human mammal not afflicted with such a disease.

Within related aspects, the present invention provides methods for inhibiting the development of a malignant disease in a patient, comprising administering to a patient bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood prepared as described above. Such bone marrow, peripheral blood or fractions may be autologous, or may be derived from a related or unrelated human or non-human animal syngeneic or allogeneic).

In other aspects, the present invention provides methods for stimulating (or priming) and/or expanding T cells, comprising contacting T cells with a WTI polypeptide under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells. Such T cells may be autologous, allogeneic, syngeneic or unrelated WTI-specific T cells, and may be stimulated in vitro or in vivo. Expanded T cells may, within certain embodiments, be present within bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood, and may (but need not) be clonal. Within certain embodiments, T cells may be present in a mammal during stimulation and/or expansion. WTl-specific T cells may be used, for example, within donor lymphocyte infusions.

Within related aspects, methods are provided for inhibiting the development of a malignant disease in a patient, comprising administering to a patient T cells.prepared as described above. Such T cells may; within certain embodiments, be autologous, syngeneic or allogeneic.

The present invention further provides, within other aspects, methods for monitoring the effectiveness of an immunization or therapy for a malignant disease associated with WT1 expressior in a patient. Such methods are based on monitoring antibody, CD4+ T cell and/or CD8+ T cell responses in the patient. Within certain such aspects, a method may comprise the steps of: incubating a first biological sample with one or more of: a WTI polypeptide; (ii) a polynucleotide encoding a WTI polypeptide; or (iii) an antigen presenting cell that expresses a WTI polypeptide, wherein the first biological sample is obtained from a patient prior to a.therapy or immunization, and wherein the incubation is performed under conditions and for a time sufficient to allow immunocomplexes to form; detecting immunocomplexes formed between the WTI polypeptide and antibodies in the biological sample that specifically bind to the WTI polypeptide; repeating steps and using a second biological sample obtained from the same patient following therapy or immunization; and (d) comparing the number of immunocomplexes detected in the first and second biological samples, and therefrom monitoring the effectiveness of the therapy or immunization in the patient.

Within certain embodiments of the above methods, the step of detecting comprises incubating the immunocomplexes with a detection reagent that is capable of binding to the immunocomplexes, wherein the detection reagent comprises a reporter group, removing unbound detection reagent, and detecting the presence or absence of the reporter group. The detection reagent may comprise, for example, a second antibody, or antigen-binding fragment thereof, capable of binding to the antibodies that specifically bind to the WTI polypeptide or a molecule such as Protein A. Within other embodiments, a reporter group is bound to the WT1 polypeptide, and the step of detecting comprises removing unbound WT1 polypeptide and subsequently detecting the presence or absence of the reporter group.

Within further aspects, methods for monitoring the effectiveness of an immunization or therapy for a malignant disease associated with WTI expression in a patient may comprise the steps of: incubating a first biological.sample with one or more of: a WTI polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide, wherein the biological sample comprises CD4+ and/or CD8+ T cells and is obtained from a patient prior to a therapy or immunization, and wherein the incubation is performed under conditions and for a time sufficient to allow specific activation, proliferation and/or lysis of T cells; (b) detecting an amount of activation, proliferation and/or lysis of the T cells; repeating steps and using a second biological sample comprising CD4+ and/or CD8+ T cells, wherein the second biological sample is obtained from the same patient following therapy or immunization; and comparing the amount of activation, proliferation and/or lysis of T cells in the first and second biological samples, and therefrom monitoring the effectiveness of the therapy or immunization in the patient.

The present invention further provides methods for inhibiting the development of a malignant disease associated with WT1 expression in a patient, comprising the steps of: incubating CD4 and/or CD8+ T cells isolated from a patient with one or more of: a WT1 polypeptide; (ii) a polynucleotide encoding a WTI polypeptide; or (iii) an antigen presenting cell that expresses a WTI polypeptide, such that the T cells proliferate; and administering to the patient an effective amount of the proliferated T cells, and therefrom inhibiting the development of a malignant disease in the patient. Within certain embodiments, the step of incubating the T cells may be repeated one or more times.

Within other aspects, the present invention provides methods for inhibiting the development of a malignant disease associated with WTI expression in a patient, comprising the steps of: incubating CD4 and/or CD8+ T cells isolated from a patient with one or more of: a WTI polypeptide; (ii) a polynucleotide encoding a WTI polypeptide; or (iii) an antigen presenting cell that expresses a WTI polypeptide, such that the T cells proliferate; cloning one or more cells that proliferated; and administering to the patient an effective amount of the cloned T cells.

Within other aspects, methods are provided for determining the presence or absence of a malignant, disease associated with WTI expression in a patient, comprising the steps of: incubating CD4 and/or CD8+ T cells isolated from a patient with one or more of: a WTI polypeptide; (ii) a polynucleotide encoding a WTI polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide; and detecting the presence or absence of specific activation of the T cells, therefrom determining the presence or absence of a malignant disease associated with WTI expression. Within certain embodiments, the step of detecting comprises detecting the presence or absence of proliferation of the T cells.

Within further aspects, the present invention provides methods for determining the presence or absence of a malignant disease associated with WTI expression in a patient, comprising the steps of: incubating a biological sample obtained from a patient with one or more of: a WTI polypeptide; (ii) a polynucleotide encoding a WTI polypeptide; or (iii) an antigen presenting cell that expresses a WTI polypeptide, wherein the incubation is performed under conditions and for a time sufficient to allow immunocomplexes to form; and detecting immunocomplexes formed between the WTI polypeptide and antibodies in the biological sample that specifically bind to the WT1 polypeptide; and therefrom determining the presence or absence of a malignant disease associated with WTI expression.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts a comparison of the mouse (MO) and human (HU) WTI protein sequences (SEQ ID NOS: 320 and 319 respectively).

Figure 2 is a Western blot illustrating the detection of WTI specific antibodies in patients with hematological malignancy (AML). Lane 1 shows molecular weight markers;, lane 2 shows.a positive control (WTI positive human leukemia cell line immunoprecipitated with a WT1 specific antibody); lane 3 shows a negative control (WT1 positive cell line immunoprecipitated with mouse sera); and lane 4 shows a WTI positive cell line immunoprecipitated with sera of a patient with AML. For lanes 2-4, the immunoprecipitate .was. separated by gel electrophoresis and probed with a WTI specific antibody.

Figure 3 is a Western blot illustrating the detection of a WTI specific antibody response.in. B6 mice immunized with TRAMP-C, a WTI positive tumor cell line. Lanes 1, 3 and 5 show molecular weight markers, and lanes 2, 4 and 6 show a WT1 specific positive control (N180, Santa Cruz Biotechnology, polypeptide spanning 180 amino acids of the N-terminal region of the WTI protein, migrating on the Western blot at 52 kD). The primary antibody used was WT180 in lane 2, sera of nonimmunized B6 mice in lane 4 and sera of the immunized B6 mice in lane 6.

Figure 4 is a Western blot illustrating the detection of WTI specific .antibodies in mice immunized with representative WT1 peptides. Lanes 1, 3 and show molecular weight markers and lanes 2, 4 and 6 show a WTI specific positive control (N180, Santa Cruz Biotechnology, polypeptide spanning 180 amino acids of the N-terminal region of the WT1 protein, migrating on the Western blot at 52 kD). The primary antibody used was WT180 in lane 2, sera of non-immunized B6 mice in lane 4 and sera of the immunized B6 mice in lane 6.

Figures 5A to 5C are graphs illustrating the stimulation of proliferative T cell responses in mice immunized with representative WTI peptides. Thymidine incorporation assays were performed using one T cell line and two different clones, as indicated, and results were expressed as cpm. Controls indicated on the x axis were no antigen (No Ag) and B6/media; antigens used were p6-22 human p 17-139 (p2) or p244-262 human (p3).

Figure 6A and 6B are histograms illustrating the stimulation of proliferative T cell responses in mice immunized with representative WTI peptides.

Three weeks after the third immunization, spleen cells of mice that had been inoculated with Vaccine A or Vaccine B were cultured with medium alone (medium) or spleen cells and medium (B6/no antigen), B6 spleen cells pulsed with the peptides p6-22 (p6), pl 17-139 (p 1 17), p244-262 (p244) (Vaccine A; Figure 6A) or p287-301 (p287), p299- 313 (p299), p421-435 (p421) (Vaccine B; Figure 6B) and spleen cells pulsed with an irrelevant control peptide (irrelevant peptide) at 25ug/ml and were assayed after 96hr for proliferation by H) thymidine incorporation. Bars represent the stimulation index which is calculated as the mean of the experimental wells divided by the mean of the control (B6 spleen cells with no antigen).

Figures 7A-7D are histograms illustrating the generation of proliferative T-cell lines and clones specific for p 17-139 and p6-22. Following in vivo immunization, the initial three in vitro stimulations (IVS) were carried out using all three peptides of Vaccine A or B, respectively. Subsequent IVS were carried out as single peptide stimulations using only the two relevant peptides pl 17-139 and p6-22.

Clones were derived from both the p6-22 and p117-139 specific T cell lines, as indicated. T cells were cultured with medium alone (medium) or spleen cells and medium (B6/no antigen), B6 spleen cells pulsed with the peptides p6-22 p 17-139 (pl 17) or an irrelevant control peptide (irrelevant peptide) at 25ug/ml and were assayed after 96hr for proliferation by thymidine incorporation. Bars represent the stimulation index which is calculated as the mean of the experimental wells divided by the mean of the control (B6 spleen cells with no antigen).

Figures 8A and 8B present the results of TSITES Analysis of human WTI (SEQ ID NO:319) for peptides that have the potential to elicit Th responses.

Regions indicated by are AMPHI midpoints of blocks, indicates residues matching the Rothbard/Taylor motif, indicates residues matching the IAd motif, and indicates residues matching the IEd motif.

Figures 9A and 9B are graphs illustrating the elicitation of WT1 peptidespecific CTL in mice immunized with WTI peptides. Figure 9A illustrates the lysis of target cells by allogeneic cell lines and Figure 9B shows the lysis of peptide coated cell lines. In each case, the lysis (as determined by standard chromium release assays) is shown at three indicated effector:target ratios. Results are provided for lymphoma cells (LSTRA and E10), as well as E10 p235-243 (E10+P235). E10 cells are also referred to herein as EL-4 cells.

Figures 10A-10D are graphs illustrating the elicitation of WTI specific CTL, which kill WTI positive tumor cell lines but do not kill WT1 negative cell lines, following vaccination of B6 mice with WTI peptide P 17. Figure 10A illustrates that T-cells of non-immunized B6 mice do not kill WTI positive tumor cell lines. Figure illustrates the lysis of the target cells by allogeneic cell lines. Figures 10C and demonstrate the lysis of WTI positive tumor cell lines, as compared to WT1 negative cell lines in two different experiments. In addition, Figures 10C and 10D show the lysis of peptide-coated cell lines (WT1 negative cell line E10 coated with the relevant WTI peptide P117) In each case, the lysis (as determined by standard chromium release assays) is shown at three indicated effector:target ratios. Results are provided for lymphoma cells (E10), prostate cancer cells (TRAMP-C), a transformed fibroblast cell line (BLK-SV40), as well as E10+p117.

Figures 11A and 11B are histograms illustrating the ability of representative peptide P117-139 specific CTL to lyse WT1 positive tumor cells. Three weeks after the third immunization, spleen cells of mice that had been inoculated with the peptides p235-243 or p117-139 were stimulated in vitro with the relevant peptide and tested for ability to lyse targets incubated with WTI peptides as well as WT1 positive and negative tumor cells. The bars represent the mean specific lysis in chromium release assays performed in triplicate with an E:T ratio of 25:1. Figure I 1A shows the cytotoxic activity of the p235-243 specific T cell line against the WTI negative cell line EL-4 (EL-4, WTI negative); EL-4 pulsed with the relevant (used for immunization as well as for restimulation) peptide p235-243 (EL-4+p235); EL-4 pulsed with the irrelevant peptides p 17-139 (EL-4+pll7), p 12 6 13 4 (EL-4+p126) or p130- 138 (EL-4+p130) and the WT1 positive tumor cells BLK-SV40 (BLK-SV40, WTI positive) and TRAMP-C (TRAMP-C, WTI positive), as indicated. Figure 11B shows cytotoxic activity of the p 17-139 specific T cell line against EL-4; EL-4 pulsed with the relevant peptide P117-139 (EL-4+pll7) and EL-4 pulsed with the irrelevant peptides p123-131 (EL-4+p123), or p128-136 (EL-4+p128); BLK-SV40 and TRAMP- C, as indicated.

Figures 12A and 12B are histograms illustrating the specificity of lysis of WT1 positive tumor cells, as demonstrated by cold target inhibition. The bars represent the mean specific lysis in chromium release assays performed in triplicate with an E:T ratio of 25:1. Figure 12A shows the cytotoxic activity of the p117-139 specific T cell line against the WTI negative cell line EL-4 (EL-4, WT1 negative); the WTI positive tumor cell line TRAMP-C (TRAMP-C, WTI positive); TRAMP-C cells incubated with a ten-fold excess (compared to the hot target) of EL-4 cells pulsed with the relevant peptide p 1 17-139 (TRAMP-C pl 17 cold target) without "Cr labeling and TRAMP-C cells incubated with EL-4 pulsed with an irrelevant peptide without "Cr labeling (TRAMP-C irrelevant cold target), as indicated. Figure 12B shows the cytotoxic activity of the pi 17-139 specific T cell line against the WT1 negative cell line EL-4 (EL-4, WTI negative); the WTI positive tumor cell line BLK-SV40 WTI positive); BLK-SV40 cells incubated with the relevant cold target (BLK-SV40 p117 cold target) and BLK-SV40 cells incubated with the irrelevant cold target (BLKirrelevant cold target), as indicated.

Figures 13A-13C are histograms depicting an evaluation of the 9mer CTL epitope within p117-139. The p 1 1 7-139 tumor specific CTL line was tested against peptides within aal 17-139 containing or lacking an appropriate H-2b class I binding motif and following restimulation with p126-134 or p130-138. The bars represent the mean specific lysis in chromium release assays performed in triplicate with an E:T ratio of 25:1. Figure 13A shows the cytotoxic activity of the p 17-139 specific T cell line against the WTI negative cell line EL-4 (EL-4, WTI negative) and EL-4 cells pulsed with the peptides p117-139 (EL-4 p117), p119-127 (EL-4 p119), p120-128 (EL-4 p120), p123-131 (EL-4 p123), p126-134 (EL-4 pl26), pl28- 13 6 (EL-4 p128), and p130-138 (EL-4 p130). Figure 13B shows the cytotoxic activity of the CTL line after restimulation with p126-134 against the WTI negative cell line EL-4, EL-4 cells pulsed with p 1 17-139 (EL-4 pl 17), p126-134 (EL-4 p126) and the WTI positive tumor cell line TRAMP-C. Figure 13C shows the cytotoxic activity of the CTL line after restimulation with p130-138 against EL-4, EL-4 cells pulsed with p 17-139 (EL-4 p117), p130-138 (EL-4 pl30) and the WTI positive tumor cell line

TRAMP-C.

DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention is generally directed to compositions and methods for the immunotherapy and diagnosis of malignant diseases.

The compositions described herein may include WTI polypeptides, WT1 polynucleotides, antigen-presenting cells (APC, dendritic cells) that express a WT1 polypeptide, agents such as antibodies that bind to a WTI polypeptide and/or immune system cells T cells) specific for WT1. WT1 Polypeptides of. the present invention generally comprise at least a portion of a Wilms Tumor gene product (WTI) or a variant thereof. Nucleic acid sequences of the subject invention generally comprise a DNA or RNA sequence that encodes all or a portion of such a polypeptide, or that is complementary to such a sequence. Antibodies are generally immune system proteins, or antigen-binding: fragments thereof, that are capable of binding to a portion of a WT1 polypeptide. T cells that may be employed within such compositions are generally T cells CD4- and/or CD8') that are specific for a WT1 polypeptide. Certain methods described herein further employ antigen-presenting cells that express a WT1 polypeptide as provided herein.

The present invention is based on the discovery that an immune response raised against a Wilms Tumor (WT) gene product WTI) can provide prophylactic and/or therapeutic benefit for patients afflicted with malignant diseases characterized by increased WTI gene expression. Such diseases include, but are not limited to, leukemias acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) and childhood ALL), as well as many cancers such as lung, breast, thyroid and gastrointestinal cancers and melanomas. The WTI gene was originally identified and isolated on the basis of a cytogenetic deletion at chromosome llpl3 in patients with Wilms' tumor (see Call et al., U.S. Patent No.

5,350,840). The gene consists of 10 exons and encodes a zinc finger transcription factor, and sequences of mouse and human WT1 proteins are provided in Figure 1 and SEQ ID NOs: 319 and 320.

WT1 POLYPEPTIDES Within the context of the present invention, a WTI polypeptide is a polypeptide that comprises at least an immunogenic portion of a native WTI a WTI protein expressed by an organism that is not genetically modified), or a variant thereof, as described herein. A WTI polypeptide may be of any length, provided that it comprises at least an immunogenic portion of a native protein or a variant thereof. In other words, a WTI polypeptide may be an oligopeptide consisting of a relatively small number of amino acid residues, such as 8-10 residues, joined by peptide bonds), a full length WTI protein present within a human or non-human animal, such as a mouse) or a polypeptide of intermediate size. Within certain embodiments, the use of WTI polypeptides that contain a small number of consecutive amino acid residues of a native WT1 polypeptide is preferred. Such polypeptides are preferred for certain uses in which the generation of a T cell response is desired. For example, such a WT1 polypeptide may contain less than 23, preferably no more than 18, and more preferably no more than 15 consecutive amino acid residues, of a native WT1 polypeptide.

Polypeptides comprising nine consecutive amino acid residues of a native WT1 polypeptide are generally suitable for such purposes. Additional sequences derived from the native protein and/or heterologous sequences may be present within any WTI polypeptide, and such sequences may (but need not) possess further immunogenic or antigenic properties. Polypeptides as provided herein may further be associated (covalently or noncovalently) with other polypeptide or non-polypeptide compounds.

An "immunogenic portion," as used herein is a portion of a polypeptide that is recognized specifically bound) by a B-cell and/or T-cell surface antigen receptor. Certain preferred immunogenic portions bind to an MHC class I or class II molecule. As used herein, an immunogenic portion is said to "bind to" an MHC class I or class II molecule if such binding is detectable using any assay known in the art. For example, the ability of a polypeptide to bind to MHC class I may be evaluated indirectly by monitoring the ability to promote incorporation of '5I labeled p2microglobulin (p2m) into MHC class I/P2m/peptide heterotrimeric complexes (see Parker et al., J. Immunol. 152:163, 1994). Alteratively; functional peptide competition assays that are known in the art may be employed. Certain immunogenic portions have one or more of the sequences recited within one or more of Tables II XIV.

Representative immunogenic portions include, but are not limited to, RDLNALLPAVPSLGGGG (human WT1 residues 6-22; SEQ ID NO:1), PSQASSGQARMFPNAPYLPSCLE (human and mouse WTI residues 117-139; SEQ ID NOs: 2 and 3 respectively), GATLKGVAAGSSSSVKWTE (human WTI residues 244-262; SEQ ID NO:4), GATLKGVAA (human WTI residues 244-252; SEQ ID NO:88), CMTWNQMNL (human and mouse WTI residues 235-243; SEQ ID NOs: 49 and 258 respectively), SCLESQPTI (mouse WTI residues 136-144; SEQ ID NO:296), SCLESQPAI (human WT1 residues 136-144; SEQ ID NO:198), NLYQMTSQL (human and mouse WT1 residues 225-233; SEQ ID NOs: 147 and 284 respectively); ALLPAVSSL (mouse WT1 residues 10-18; SEQ ID NO:255); or RMFPNAPYL (human and mouse WTI residues 126-134; SEQ ID NOs: 185 and 293 respectively).

Further immunogenic portions are provided herein, and others may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Representative techniques for identifying immunogenic portions include screening polypeptides for the ability to react with antigen-specific antisera and/or Tcell lines or clones. An immunogenic portion of a native WT1 polypeptide is a portion that reacts with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length WTI in an ELISA and/or T-cell reactivity assay).

In other words, an immunogenic portion may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane; Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

Alternatively, immunogenic portions may be identified using computer analysis, such as the Tsites program (see Rothbard and Taylor, EMBO J 7:93-100, 1988; Deavin et al., Mol. Immunol. 33:145-155, 1996), which searches for peptide motifs that have the potential to elicit Th responses. CTL peptides with motifs appropriate for binding to murine and human class I or class II MHC may be identified according to BIMAS (Parker et al., J. Immunol. 152:163, 1994) and other HLA peptide binding prediction analyses. To confirm immunogenicity, a peptide may be tested using an HLA A2 transgenic mouse model and/or an in vitro stimulation assay using dendritic cells, fibroblasts or peripheral blood cells.

As noted above, a composition may comprise a variant of a native WTI protein. A polypeptide "variant," as used herein, is a polypeptide that differs from a native polypeptide in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the polypeptide is retained the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished relative to the native polypeptide). In other words, the ability of a variant to react with antigen-specific antisera and/or T-cell lines or clones may be enhanced or unchanged, relative to the native polypeptide, or may be diminished by less than 50%, and preferably less than 20%, relative to the native polypeptide. Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antisera and/or T-cells as described herein. It has been found, within the context of the present invention, that a relatively small number of substitutions 1 to 3) within an immunogenic portion of a WTI polypeptide may serve to enhance the ability of the polypeptide to elicit an immune response. Suitable substitutions may generally be identified by using computer programs, as described above, and the effect confirmed based on the reactivity of the modified polypeptide with antisera and/or T-cells as described herein. Accordingly, within certain preferred embodiments, a WTI polypeptide comprises a variant in which 1 to 3 amino acid resides within an immunogenic portion are substituted such that the ability to react with antigen-specific antisera and/or T-cell lines or clones is statistically greater than that for the unmodified polypeptide. Such substitutions are preferably located within an MHC binding site of the polypeptide, which may be identified as described above. Preferred substitutions allow increased binding to MHC class I or class II molecules.

Certain variants contain conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged: Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: ala, pro, gly, glu, asp, gin, asn, ser, thr; cys, ser, tyr, thr; val, ile, leu, met, ala, phe; lys, arg, his; and phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

As noted above, WT1 polypeptides may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or posttranslationally directs transfer of the protein. A polypeptide may also, or alternatively, be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

WTI polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by a WTI polynucleotide as described herein may be readily prepared from the polynucleotide. In general, any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant WT1 polypeptides. Expression may be achieved in any appropriate'host'cell that has'beeh transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.

Supematants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. The concentrate may then be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide. Such techniques may be used to prepare native polypeptides or variants thereof. For example, polynucleotides that encode a variant of a native polypeptide may generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides.

Certain portions and other variants may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, polypeptides having fewer than about 500 amino acids, preferably fewer than about 100 amino acids, and more preferably fewer than about 50 amino acids, may be synthesized.

Polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc.

85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions.

In general, polypeptides and polynucleotides as described herein are isolated. An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated -if, for example, it is cloned into a vector that is not a part of the natural environment.

Within further aspects, the present invention provides mimetics of WTI polypeptides. Such mimetics may comprise amino acids linked to one or more amino acid mimetics one or more amino acids within the WTI protein may be replaced by an amino acid mimetic) or may be entirely nonpeptide mimetics. An amino acid mimetic is a compound that is conformationally similar to an amino acid such that it can be substituted for an amino acid within a WTI polypeptide without substantially diminishing the ability to react with antigen-specific antisera and/or T cell lines or clones. A nonpeptide mimetic is a compound that does not contain amino acids, and that has an overall conformation that is similar to a WT1 polypeptide such that the ability of the mimetic to react with WTI-specific antisera and/or T cell lines or clones is not substantially diminished relative to the ability of a WTI polypeptide. Such mimetics may be designed based on standard techniques nuclear magnetic resonance and computational techniques) that evaluate the three dimensional structure of a peptide sequence. Mimetics may be designed where one or more of the side chain functionalities of the WT1 polypeptide are replaced by groups that do not necessarily have the same size or volume, but have similar chemical and/or physical properties which produce similar biological responses. It should be understood that, within embodiments described herein, a mimetic may be substituted for a WTI polypeptide.

WTI POLYNUCLEOTIDES Any polynucleotide that encodes a WT1 polypeptide as described herein is a WTI polynucleotide encompassed by the present invention. Such polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

WTI polynucleotides may encode a native WTI protein, or may encode a variant of WT1 as described herein. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the immunogenicity of the encoded polypeptide is not diminished, relative to a native WTI protein. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein. Preferred variants contain nucleotide substitutions, deletions, insertions and/or additions at no more than 20%, preferably at no more than 10%, of the nucleotide positions that encode an immunogenic portion of a native WTI sequence. Certain variants- are substantially homologous to a native gene, or a portion thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a WTI polypeptide (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH hybridizing at 50 0 C-65 0 C, 5 X SSC, overnight; followed by washing twice at 65*C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS). Such hybridizing DNA sequences are also within the scope of this invention.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a WTI polypeptide. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.

Once an immunogenic portion of WTI is identified, as described above, a WTI polynucleotide may be prepared using any of a variety of techniques. For example, a WTI polynucleotide may be amplified from cDNA prepared from cells that express WT1. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequence of the immunogenic portion and may be purchased or synthesized. For example, suitable primers for PCR amplification of a human WT1 gene include: first step P18: 1434-1414: 5' GAG AGT CAG ACT TGA AAG CAGT 3' (SEQ ID and P135: 5' CTG AGC CTC AGC AAA TGG GC 3' (SEQ ID NO:6); second step P136: 5' GAG CAT GCA TGG GCT CCG ACG TGC GGG 3' (SEQ ID NO:7) and P137: 5' GGG GTA CCC ACT GAA CGG TCC CCG A 3' (SEQ ID NO:8).

Primers for PCR amplification of a mouse WT1 gene include: first step P138: 5' TCC GAG CCG CAC CTC ATG 3' (SEQ ID NO:9) and P139: 5' GCC TGG GAT GCT GGA CTG 3' (SEQ ID NO:10), second step P140: 5' GAG CAT GCG ATG GGT TCC GAC GTG CGG 3' (SEQ ID NO:11) and P141: 5' GGG GTA CCT CAA AGC GCC ACG TGG AGT TT 3' (SEQ ID NO:12).

An amplified portion may then be used to isolate a full length gene from a human genomic DNA library or from a suitable cDNA library, using well known techniques. Alternatively, a full length gene can be constructed from multiple PCR fragments. WTI polynucleotides may also be prepared by synthesizing oligonucleotide components, and ligating components together to generate the complete polynucleotide.

WTI polynucleotides may also be synthesized by any method known in the art, including chemical synthesis solid phase phosphoramidite chemical synthesis). Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a WTI polypeptide, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may be administered to a patient such that the encoded polypeptide is generated in vivo by transfecting antigen-presenting cells such as dendritic cells with a cDNA construct encoding a WTI polypeptide, and administering the transfected cells to the patient).

Polynucleotides that encode a WTI polypeptide may generally be used for production of the polypeptide, in vitro or in vivo. WTI polynucleotides that are complementary to a coding sequence antisense polynucleotides) may also be used as a probe or to inhibit WTI expression. cDNA constructs that can be transcribed into antisense RNA may also be introduced into cells of tissues to facilitate the production of antisense RNA.

Any polynucleotide may be further modified to increase stability in vivo.

Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art. cDNA constructs within such a vector may be used, for example, to transfect human or animal cell lines for use in establishing WTI positive tumor models which may be used to perform tumor protection and adoptive immunotherapy experiments to demonstrate tumor or leukemia-growth inhibition or lysis of such cells.

Other therapeutic formulations for polynucleotides include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

ANTIBODIES AND FRAGMENTS THEREOF The present invention further provides binding agents, such as antibodies and antigen-binding fragments thereof, that specifically bind to a WTI polypeptide. As used herein, an agent is said to "specifically bind" to a WTI polypeptide if it reacts at a detectable level (within, for example, an ELISA) with a WT1 polypeptide, and does not react detectably with unrelated proteins under similar conditions. As used herein, "binding" refers to a noncovalent association between two separate molecules such that a "complex" is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations. In general, two compounds are said to "bind," in the context of the present invention, when the binding constant for complex formation exceeds about 10' L/mol. The binding constant maybe determined using methods well known in the art.

Any agent that satisfies the above requirements may be a binding agent.

In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Certain antibodies are commercially available from, for example, Santa Cruz Biotechnology (Santa Cruz, CA). Alternatively, antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described-herein; or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals mice, rats, rabbits, sheep or goats): In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J.

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

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

Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.

Monoclonal antibodies and fragments thereof may be coupled to one or more therapeutic agents. Suitable agents in this regard include radioactive tracers and chemotherapeutic agents, which may be used, for example, to purge autologous bone marrow in vitro). Representative therapeutic agents include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include 0Y, 1 25 I, 13', 1 8 6 Re, 8 Re, 21 'At, and 1 2 Bi. Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein. For diagnostic purposes, coupling of radioactive agents may be used to facilitate tracing of metastases or to determine the location of WTI-positive tumors.

A therapeutic agent may be coupled covalently bonded) to a suitable monoclonal antibody either directly or indirectly via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonylcontaining group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group a halide) on the other.

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

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

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

It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody.

Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used. A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins U.S. Patent No.

4,507,234, to Kato et peptides and polysaccharides such as aminodextran

U.S.

Patent No. 4,699,784, to Shih et A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle U.S. Patent Nos.

4,429,008 and 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Patent No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. A radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide. For example, U.S. Patent No. 4,673,562, to Davison et al. discloses representative chelating compounds and their synthesis.

A variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody.

Also provided herein are anti-idiotypic antibodies that mimic an immunogenic portion of WTI. Such antibodies may be raised against an antibody, or antigen-binding fragment thereof, that specifically binds to an immunogenic portion of WT1, using well known techniques. Anti-idiotypic antibodies that mimic an immunogenic portion of WTI are those antibodies that bind to an antibody, or antigenbinding fragment thereof, that specifically binds to an immunogenic portion of WTI, as described herein.

T CELLS Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for.WTl-- Such:cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be present within (or isolated from) bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood of a mammal, such as a patient, using a commercially available cell separation system, such as the CEPRATE T M system, available from CellPro Inc., Bothell WA (see also U.S.

Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human animals, cell lines or cultures.

T cells may be stimulated with WTI polypeptide, polynucleotide encoding a WTI polypeptide and/or an antigen presenting cell (APC) that expresses a WTI polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the WTI polypeptide.

Preferably, a WTI polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of antigen-specific T cells. Briefly, T cells, which may be isolated from a patient or a related or unrelated donor by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes), are incubated with WTI polypeptide. For example, T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37 0 C with WTI polypeptide to 25 utg/ml) or cells synthesizing a comparable amount of WTI polypeptide. It may be desirable to incubate a separate aliquot of a T cell sample in the absence of WTI polypeptide to serve as a control.

T cells are considered to be specific for a WTI polypeptide if the T cells kill target cells coated with a WTI polypeptide or expressing a gene encoding such a polypeptide. T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA.. synthesis by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA).

Other ways to detect T cell proliferation include measuring increases in interleukin-2 (IL-2) production, Ca 2 flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium. Alternatively, synthesis of lymphokines (such as interferongamma) can be measured or the relative number of T cells that can respond to a WTI polypeptide may be quantified. Contact with a WT1 polypeptide (200 ng/ml 100 pg/ml, preferably' 100 ng/ml 25 tg/ml) for 3 7 days should result in at least a two fold increase in proliferation of the T cells and/or contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release TNF or IFN-y) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998). WT1 specific T cells may be expanded using standard techniques. Within preferred embodiments, the T cells are derived from a patient or a related or unrelated donor and are administered to the patient following stimulation and expansion.

T cells that have been activated in response to a WTI polypeptide, polynucleotide or WTI-expressing APC may be CD4' and/or CD8'. Specific activation of CD4+ or CD8 T cells may be detected in a variety of ways. Methods for detecting specific T cell activation include detecting the proliferation of T cells, the production of cytokines lymphokines), or the generation of cytolytic activity generation of cytotoxic T cells specific for WTI). For CD4 T cells, a preferred method for detecting specific T cell activation is the detection of the proliferation of T cells. For CD8+ T cells, a preferred method for detecting specific T cell activation is the detection of the generation of cytolytic activity.

For therapeutic purposes, CD4 or CD8+ T cells that proliferate in response to the WTI polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to WT1 polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that.synthesize a WTI polypeptide. The addition of stimulator cells is preferred where generating CD8 T cell responses. T cells can be grown to large numbers in vitro with retention of specificity in response to intermittent restimulation with WT1 polypeptide. Briefly, for the primary in vitro stimulation (IVS), large numbers of lymphocytes greater than 4 x 107) may be placed in flasks with media containing human serum. WTI polypeptide peptide at 10 fg/ml) may be added directly, along with tetanus toxoid 5 ptg/ml). The flasks may then be incubated 37°C for 7 days). For a second IVS, T cells are then harvested and placed in new flasks with 2-3 x 107 irradiated peripheral blood mononuclear cells. WTI polypeptide p.g/ml) is added directly.. The flasks are incubated at 37 0 C for 7 days. On day 2 and day 4 after the second IVS, 2-5 units of interleukin-2 (IL-2) may be added. For a third IVS, the T cells may be placed in wells and stimulated with the individual's own EBV transformed B cells coated with the peptide. IL-2 may be added on days 2 and 4 of each cycle. As soon as the cells are shown to be specific cytotoxic T cells, they may be expanded using a 10 day stimulation cycle with higher IL-2 (20 units) on days 2, 4 and 6.

Alternatively, one or more T cells that proliferate in the presence of WT1 polypeptide can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution. Responder T cells may be purified from the peripheral blood of sensitized patients by density gradient centrifugation and sheep red cell rosetting and established in culture by stimulating with the nominal antigen in the presence of irradiated autologous filler cells. In order to generate CD4+ T cell lines, WTI polypeptide is used as the antigenic stimulus and autologous peripheral blood lymphocytes (PBL) or lymphoblastoid cell lines (LCL) immortalized by infection with Epstein Barr virus are used as antigen presenting cells. In order to generate CD8+ T cell lines, autologous antigen-presenting cells transfected with an expression vector which produces WTI polypeptide may be used as stimulator cells. Established T cell lines may be cloned 2-4 days following antigen stimulation by plating stimulated T cells at a frequency of 0.5 cells per well in 96-well flat-bottom plates with 1 x 106 irradiated PBL or LCL cells and recombinant interleukin-2 (rIL2) (50 U/ml). Wells with established clonal growth may be identified at approximately 2-3 weeks after initial plating and restimulated with appropriate antigen in the presence of autologous antigen-presenting cells, then subsequently expanded by the addition of low doses of rIL2 (10 U/ml) 2-3 days following antigen stimulation. T cell clones may be maintained in 24-well plates by periodic restimulation with antigen and rIL2 approximately every two weeks.

Within certain embodiments, allogeneic T-cells may be primed sensitized to WTI) in vivo and/or in vitro. Such priming may be achieved by contacting T cells with a WT1 polypeptide, a polynucleotide encoding such a polypeptide or a cell producing such a polypeptide under conditions and for a time sufficient to permit the priming of T cells. In general, T cells are considered to be primed if, for example, contact with a WTI polypeptide results in proliferation and/or activation of the T cells, as measured by standard proliferation, chromium release and/or cytokine release assays as described herein. A stimulation index of more than two fold increase in proliferation or lysis, and more than three fold increase in the level of cytokine, compared to negative controls, indicates T-cell specificity. Cells primed in vitro may be employed, for example, within a bone marrow transplantation or as donor lymphocyte infusion.

PHARMACEUTICAL COMPOSITIONS AND VACCINES Within certain aspects, polypeptides, polynucleotides, antibodies and/or T cells may be incorporated into pharmaceutical compositions or vaccines.

Alternatively, a pharmaceutical composition may comprise an antigen-presenting cell a dendritic cell) transfected with a WTI polynucleotide such that the antigen presenting cell expresses a WTI polypeptide. Pharmaceutical compositions comprise one or more such compounds or cells and a physiologically acceptable carrier or excipient. Certain vaccines may comprise one or more such compounds or cells and a non-specific immune response.enhancer, such as an adjuvant or a liposome (into which the compound is incorporated). Pharmaceutical compositions and vaccines may additionally contain a delivery system, such as biodegradable microspheres which are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.

Within certain embodiments, pharmaceutical compositions and vaccines are designed to elicit T cell responses specific for a WTI polypeptide in a patient, such as a human. In general, T cell responses may be favored through the use of relatively short polypeptides comprising less than 23 consecutive amino acid residues of a native WTI polypeptide, preferably 4-16 consecutive residues, more preferably 8-16 consecutive residues and still more preferably 8-10 consecutive residues. Alternatively, or in addition, a vaccine may comprise a non-specific immune response enhancer that preferentially enhances a T cell response. In other words, the immune response enhancer may enhance the level of a T cell response to a WT1 polypeptide by an amount that is proportionally greater than the amount by which an antibody response is enhanced. For example, when compared to a standard oil based adjuvant, such as CFA, an immune response enhancer that preferentially enhances a T cell response may enhance a proliferative T cell response by at least two fold, a lytic response by at least and/or T cell activation by at least two fold compared to WTI-megative control cell lines, while not detectably enhancing an antibody response. The amount by which a T cell or antibody response to a WTI polypeptide is enhanced may generally be determined using any representative technique known in the art, such as the techniques provided herein.

A pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. As noted above, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems and mammalian expression systems. Appropriate nucleic acid expression systems contain the necessary DNA, cDNA or RNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

As noted above, a pharmaceutical composition or vaccine may comprise an antigen-presenting cell that expresses a WTI polypeptide. For therapeutic purposes, as described herein, the antigen presenting cell is preferably an autologous dendritic cell. Such cells may be prepared and transfected using standard techniques, such as those described by Reeves et al., Cancer Res. 56:5672-5677, 1996; Tuting et al., J.

Immunol. 160:1139-1147, 1998; and Nair et al., Nature Biotechnol. 16:364-369, 1998).

Expression of a WT1 polypeptide on the surface of an antigen-presenting cell may be confirmed by in vitro stimulation and standard proliferation as well as chromium release assays, as described herein.

While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.

For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum; cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions of this invention. For certain topical applications, formulation as a cream or lotion, using well known components, is preferred.

Such compositions may also comprise buffers neutral buffered saline or phosphate buffered saline), carbohydrates glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants aluminum hydroxide) and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.

Any of a variety of non-specific immune response enhancers, such as adjuvants, may be employed in the vaccines of this invention. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable nonspecific immune response enhancers include alum-based adjuvants Alhydrogel, Rehydragel, aluminum phosphate, Algammulin, aluminum hydroxide); oil based adjuvants (Freund's adjuvant Specol, RIBI, TiterMax, Montanide ISA50 or Seppic MONTANIDE ISA 720; cytokines GM-CSF or Flat3-ligand); microspheres; nonionic block copolymer-based adjuvants; dimethyl dioctadecyl ammoniumbromide (DDA) based adjuvants AS-1, AS-2 (Smith Kline Beecham); Ribi Adjuvant system based adjuvants; QS21 (Aquila); saponin based adjuvants (crude saponin, the saponin Quil A muramyl dipeptide (MDP) based adjuvants such as SAF (Syntex adjuvant in its microfluidized form dimethyl-dioctadecyl ammonium bromide (DDA); human complement based adjuvants m. vaccae and derivatives; immune stimulating complex (iscom) based adjuvants; inactivated toxins; and attenuated infectious agents (such as M. tuberculosis).

As noted above, within certain embodiments, immune response enhancers are chosen for their ability to preferentially elicit or enhance a T cell response CD4' and/or CD8') to a WTI polypeptide. Such immune response enhancers are well known in the art, and include (but are not limited to) Montanide ISA50, Seppic MONTANIDE ISA 720, cytokines GM-CSF, Flat3-ligand), microspheres, dimethyl dioctadecyl ammoniumbromide (DDA) based adjuvants, AS-1 (Smith Kline Beecham), AS-2 (Smith Kline Beecham), Ribi Adjuvant system based adjuvants, QS21 (Aquila), saponin based adjuvants (crude saponin, the saponin Quil Syntex adjuvant in its microfluidized form (SAF-m), MV, ddMV (Genesis), immune stimulating complex (iscom) based adjuvants and inactivated toxins.

The compositions and vaccines described herein may be administered as part of a sustained release formulation a formulation such as a capsule or sponge that effects a slow release- of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide, antibody or cell dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

THERAPY OF MALIGNANT DISEASES In further aspects of the present invention, the compositions and vaccines described herein may be used to inhibit the development of malignant diseases progressive or metastatic diseases or diseases characterized by small tumor burden such as minimal residual disease). In general, such methods may be used to prevent, delay or treat a disease associated with WTI expression. In other words, therapeutic methods provided herein may be used to treat an existing- WTI -associated disease, or may be used to prevent or delay the onset of such a disease in a patient who is free of disease or who is afflicted with a disease that is not yet associated with WTI expression.

As used herein, a disease is "associated with WTI expression" if diseased cells tumor cells) at some time during the course of the disease generate detectably higher levels-of a WTI polypeptide than normal cells of the same tissue.

Association of WTI expression with a malignant disease does not require that WTI be present on a tumor. For example, overexpression of WTI may be involved with initiation of a tumor, but the protein expression may subsequently be lost.

Alternatively, a malignant disease that is not characterized by an increase in WTI expression may, at a later time, progress to a disease that is characterized by increased WTI expression. Accordingly, any malignant disease in which diseased cells formerly expressed, currently express or are expected to subsequently express increased levels of WTi1 is considered to be "associated with WTI expression.

Immunotherapy may be performed using any of a variety of techniques, in which compounds or cells provided herein function to remove WTI-expressing cells from a patient. Such removal may take place as a result of enhancing or inducing an immune response in a patient specific for WTI or a cell expressing WTI. Alternatively, WTI -expressing cells may be removed ex vivo by treatment of autologous bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood). Fractions of bone marrow or peripheral blood may be obtained using any standard technique in the art.

Within such methods, pharmaceutical compositions and vaccines may be administered to a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A patient may or may not be afflicted with a malignant disease. Accordingly, the above pharmaceutical compositions and vaccines may be used to prevent the onset of a disease prophylactically) or to treat a patient afflicted with a disease to prevent or delay progression and/or metastasis of an existing disease). A patient afflicted with a disease may have a minimal residual disease a low tumor burden in a leukemia patient in complete or partial remission or a cancer patient following reduction of the tumor burden after surgery radiotherapy and/or chemotherapy). Such a patient may be immunized to inhibit a relapse prevent or delay the relapse, or decrease the severity of a relapse). Within certain preferred embodiments, the patient is afflicted with a leukemia AML, CML, ALL or childhood ALL), a myelodysplastic. syndrome (MDS) or a cancer gastrointestinal, lung, thyroid or breast cancer or a melanoma), where the cancer or leukemia is WTI positive reacts detectably with an anti-WTl antibody, as provided herein or expresses WT1 mRNA at a level detectable by RT-PCR, as described herein) or suffers from an autoimmune disease directed against WTI-expressing cells.

The compositions provided herein may be used alone or in combination with conventional therapeutic regimens such as surgery, irradiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

As discussed in greater detail below, binding agents and T cells as provided herein may be used for purging of autologous stem cells. Such purging may be beneficial prior to, for example, bone marrow transplantation or transfusion of blood or components thereof. Binding agents, T cells, antigen presenting cells (APC) and compositions provided herein may further be used for expanding and stimulating (or priming) autologous, allogeneic, syngeneic or unrelated WTI-specific T-cells in vitro and/or in vivo. Such WTl-specific T cells may be used, for example, within donor lymphocyte infusions.

Routes and frequency of administration, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques.

In general, the pharmaceutical compositions and vaccines may be administered by injection intracutaneous, intramuscular, intravenous or subcutaneous), intranasally by aspiration) or orally. In some tumors, pharmaceutical compositions or vaccines may be administered locally (by, for example, rectocoloscopy, gastroscopy, videoendoscopy, angiography or other methods known in the art). Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter; Alternate protocols may be appropriate for individual patients.

A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response that is at least 10-50% above the basal untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome more frequent complete or partial remissions, or longer disease-free and/or overall survival) in vaccinated patients as compared to non-vaccinated patients.

In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 100 plg to 5 mg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome more frequent complete or partial remissions, or longer disease-free and/or overall survival) in treated patients as compared to non-treated patients.

Increases in preexisting immune responses to WT1 generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.

Within further aspects, methods for inhibiting the development of a malignant disease associated with WTI expression involve the administration of autologous T cells that have been activated in response to a WTI polypeptide or WTIexpressing APC, as described above. Such T cells may be CD4' and/or CD8', and may be proliferated as described above. The T cells may be administered to the individual in an amount effective to inhibit the development of a malignant disease. Typically, about 1 x 109 to I x 10 T cells/M are administered intravenously, intracavitary or in the bed of a resected tumor. It will be evident to those skilled in the art that the number of cells and the frequency of administration will be dependent upon the response of the patient.

Within certain embodiments, T cells may be stimulated prior to an autologous bone marrow transplantation. Such stimulation may take place in vivo or in vitro. For in vitro stimulation, bone marrow and/or peripheral blood (or a fraction of bone marrow or peripheral blood) obtained from a-patient may be contacted with a WTI polypeptide, a polynucleotide encoding a WT1 polypeptide and/or an APC that expresses a WTI polypeptide under conditions and for a time sufficient to permit the stimulation of T cells as described above. Bone marrow, peripheral blood stem cells and/or WTI-specific T cells may then be administered to a patient using standard techniques.

Within related embodiments, T cells of a related or unrelated donor may be stimulated prior to a syngeneic or allogeneic (related or unrelated) bone marrow transplantation. Such stimulation may take place in vivo or in vitro. For in vitro stimulation, bone marrow and/or peripheral blood (or a fraction of bone marrow or peripheral blood) obtained from a related or unrelated donor may be contacted with a WTI polypeptide, WT1 polynucleotide and/or APC that expresses a WTI polypeptide under conditions and for a time sufficient to permit the stimulation of T cells as described above. Bone marrow, peripheral blood stem cells and/or WTl-specific T cells may then be administered to a patient using standard techniques.

Within other embodiments, WTI-specific T cells as described herein may be used to remove cells expressing WTI from autologous bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood CD34' enriched peripheral blood (PB) prior to administration to a patient). Such methods may be performed by contacting bone marrow or PB with such T cells under conditions and for a time sufficient to permit the reduction of WTI expressing cells to less than 10%, preferably less than 5% and more preferably less than of the total number of myeloid or lymphatic cells in the bone marrow or peripheral blood. The extent to which such cells have been removed may be readily determined by standard methods such as, for example, qualitative and quantitative PCR analysis, morphology, immunohistochemistry and FACS analysis. Bone marrow or PB (or a fraction thereof) may then be administered to a patient using standard techniques.

DIAGNOSTIC METHODS The present invention further provides methods for detecting a malignant disease associated with WT1 expression, and for monitoring the effectiveness of an immunization or therapy for such a disease. Such methods are based on the discovery, within the present invention, that an immune response specific for WTI protein can be detected in patients afflicted with such diseases, and that methods which enhance such immune responses may provide a preventive or therapeutic benefit.

To determine the presence or absence of a malignant disease associated with WTI expression, a patient may be tested for the level of T cells specific for WTl.

Within certain methods, a biological sample comprising CD4' and/or CD8' T cells isolated from a patient is incubated with a WTI polypeptide, a polynucleotide encoding a WTI polypeptide and/or an APC that expresses a WT1 polypeptide, and the presence or absence of specific activation of the T cells is detected, as described herein. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37 0 C with WTI polypeptide 5 25 .tg/ml).

It may be desirable to incubate another aliquot of a T cell sample in the absence of WT polypeptide to serve as a control. For CD4" T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8' T cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in diseasefree patients indicates the presence of a malignant disease associated with WTI expression. Further correlation may be made, using methods well known in the art, between the level of proliferation and/or cytolytic activity and the predicted response to therapy. In particular, patients that display a higher antibody, proliferative and/or lytic response may be expected to show a greater response to therapy.

Within other methods, a biological sample obtained from a patient is tested for the level of antibody specific for WTI. The biological sample is incubated with a WTI polypeptide, a polynucleotide encoding a WT1 polypeptide and/or an APC that expresses a WTI polypeptide under conditions and for a time sufficient to allow immunocomplexes to form. Immunocomplexes formed between the WTI polypeptide and antibodies in the biological sample that specifically bind to the WTI polypeptide are then detected. A biological sample for use within such methods may be any sample obtained from a patient that would be expected to contain antibodies. Suitable biological samples include blood, sera, ascites, bone marrow, pleural effusion, and cerebrospinal fluid.

The biological sample is incubated with the WTI polypeptide in a reaction mixture under conditions and for a time sufficient to permit immunocomplexes to form between the polypeptide and antibodies specific for WTI. For example, a biological sample and WTI polypeptide may be incubated at 4°C for 24-48 hours.

Following the incubation, the reaction mixture is tested for the presence of immunocomplexes. Detection of immunocomplexes formed between the WTI polypeptide and antibodies present in the biological sample may be accomplished by a variety of known techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISA). Suitable assays are well known in the art and are amply described in the scientific and patent literature Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Assays that may be used include, but are not limited to, the double monoclonal antibody sandwich immunoassay technique of David et al. Patent 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide etal., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh, 1970); the "western blot" method of Gordon et al. Patent 4,452,901); immunoprecipitation of labeled ligand (Brown etal., J. Biol. Chem. 255:4980-4983, 1980); enzyme-linked immunosorbent assays as described by, for example, Raines and Ross Biol. Chem.

257:5154-5160, 1982); immunocytochemical techniques, including the use of fluorochromes (Brooks et al., Clin. Exp. Immunol. 39: 477, 1980); and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad. Sci. USA 81:2396-2400, 1984). Other immunoassays include, but are not limited to, those described in U.S. Patent Nos.: 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876.

For detection purposes, WT1 polypeptide may either be labeled or unlabeled. Unlabeled WTI polypeptide may be used in agglutination assays or in combination with labeled detection reagents that bind to the immunocomplexes anti-immunoglobulin, protein G, protein A or a lectin and secondary antibodies, or antigen-binding fragments thereof, capable of binding to the antibodies that specifically bind to the WTI polypeptide). If the WTI polypeptide is labeled, the reporter group may be any suitable reporter group known in the art, including radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.

Within certain assays, unlabeled WTI polypeptide is immobilized on a solid support. The solid support may be any material known to those of ordinary skill in the art to which the polypeptide may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.

Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S.

Patent No. 5,359,681. The polypeptide may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent).

Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the WTI polypeptide, in a suitable buffer, with the solid support for a suitable amount of time.

The contact time varies with temperature, but is typically between about 1 hour and about I day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of polypeptide ranging from about ng to about 10 [ig, and preferably about 100 ng to about 1-pig, is sufficient to immobilize an adequate amount of polypeptide.

Following immobilization, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin, Tween 20TM (Sigma Chemical Co., St.

Louis, MO), heat-inactivated normal goat serum (NGS), or BLOTTO (buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent).

The support is then incubated with a biological sample suspected of containing specific antibody. The sample can be applied neat, or, more often, it can be diluted, usually in a buffered solution which contains a small amount by weight) of protein, such as BSA, NGS, or BLOTTO. In general, an appropriate contact time incubation time) is a period of time that is sufficient to detect the presence of antibody that specifically binds WTI within a sample containing such an antibody. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound antibody. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time.

At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20

TM

A detection reagent that binds to the immunocomplexes and that comprises a reporter group may then be added. The detection reagent is incubated with the immunocomplex for an amount of time sufficient to detect the bound antibody. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate.

Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups horseradish peroxidase, beta-galactosidase, alkaline phosphatase and glucose oxidase) may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.

Regardless of the specific method employed, a level of bound detection reagent that is at least two fold greater than background the level observed for a biological sample obtained from a disease-free individual) indicates the presence of a malignant disease associated with WTI expression.

In general, methods for monitoring the effectiveness of an immunization or therapy involve monitoring changes in the level of antibodies or T cells specific for WT1 in the patient. Methods in which antibody levels are monitored may comprise the steps of: incubating a first biological sample, obtained from a patient prior to a therapy or immunization, with a WTI polypeptide, wherein the incubation is performed under conditions and for a time sufficient to allow immunocomplexes to form; (b) detecting immunocomplexes formed between the WTI polypeptide and antibodies in the biological sample that specifically bind to the WTI polypeptide; repeating steps and using a second biological sample taken from the patient following therapy or immunization; and comparing the number of immunocomplexes detected in the first and second biological samples. Alternatively, a polynucleotide encoding a WT1 polypeptide, or an APC expressing a WTI polypeptide may be employed in place of the WTI polypeptide. Within such methods, immunocomplexes between the WTI polypeptide encoded by the polynucleotide, or expressed by the APC, and antibodies in the biological sample are detected.

Methods in which T cell activation and/or the number of WTI specific precursors are monitored may comprise the steps of: incubating a first biological sample comprising CD4+ and/or CD8+ cells bone marrow, peripheral blood or a fraction thereof), obtained from a patient prior to a therapy or immunization, with a WTI polypeptide, wherein the incubation is performed under conditions and for a time sufficient to allow specific activation, proliferation and/or lysis of T cells; detecting an amount of activation, proliferation and/or lysis of the T cells; repeating steps (a) and using a second biological sample comprising CD4+ and/or CD8+ T cells, and taken from the same patient following therapy or immunization; and comparing the amount of activation, proliferation and/or lysis of T cells in the first and second biological samples. Alteratively, a polynucleotide encoding a WT1 polypeptide, or an APC expressing a WT1 polypeptide may be employed in place of the WTI polypeptide.

A biological sample for use within such methods may be any sample obtained from a patient that would be expected to contain antibodies, CD4+ T cells and/or CD8+ T cells: Suitable biological samples include blood, sera, ascites, bone marrow, pleural effusion and cerebrospinal fluid. A first biological sample may be obtained prior to initiation of therapy or immunization or part way through a therapy or vaccination regime. The second biological sample should be obtained in a similar manner, but at a time following additional therapy or immunization. The second biological sample may be obtained at the completion of, or part way through, therapy or immunization, provided that at least a portion of therapy or immunization takes place between the isolation of the first and second biological samples.

Incubation and detection steps for both samples may generally be performed as described above. A statistically significant increase in the number of immunocomplexes in the second sample relative to the first sample reflects successful therapy or immunization.

The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1 Identification of an Immune Response to WTI in Patients with Hematological Malignancies This Example illustrates the identification of an existent immune response in patients with a hematological malignancy.

To evaluate the presence of preexisting WTI specific antibody responses in patients, sera of patients with AML, ALL. CML and severe aplastic anemia were analyzed using Western blot analysis. Sera were tested for the ability to immunoprecipitate WTI from the human leukemic cell line K562 (American Type Culture Collection, Manassas, VA). In each case, immunoprecipitates were separated by gel electrophoresis, transferred to membrane and probed with the anti WT-1 antibody WTI80 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). This Western blot analysis identified potential WT1 specific antibodies in patients with hematological malignancy. A representative Western blot showing the results for a patient with AML is shown in Figure 2. A 52 kD protein in the immunoprecipitate generated using the patient sera was recognized by the WTI specific antibody. The 52 kD protein migrated at the same size as the positive control.

Example 2 Induction of Antibodies to WTI in Mice Immunized with Cell Lines Expressing WTI This Example illustrates the use of cells expressing WT1 to induce a WTI specific antibody response in vivo.

Detection of existent antibodies to WTI in patients with leukemia strongly implied that it is possible to immunize to WTI protein to elicit immunity to WTI. To test whether immunity to WT1 can be generated by vaccination, mice were injected with TRAMP-C, a WTI positive tumor cell line of B6 origin. Briefly, male B6 mice were immunized with 5 x 10 6 TRAMP-C cells subcutaneously and boosted twice with 5 x 10 6 cells at three week intervals. Three weeks after the final immunization, sera were obtained and single cell suspensions of spleens were prepared in RPMI 1640 medium (GIBCO) with 25pM P-2-mercaptoethanol, 200 units of penicillin per ml, L-glutamine, and 10% fetal bovine serum.

Following immunization to TRAMP-C, a WTI specific antibody response in the immunized animals was detectable. A representative Western blot is shown in Figure 3. These results show that immunization to WT1 protein can elicit an immune response to WTI protein.

Example 3 Induction of Th and Antibody Responses in Mice Immunized with WTI Peptides This Example illustrates the ability of immunization with WT1 peptides to elicit an immune response specific for WTI.

Peptides suitable for eliciting Ab and proliferative T cell responses were identified according to the Tsites program (Rothbard and Taylor, EMBO J. 7:93-100, 1988; Deavin et al., Mol Immunol. 33:145-155, 1996), which searches for peptide motifs that have the potential to elicit Th responses. Peptides shown in Table I were synthesized and sequenced.

Table I WTI Peptides Peptide Sequence Comments Mouse: p6-22 RDLNALLPAVSSLGGGG 1 mismatch relative to (SEQ ID NO:13) human WTI sequence Human: p6-22 RDLNALLPAVPSLGGGG (SEQ ID NO:1) Human/mouse:

PSQASSGQARMFPNAPYLPSCLE

p1 17-139 (SEQ ID NOs: 2 and 3) Mouse: p244-262 GATLKGMAAGSSSSVKWTE 1 mismatch relative to (SEQ ID NO:14) human WTI sequence Human: p244-262 GATLKGVAAGSSSSVKWTE (SEQ ID NO:4) Human/mouse: RIHTHGVFRGIQDVR p287-301 (SEQ ID NOs: 15 and 16) Mouse: p299-313 VRRVSGVAPTLVRS 1 mismatch relative to (SEQ ID NO: 17) human WTI sequence Human/mouse:

CQKKFARSDELVRHH

p 4 2 1-435 (SEQ ID NOs: 19 and For immunization, peptides were grouped as follows: Group A: p6-22 human: 10.9mg in ml (l0pl 100pg) pi 17-139 human/mouse: 7.6mg in Iml (14l 100pg) p244-262 human: 4.6.mg in Iml (22pl 100pg) Group B: p287-301 human/mouse: 7.2mg in Imi (14pl 100lpg) mouse p299-313: 6.6.mg in Iml (15pl 100pg) p421-435 human/mouse: 3.3mg in Iml (30pl 100pg) Control: (FBL peptide 100g) CFA/IFA Control: (CD45 peptide 100p g) CFA/IFA Group A contained peptides present within the amino' terminus portion of WT1 (exon 1) and Group B contained peptides present within the carboxy terminus, which contains a four zinc finger region with sequence homology to other DNA-binding proteins. Within group B, p287-301 and p 2 9 9 3 13 were derived from exon 7, zinc finger 1, and p421-435 was derived from exon 10, zinc finger IV.

B6 mice were immunized with a group of WTI peptides or with a control peptide. Peptides were dissolved in Iml sterile water for injection, and B6 mice were immunized 3 times at time intervals of three weeks. Adjuvants used were CFA/IFA, GM-CSF, and Montinide. The presence of antibodies specific for WT1 was then determined as described in Examples 1 and 2, and proliferative T cell responses were. evaluated using a standard thymidine incorporation assay, in which cells were cultured in the presence of antigen and proliferation was evaluated by measuring incorporated radioactivity (Chen et al., Cancer Res. 54:1065-1070, 1994). In particular, lymphocytes were cultured in 96-well plates at 2x10 5 cells per well with 4x10 irradiated (3000 rads) syngeneic spleen cells and the designated peptide.

Immunization of mice with the group of peptides designated as Group A elicited an antibody response to WTI (Figure No antibodies were detected following immunization to Vaccine B, which is consistent with a lack of helper T cell response from immunization with Vaccine B. PI17-139 elicited proliferative T cell responses (Figures 5A-5C). The stimulation indices (SI) varied between 8 and 72.

Other peptides (P6-22 and P299-313) also were shown to elicit proliferative T cell responses. Immunization with P6-22 resulted in a stimulation index (SI) of 2.3 and immunization with P299-313 resulted in a SI of 3.3. Positive controls included ConA stimulated T cells, as well as T cells stimulated with known antigens, such as CD45 and FBL, and allogeneic T cell lines (DeBruijn et al., Eur. J. Immunol. 21:2963-2970, 1991).

Figures 6A and 6B show the proliferative response observed for each of the three peptides within vaccine A (Figure 6A) and vaccine B (Figure 6B). Vaccine A elicited proliferative T cell responses to the immunizing peptides p6-22 and p117-139, with stimulation indices (SI) varying between 3 and 8 (bulk lines). No proliferative response to p244-262 was detected (Figure 6A).

Subsequent in vitro stimulations were carried out as single peptide stimulations using only p6-22 and pI 17-139. Stimulation of the Vaccine A specific T cell line with p117-139 resulted in proliferation to p117-139 with no response to p6-22 (Figure7A). Clones derived from the line were specific for p117-139 (Figure 7B). By contrast, stimulation of the Vaccine A specific T cell line with p6-22 resulted in proliferation to p6-22 with no response to p117-139 (Figure 7C). Clones derived from the line were specific for p6-22 (Figure 7D).

These results show that vaccination with WTI peptides can elicit antibody responses to WT1 protein and proliferative T cell responses to the immunizing peptides.

Example 4 Induction of CTL Responses in Mice Immunized with WTI Pentides This Example illustrates the ability of WTI peptides to elicit CTL immunity.

Peptides (9-mers) with motifs appropriate for binding to class I MHC were identified using a BIMAS HLA peptide binding prediction analysis (Parker et al., J. Immunol. 152:163, 1994). Peptides identified within such analyses are shown in Tables II XLIV. In each of these tables, the score reflects the theoretical binding affinity (half-time of dissociation) of the peptide to the MHC molecule indicated.

Peptides identified using the Tsites program (Rothbard and Taylor, EMBO J. 7:93-100, 1988; Deavin et al., Mol. Immunol. 33:145-155, 1996), which searches for peptide motifs that-have, the potential to elicit-Th responses are further shown in Figures 8A and 8B, and Table XLV.

Table II Results of BIMAS HLA Peptide Binding Prediction Analysis for 71, Binding of Human WTI Peptides to Human HLA Al Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 137 CLESQPAIR (SEQ ID 18.000 NO:47) 2 80 GAEPHEEQC (SEQ 9.000 ID NO:87) 3 40 FAPPGASAY (SEQ 5.000 ID NO:74) 4 354 QCDFKDCER (SEQ 5.000 ID NO:162) 2 GSDVRDLNA (SEQ 3.750 ID NO:101) 6 152 VTFDGTPSY (SEQ ID 2.500 NO:244) 7 260 WTEGQSNHS (SEQ 2.250 ID NO:247) 8 409 TSEKPFSCR (SEQ ID 1.350 NO:232) 9 73 KQEPSWGGA (SEQ 1.350 ~~~IDNO: 125) 386 KTCQRKFSR (SEQ 1.250 NO: 128)__ 11 37 VLDFAPPGA (SEQ 1.000 NO:24 1) 12 325 CAYPGCNKR (SEQ 1.000 ID NO:44) 13 232 QLECMTWNQ (SEQ 0.900 NO: 167) 14 272 ESDNHTTPI (SEQ ID 0.750 NO:71) 366 RSDQLKRHQ (SEQ 0.750 ID NO: 193)__ 16 222 SSDNLYQMT (SEQ 0.750 NO:2 17) 17 427 RSDELVRHH (SEQ 0.750 ID NO: 19 1) 18 394 RSDHLKTHT (SEQ 0.750 NO: 192) 19 317 TSEKRPFMC (SEQ 0.675 213 QALLLRTPY (SEQ ID 0.500 160) Table III Results of BIMAS HLA Pep~tide Bindingr Prediction Analysis for Binding of Human WITI Peptides to Human HLA A 0201* Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 126 RMFPNAPYL (SEQ 3 13.968 NO: 185)__ 2 187 SLGEQQYSV (SEQ 285.163 NO:214) 3 10 ALLPAVPSL (SEQ ID 18 1.794 4 242 NLGATLKGV (SEQ 159.970 NO:146) 225 NLYQMTSQL (SEQ 68.360 NO: 147) 6 292 GVFRGIQDV (SEQ 5 1.790 NO: 103) 7 191 QQYSVPPPV (SEQ 22.566 8 280 ILCGAQYRI (SEQ ID 17.736 NO: 116) 9 235 CMTWNQMNL (SEQ 15.428 441 NMTKLQLAL (SEQ 15.428 IDNO: 149)__ 11 7 DLNALLPAV (SEQ 11.998 ~~ID 12 227 YQMTSQLEC (SEQ 8.573 NO:251) 13 239 NQMNLGATL (SEQ 8.0 14 IDNO: 14 309 TLVRSASET (SEQ ID 7.452 408 KTSEKPFSC (SEQ ID 5.743 ~~NO:129) 16 340 LQMHSRKHT (SEQ 4.752 ID NO: 139) 17 228 QMTSQLECM (SEQ 4.044 ID NO: 169) 18 93 TVHFSGQFT (SEQ ID 3.586 NO:235) 19 37 VLDFAPPGA (SEQ 3.378 ID NO:241) 86 EQCLSAFTV (SEQ ID 3.068 Table IV Results of BIMAS HLA Peptide Binding Prediction Analysis for Bindirng of Human WVTI Petides to Human HLA A 0205 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Containing Rank Start Position Listing This Subsequence) 110 ALLPAVPSL (SEQ ID 42.000 NO :34) 2 292 GVFRGIQDV (SEQ ID 24.000 NO: 103) 3 126 RMFPNAPYL (SEQ ID 21.000 NO: 185) 4 225 NLYQMTSQL (SEQ 21.000 NO: 147) 239 NQMNLGATL (SEQ 16.800 151) 6 302 RVPGVAPTL (SEQ ID 14.000 NO: 195) 7 441 NMTKLQLAL (SEQ 7.000 NO: 149) 8 235 CMTWNQMNL (SEQ 7.000 NO:49) 9 187 SLGEQQYSV (SEQ ID 6.000 NO:214) 191 QQYSVPPPV (SEQ ID 4.800 NO :171) 11 340 LQMHSRKI-T (SEQ 4.080 ~~~IDNO: 12 242 NLGATLKGV (SEQ 4.000 NO: 146)__ 13 227 YQMTSQLEC (SEQ ID 3.600 ~~~NO:25 1) 14 194 SVPPPVYGC (SEQ ID 2.000 93 TVHFSGQFT (SEQ ID 2.000 16 280 LLCGAQYRI (SEQ ID 1.700 116) 17 98 GQFTGTAGA (SEQ ID 1.200 18 309 TLVRSASET (SEQ ID 1.000 19 81 AEPHEEQCL (SEQ ID 0.980 NO 73 KQEPSWGGA (SEQ 0.960 ID NO: 125) Table V Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WTI Peptides to Human HLA A24 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 302 RVPGVAPTL (SEQ 16.800 I NO: 195) 2 218 RTPYSSDNL (SEQ ID 12.000 194) 3 356 DFKDCERRF (SEQ 12.000 4 126 RMFPNAPYL (SEQ 9.600 NO: 185) 326 AYPGCNKRY (SEQ 7.500 ID NO:42) 6 270 GYESNHT (SEQ ID 7.500 NO: 106)T 7 239 NQMNLGATL (SEQ 7.200 NO: 151) 8 10 ALLPAVPSL (SEQ ID 7.200 NO:34) 9 10 NAPYLPSCL (SEQ ID 7.200 144) 329 GCNKRYFKL (SEQ 6.600 11 417 RWPSCQKKF (SEQ 6.600 NO: 196) 12 47 AYGSLGGPA (SEQ 6.000 ID NOA41) 13 180 DPMGQQGSL (SEQ 6.000 ID NO:59) 14 4 DVRDLNALL (SEQ 5.760 NO:62) 285 QYRIHTHGV (SEQ 5.000 NO: 175) 16 192 QYSVPPPVY (SEQ 5.000 NO: 176) 17 207 DSCTGSQAL (SEQ 4.800 NO:61) 18 441 NMTKLQLAL (SEQ 4.800 NO: 149) 19 225 NLYQMTSQL (SEQ 4.000 NO: 147)__ 235 CMTWNQMNL (SEQ 4.000 Table VI Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WTIl Perntides to Human HLA A3 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 436 NMHQRNMTK (SEQ 40.000 ID NO: 148) 2 240 QMNLGATLK (SEQ 20.000 NO: 168) 3 88 CLSAFTVHF (SEQ ID 6.000 4 126 RMFPNAPYL (SEQ 4.500 NO: 185) 169 AQFPNHSFK (SEQ 4.500 ID 6 10 ALLPAVPSL (SEQ ID 4.050 7 137 CLESQPAIR (SEQ ID 4.000 8 225 NLYQMTSQL (SEQ 3.000 NO: 147) 9 32 AQWAPVLDF (SEQ 2.700 NO:37)__ 280 ILCGAQYRI (SEQ ID 2.700 NO: 116) 11 386 KTCQRKFSR (SEQ 1.800 ID NO: 128) 12 235 CMTWNQMNL (SEQ 1.200 NO:49) 13 441 NMTKLQLAL (SEQ 1.200 NO: 149) 14 152 VTFDGTPSY (SEQ ID 1.000 ____NO:244) 187 SLGEQQYSV (SEQ 0.900 NO:214) 16 383 FQCKTCQRK (SEQ 0.600 17 292 GVFRGIQDV (SEQ 0.450 NO: 103) 18 194 SVPPPVYGC (SEQ ID 0.405 19 287 RIHTHGVFR (SEQ ID 0.400 Table VII Results of BIMAS HLA Peptide Binding~ Prediction Analysis for Bindinof Humn WTI Pentides to Human H-LA A68 I Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 100 FTGTAGACR (SEQ 100.000 NO:84) 2 386 KTCQRKFSR (SEQ 5 0.000 ID NO: 128) 3 368 DQLKRHQRR (SEQ 30.000 4 312 RSASETSEK (SEQ ID 18.000 190)__ 337 LSHLQMHSR (SEQ 15.000 NO: 14 1) 6 364 FSRSDQLKR (SEQ ID 15.000 NO 7 409 TSEKPFSCR (SEQ ID 15.000 NO:232) 8 299 DVRRVPGVA (SEQ 12.000 ID NO:63) 9 4 DVRDLNALL (SEQ 12.000 ID NO:62) 118 SQASSGQAR (SEQ 10.000 ID NO:216) I1 343 HSRKHTGEK (SEQ 9.000 NO: I11) 12 169 AQFPNHSFK (SEQ 9.000 NO:36) 13 292 GVFRGIQDV (SEQ 8.000 NO: 103) 14 325 CAYPGCNKR (SEQ 7.500 NO:44) 425 FARSDELVR (SEQ-- 7.500 16 354 QCDFKDCER (SEQ 7.500 NO: 162) 17 324 __MCAYPGCNK (SEQ 6.000 NO: 142) 18 251 AAGSSSSVK (SEQ 6.000 NO:28) 19 379 GVKPFQCKT (SEQ 6.000 ID NO: 104) 137 CLESQPAIR (SEQ ID 5.000 Table VIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Bindinp, of Human WTI Peptides to Human HLA A 1101 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 386 KTCQRKFSR (SEQ 1.800 IDNO: 128) 2 169 AQFPNHSFK (SEQ 1.200 ~~ID NO:36) 3 436 NMHQRNMTK (SEQ 0.800 NO: 148) 4 391 KFSRSDHLK (SEQ 0.600 NO: 120) 373 HQRR.HTGVK (SEQ 0.600 ID NO: 109) 6 383 FQCKTCQRK (SEQ 0.600 NO:80)__ 7 363 RFSRSDQLK (SEQ ID 0.600 178) 8 240 QMNLGATLK (SEQ 0.400 ID NO:168) 9 287 RIHTHGVFR (SEQ ID 0.240 100 FTGTAGACR (SEQ 0.200 NO:84)__ 1 1 324 MCAYPGCNK (SEQ 0.200 ~~~IDNO: 12 251 AAGSSSSVK (SEQ 0.200 13 415 SCRWPSCQK (SEQ 0.200 ID NO:201) 14 118 SQASSGQAR (SEQ 0.120 16) 292 GVFRGIQDV (SQ0.120 103)__ 16 137 CLESQPAIR (SEQ ID 0.080 17 425 FARSDELVR (SEQ 0.080 18 325 CAYPGCNKR (SEQ 0.080 NO:44)__ 19 312 RSASETSEK (SEQ ID 0.060 190) 65 PPPPHSFI (SEQ ID 0.060 NO: I56)K Table IX Results of BIMAS HLA Pevtide Bindiny, Prediction Analvsis for Binding of Human WTI Peptides to Human HLA A 3101 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 386 KTCQRKFSR (SEQ 9.000 NO: 128) 2 287 RIHTHGVFR (SEQ ID 6.000 182) 3 137 CLESQPAIR (SEQ ID 2.000 4 118 SQASSGQAR (SEQ 2.000 NO:216) 368 DQLKRHQRR (SEQ 1.200 6 100 FTGTAGACR (SEQ 1.000 NO:84) 7 293 VFRGIQDVR (SEQ ID 0.600 8 325 CAYPGCNKR (SEQ 0.600 ID NO:44) 9 169 AQFPNHSFK (SEQ 0.600 NO:36) 279 PILCGAQYR (SEQ ID 0.400 11 436 NMHQRNMTK (SEQ 0.400 NO: 148) 12 425 FARSDELR (SEQ 0.400 13 32 AQWAPVLDF (SEQ 0.240 ID NO:37) 14 240 QMNLGATLK (SEQ 0.200 NO: 168)__ 354 QCDFKDCER (SEQ 0.200 NO: 162)__ 16 373 HQRR.HTGVK (SEQ 0.200 NO: 109) 17 383 FQCKTCQRK (SEQ 0.200 18 313 SASETSEKR (SEQ ID 0.200 197) 19 358 KDCERRFSR (SEQ 0.180 NO: 118)__ 391 KFSRSDHLK (SEQ 0.180 120)__ Table X Results of BIMAS HLA Pentide Binding~ Prediction Analysis for Binding of Human WTI Peptides to Human HLA A 3302 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 337 LSHLQMHSR (SEQ 15.000 ID NO: 141) 2 409 TSEKPFSCR (SEQ ID 15.000 3 364 FSRSDQLKR (SEQ ID) 15.000 4 137 CLESQPAIR (SEQ ID 9.000 368 DQLKRHQRR (SEQ 9.000 6 287 RIHTHGVFR. (SEQ ID 4.500 182) 7 210 TGSQALLLR (SEQ ID 3.000 8 425 FARSDELVR (SEQ 3.000 9 313 SASETSEKR (SEQ ID 3.000 197) 293 VFRGIQDVR (SEQ ID 3.000 I1 354 QCDFKDCER (SEQ 3.000 ID 12 100 FTGTAGACR (SEQ 3.000 NO:84) 13 118 SQASSGQAR (SEQ 3.000 14 325 CAYPGCNKR (SEQ 3.000 207 DSCTGSQAL (SEQ 1.500 NO:61) 16 139 ESQPAIRNQ (SEQ ID 1.500 17 299 DVRRVPGVA (SEQ 1.500 NO:63) 18 419 PSCQKKFAR (SEQ 1.500 NO: 159) 19 272 ESDNHTTPI (SEQ ID 1.500 4 DVRDLNALL (SEQ 1.500 Table XI Results of BIMAS HLA Peptide Binding Prediction Analysis for B indine, of Human WTI Peptides to Human HLA B 14 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 362 RRFSRSDQU(SEQ ID 1000.000 2 332 KRYFKLSHL (SEQ 300.000 NO: 127) 3 423 KKFARSDEL (SEQ 150.000 NO: 122) 4 390 RKFSRSDHL (SEQ ID 150.000 NO: 183) 439 QRNMTKLQL (SEQ 20.000 IDNO: 173) 6 329 GCNKRYFKL (SEQ 10.000 7 10 ALLPAVPSL (SEQ ID- 10.000 NO-:34) 8 180 DPMGQQGSL (SEQ 9.000 9 301 RRV5N-PGVAPT (SEQ 6.000 NO:1-8 9) 126 RMPAPYL (SEQ- 5.000 NO: 185) 11 371 KRHQRRHTG (SEQ 5.000 IDNO: 126) 12 -225 NLYQMTsQL (SEQ 5.000 NO: 147) 13 144 IRNQGSTv (SEQ ID 4.000 13f NO: 14 429 DEL VR-HNM (SEQ 3.000 ID NO:53) 437 MHQRNMTKI (SEQ- 3.000 NO: 143) 16 125 ARMFNAPY (SEQ -3.000 NO:38) 17 239 NQMNLGATL (SEQ- 3.000 NO: 15 1) 18 286 YRJHTHGVF (SEQ ID 3.000 19 174 HSFKI-EDPM (SEQ 3.000 ID NO:I10)__ 372 IRHQRRH-TGV (SEQ 3.000 NO: 18 Table XII Results of BIMAS HLA Peptide Bindin Prediction Analysis for Bindine of Human WTI Pcrptides to Human HLA Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 81 AEPHEEQCL (SEQ ID 40.000 2 429 DEL VRHHNM (SEQ 24.000 NO:53) 3 410 SEKPFSCRW (SEQ 20.000 IDNO:207) 4 318 SEKRPFCA (SEQ 15.000 233 LECMTWNQM (SEQ 12.000 IDNO:13 1) 6 3 SDVRDLNAL (SEQ 10.000 ~~ID 7 349 GEKPYQCDF (SEQ 8.000 NO:91) 8 6 RDLNALLPA (SEQ 5.000 NO: 177) 9 85 EEQCLSAFT (SEQ ID 4.000 315 SETSEKRPF (SEQ ID 4.000 11 261 TEGQSNHST (SEQ ID 4.000 1) 12 23 GCALPVSGA (SEQ 3.000 NO:89) 13 38 LDFAPPGAS (SEQ ID 3.000 130) 14 273 SDNHTTPIL (SEQ ID 2.500 206 TDSCTGSQA (SEQ 2.500 NO:220) 16 24 CALPVSGAA (SEQ 2.000 NO:43) 17 98 GQFTGTAGA (SEQ 2.000 ID NO:99) 18 30 GAAQWAPVL (SEQ 2.000 NO:86) 19 84 HEEQCLSAF (SEQ ID 2.000 NO:107) 26 LTPVSGAAQW (SEQ 2.000 ID NO:138) Table XIII Results of BIMAS HLA Peptide Binding~ Prediction Analysis for Binding of Human WTI Pentides to Human HLA Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 181 AEPHEEQCL (SEQ ID 160.000 2 3 SDVRDLNAL (SEQ 40.000 IDNO:206) 3 429 DEL VRHNNM (SEQ 40.000 NO:53)__ 4 233 LECMTWNQM (SEQ 22.000 13 1) 273 SDNHTTPIL (SEQ ID 20.000 NO:204) 6 209 CTGSQALLL (SEQ ID 8.000 7 30 GAAQWAPVL (SEQ 8.000 ID NO:86)__ 8 318 SEKRPFMCA (SEQ 8.000 ID NO:208) 9 180 DPMGQQGSL (SEQ 8.000 ID NO:59) 138 LESQPAIRN (SEQ ID 5.280 11 239 NQMNLGATL (SEQ 4.400 NO: 151) 12 329 GCNKRYFKI (SEQ 4.400 13 130 NAPYLPSCL (SEQ ID 4.400 NO: 144) 14 85 EEQCLSAFT (SEQ ID 4.400 208 SCTGSQALL (SEQ ID 4.000 16 207 DSCTGSQAL (SEQ 4.000 NO:61) 17 218, RTPYSSDNL (SEQ ID 4.000 194) 18 261 TEGQSNHST (SEQ ID 4.000 19 18 LGGGGGCAL (SEQ 4.000 NO: 134) 221 YSSDNLYQM (SEQ 2.200 Table XIV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding, of Human WTI Peptides to Human HLA B61 Score (Estimate of Half Time of Subsequence- Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 318 SEKRPFMCA (SEQ 20.000 2 429 DEL VRHHNM (SEQ 16.000 NO:53) 3 298 QDVRRVPGV (SEQ 10.000 164) 4 81 AEPHEEQCL (SEQ ID 8.000 233 LECMTWNQM (SEQ 8.000 ID NO:]13 1) 6 6 RDLNALLPA (SEQ 5.500 NO: 177) 7 85 EEQCLSAFT (SEQ ID 4.000 8 261 TEGQSNHST (SEQ ID 4.000 NO:221) 9 206 TDSCTGSQA (SEQ 2.500 NO:220) 295 RGIQDVRRV (SEQ 2.200 179)__ 11 3 SDVRDLNAL (SEQ 2.000 0:206)__ 12 250 VAAGSSSSV (SEQ 2.000 ID NO:236) 13 29 SGAAQWAPV (SEQ 2.000 NO:21 1) 14 315 SETSEKRPF (SEQ ID 1.600 138 LESQPAIRN (SEQ ID 1.200 132)__ 16 244 GATLKGVAA (SEQ 1.100 NO:88) 17 20 GGGGCALPV (SEQ 1.100 NO:92)__ 18 440 RNMTKLQLA (SEQ 1.100 NO: 186) 19 23 GCALPVSGA (SEQ 1.100 NO:89)__ 191 QQYSVPPPV (SEQ 1.000 Table XV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding, of Human WTI Pentides, to Human HLA B62 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 146 NQGYSTVTF (SEQ 211.200 NO: 150) 2 32 AQWAPVLDF (SEQ 96.000 NO:37) 3 263 GQSNHSTGY (SEQ 96.000 100)__ 4 88 CLSAFTVHF (SEQ ID 96.000 17 SLGGGGGCA (SEQ 9.600 NO:215) 6 239 NQMNLGATL (SEQ 8.800 NO: 15 1) 7 191 QQYSVPPPV (SEQ 8.000 IDNOA 7 1) 8 98 GQFTGTAGA (SEQ 8.000 NO:99) 9 384 QCKTCQRKF (SEQ 6.000 NO: 40 FAPPGASAY (SEQ 4.800 11 227 YQMTSQLEC (SEQ 4.800 NO:25 12 187 SLGEQQYSV (SEQ 4.400 ID NO:2 13 86 EQCLSAFTV (SEQ ID 4.400 14 152 VTFDGTPSY (SEQ ID 4.400 NO:244) 101 TGTAGACRY (SEQ 4.000 NO:224) 16 242 NLGATLKGV (SEQ 4.000 ID NO: 146) 17 92 FTVHFSGQF (SEQ ID 4.000 18 7 DLNALLPAV (SEQ 4.000 ID 19 123 GQARMFPNA (SEQ 4.000 NO:98) 280 ILCGAQYRI (SEQ ID 3.120 NO: 116)__ Table XVI.

Results of BIMAS HLA Pentide Bindina Prediction Analysis for Binding of Human WTI Pentides to Human HLA B7 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 180 DPMGQQGSL (SEQ 240.000 NO:59)__ 2 4 DVRDLNALL (SEQ 200.000 IDNO:62) 3 302 RVPGVAPTL (SEQ 20.000 NO. 195) 4 30 GAAQWAPVL (SEQ 12.000 NO:86) 239 NQMNLGATL (SEQ 12.000 ID NO: 151)__ 6 130 NAPYLPSCL (SEQ ID 12.000 NO: 144) 7 10 ALLPAVPSL (SEQ ID 12.000 8 299 DVRRVPGVA (SEQ 5.000 NO:63) 9 208 SCTGSQALL (SEQ ID 4.000 303 VPGVAPTLV (SEQ 4.000 IDNO:242) I11 18 LGGGGGCAL (SEQ 4.000 134) 12 218 RTPYSSDNL (SEQ ID 4.000 NO: 194) 13 207 DSCTGSQAL (SEQ 4.000 ~~~IDNO:61) 14 209 CTGSQALLL (SEQ ID 4.000 329 GCNKRYFKL (SEQ 4.000 NO:90)__ 16 235 CMTWNQMNL (SEQ 4.000 IDNO:49) 17 441 NMTKLQLAL (SEQ 4.000 NO: 149) 18 126 RMFPNAPYL (SEQ 4.000 NO: 185)__ 19 225 NLYQMTSQL (SEQ 4.000 Table XVII Results of BIMAS HLA Pentide Binding Prediction Analysis for Binding of Human W71 Peptides to Human HLA B8 Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 329 GCNKRYFKL (SEQ ID 2 4 DVRDLNALL (SEQ NO:62)__ 3 316 ETSEKRPFM (SEQ ID 3.000 4 -10 DPMGQQGSL (SEQ 1.600 NO:59) 208 SCTGSQALL (SEQ ID) 0.800 6 130 NAPYLPSCL (SEQ ID 0.800 144) 7 244 GATLKGVAA (SEQ 0.800 ID NO: 88) 8 30 GAAQWAPVL (SEQ 0.800 ID NO:86) 9 299 DVRRVPGVA (SEQ 0.400 NO:63) 420 SCQKKFARS (SEQ- 0.400 NO:200)__ 11 387 TCQRKFSRS (SEQ ID 0.400 19) 12 225 NLYQMTSQL (SEQ 0.400 NO: 147)__ 13 141 QPAIRNQGY (SEQ 0.400 NO: 170)__ 14 10 ALLPAVPSL (SEQ ID 0.400 207 DSCTGSQAL (SEQ 0.400 NO:61) 16 384 QCKTCQRKF (SEQ 0.400 NO: 163) 17 136 SCLESQPAI (SEQ ID 0.300 NO: 198) 18 347 HTGEKPYQC (SEQ 0.300 NO: 112) 19 401 HTRTHTGKT (SEQ 0.200 IDNO: 114) 332 KRYFKLSHL (SEQ 0.200 127) Table XVIII Results of BJMAS HLA Peptide Bindingt Prediction Analysis for Binding of Human WTI PeR~tides to Human HLA B 2702 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 332 KRYFKLSHL (SEQ 900.000 IDNO: 2 362 RRFSRSDQL (SEQ ID 900.000 NO: 187)__ 3 286 YRIHTHGVF (SEQ ID 200.000 NO:252) 4 125 AR.MFPNAPY (SEQ 200.000 DNO:38) 375 RRHTGVKPF (SEQ 180.000 ID, NO: 188)__ 6 32 AQWAPVLDF (SEQ 100.000 NO:37)__ 7 301 RRVPGVAPT (SEQ 60.000 189)__ 8 439 QRNMTKLQL (SEQ 60.000 NO: 173)__ 9 126 RMFPNAPYL (SEQ 22.500 NO: 185) 426 ARSDELVRH (SEQ 20.000 NO:39) 11 146 NQGYSTVTF (SEQ 20.000 NO: 150) *12 144 IRNQGYSTV (SEQID, 20.000 117)__ 13 389 QRKFSRSDH (SEQ 20.000 ID,_NO: 172)__ 14 263 GQSNHSTGY (SEQ 20.000 NO:l100) 416 CRWPSCQKK (SEQ 20.000 16 191 QQYSVPPPV (SEQ 10.000 NO: 17 1) 17 217 LRTPYSSDN (SEQ ID 10.000 140) 18 107 CRYGPFGPP (SEQ ID 10.000 NO:51)__ 19 98 GQFTGTAGA (SEQ 10.000 NO:99) 239 NQMNLGATL (SEQ 6.000 J ID NO: 151)__ Table XIX Results of BIMAS HLA Peptide Bindiniz Prediction Analysis for Binding of Human WTI Peptides to Human HLA B 2705 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 332 KRYFKLSHL (SEQ 30000.000 IDNO: 127) 1_ 362 NO: 187) 30000.000 3 416 CRWPSCQKK (SEQ 10000.000 4 439 QRNMTKLQL (SEQ 2000.000 NO: 286 YRIHTHGVF (SEQ ID 1000.000 NO:252) 6 125 ARMFPNAPY (SEQ 1000.000 ID NO:38) 7 294 FRGI QDVRR (SEQ ID 1000.000 8 432 VRI-HNMHQR (SEQ 1000.000 _____IDNO:243) 9 169 AQFPNHSFK (SEQ 1000.000 NO:36) 375 RRI-TGVKPF (SEQ 900.000 NO: 188) 11 126 RMFPNAPYL (SEQ 750.000 NO:185) 12 144 IRNQGYSTV (SEQ ID 600.000 117) 13 301 RRVPGVAPT (SEQ 600.000 NO: 189) 14 32 AQWAPVLDF (SEQ 500.000 ID NO:37)__ 191 QQYSVPPPV (SEQ 300.000 NO: 171) 16 373 HQRRHTGVK (SEQ 200.000 NO: 109) 17 426 ARSDELVRH (SEQ 200.000 ID NO:39) 18 383 FQCKTCQRK (SEQ 200.000 ID 19 239 NQMNLGATL (SEQ 200.000 ID NO: 151) 389 QRKFSRSDH (SEQ 200.000 ID NO: 172) Table XX Results of BIMAS HLA Peptide Bindinp, Prediction Analysis for Bindinpg of Human WTI Peptides to Human HLA B 3501 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 278 TPILCGAQY (SEQ ID 40.000 2 141 QPAIRNQGY (SEQ 40.000 ID NO: 170) 3 219 TPYSSDNLY (SEQ ID 40.000 NO:23 4 327 YPGCNKRYF (SEQ 20.000 ID NO:250) 163 TPSHHAAQF (SEQ 20.000 ID NO:228) 6 180 DPMGQQGSL (SEQ 20.000 NO:59) 7 221 YSSDNLYQM (SEQ 20.000 ID NO:253) 8 26 LPVSGAAQW (SEQ 10.000 ID NO: 138) 9 174 HSFKiHEDPM (SEQ 10.000 ID NO:1I 82 EPHEEQCLS (SEQ ID) 6.000 11 213 QALLLRTPY (SEQ ID 6.000 160) 12 119. QASSGQARM (SEQ 6.000 ID NO: 16 1) 13 4 DVRDLNALL (SEQ 6.000 ID 14 40 FAPPGASAY (SEQ 6.000 ID NO:74) 120 ASSGQARMF (SEQ 5.000 16 207 DSCTGSQAL (SEQ 5.000 NO:61) 17 303 VPGVAPTLV (SEQ 4.000 NO:242) 18 316 ETSEKRPFM (SEQ ID 4.000 19 152 VTFDGTPSY (SEQ ID 4.000 412 KPFSCRWPS (SEQ ID, 4.000 123)__ Table XXI Results of BIMAS HLA Peptide Binding Prediction Analysis for Bindina of Human WTI Peptides to Human HLA.B 3.701 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 3 SDVRDLNAL (SEQ 40.000 ID NO:206) 2 273 SDNHTTPIL (SEQ ID 40.000 3 81 AEPHEEQCL (SEQ ID 10.000 ,4 298 QDVRRVPGV (SEQ 8.000 164) 428 SDELVRHHN (SEQ 6.000 NO:203) 6 85 EEQCLSAFT (SEQ ID 5.000 7 208 SCTGSQALL (SEQ ID 5.000 8 4 DVRDLNALL (SEQ 5.000 9 209 ICTGSQALLL (SEQ IDI 5.000 I NO:52) 38 LDFAPPGAS (SEQ ID 4.000 NO: 130) 11 223 SDNLYQMTS (SEQ 4.000 NO:205) 12 179 EDPMGQQGS (SEQ 4.000 ID NO:64) 13 206 TDSCTGSQA (SEQ 4.000 14 6 RDLNALLPA (SEQ 4.000 ID NO: 177) 84 HEEQCLSAF (SEQ ID 2.000 NO: 107) 16 233 LECMTWNQM (SEQ 2.000 NO: 13 1) 17 429 DEL VRHHNM (SEQ 2.000 18 315 SETSEKRPF (SEQ-ID 2.000 19 349 GEKPYQCDF (SEQ 2.000 ID NO:91) 302 RVPGVAPTL (SEQ 1.500 195)__ Table XXII Results of BIMAS HLA Pelptide Binding- Prediction Analysis for Binding of Human WTI Peptides to Human HLA B 3801 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 437 MHQRNMTKL (SEQ 36.000 ID NO: 143) 2 434 HHNMHQRNM (SEQ 6.000 ID NO: 108) 3 372 RHQRRHTGV (SEQ 6.000 ID NO: 181) 4 180 DPMGQQGSL (SEQ 4.000 ID NO:59) 433 RH-HNMHQRN (SEQ 3.900 ID NO: 180) 6 165 SHHAAQFPN (SEQ 3.900 ID NO:213) 7 202 CHTPTDSCT (SEQ ID 3.000 8 396 DHLKTHTRT (SEQ 3.000 NO:57) 9 161 GHTPSHHAA (SEQ 3.000 NO:94) 302 RVPGVAPTL (SEQ 2.600 195)__ 11 417 RWPSCQKKF (SEQ 2.400 NO: 12 327 YPGCNKRYF (SEQ 2.400 NO:250) 13 208 SCTGSQALL (SEQ ID 2.000 14 163 TPSHHAAQF (SEQ 2.000 ~~ID NO:228) 120 ASSGQARMF (SEQ 2.000 ID 16 18 LGGGGGCAL (SEQ 2.000 NO: 134) 17 177 KHEDPMGQQ (SEQ 1.800 NO: 121) 18 83 PHEEQCLSA (SEQ ID 1.800 19 10 ALLPAVPSL (SEQ ID 1.300 225 NLYQMTSQL (SEQ 1.300 NO: 147) Table XXIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WrTI Peptides to Human HLA B 3901 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 437 MHQRNMTKL (SEQ 135.000 NO: 143) 2 332 KRYFKLSHL (SEQ 45.000 NO: 127) 3 434 HHNMHQRNM (SEQ 30.000 NO: 108) 4 362 RRFSRSDQL (SEQ ID 30.000 187) 372 RHQRRHTGV (SEQ 30.000 NO: 18 1) 6 10 ALLPAVPSL (SEQ ID 9.000 7 439 QRNMTKLQL (SEQ 7.500 ID NO: 173) 8 390 RKFSRSDHL (SEQ ID 6.000 183) 9 396 DHLKTHTRT (SEQ 6.000 NO:57) 239 NQMNLGATL (SEQ 6.000 11 423 KKFARSDEL (SEQ 6.000 ID NO: 122) 12 126 RMFPNAPYL (SEQ 6.000 NO: 185)__ 13 225 NLYQMTSQL (SEQ 6.000 NO: 147)__ 14 180 DPMGQQGSL (SEQ 6.000 144 IRNQGYSTV (SEQ ID 5.000 117)__ SCLESQPAI (SEQ ID NO: 198) 4.000 17 292 GVFRGIQDV (SEQ 3.000 NO: 103) 18 302 RVPGVAPTL (SEQ 3.000 NO: 195) 19 208 SCTGSQALL (SEQ ID 3.000 207 DSCTGSQAL (SEQ 3.000 I Table XXIV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WTI Peptides to Human HLA B 3902 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 239 NQMINLGATL (SEQ 24.000 NO: 15 1) 2 390 RKFSRSDHL (SEQ ID 20.000 183) 3 423 KKFARSDEL (SEQ 20.000 NO: 122) 4 32 AQWAPVLDF (SEQ 5.000 146 NQGYSTVTF (SEQ 5.000 NO: 150) 6 130 NAPYLPSCL (SEQ ID 2.400 NO: 144) 7 225 NLYQMTSQL (SEQ 2.400 NO: 147) 8 30 GAAQWAPVL (SEQ 2.400 NO:86) 9 441 NMTKLQLAL (SEQ 2.400 NO: 149) 302 RVPGVAPTL (SEQ 2.400 ID NO: 195) 11 126 RMFPNAPYL (SEQ 2.000 NO: 185) 12 218 RTPYSSDNL (SEQ ID 2.000 194) 13 209 CTGSQALLL (SEQ ID 2.000 14 332 KRYFKLSHL (SEQ 2.000 NO: 127) 180 DPMGQQGSL (SEQ 2.000 NO:59)__ 16 437 MHQRNMTKL (SEQ 2.000 NO: 143) 17 207 DSCTGSQAL (SEQ 2.000 ID NO:61) 18 208 SCTGSQALL (SEQ ID) 2.000 19 329 GCNKRYFKL (SEQ 2.000 NO:90)__ 10 ALLPAVPSL (SEQ ID 2.000 Table XXV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WTI Peptides to Human HLA B 4403 (99[:ON cli 000,C bas) A,00301iso0 98 o (9oz:ON (Ii ooocf b9S) 1VM(IlIAUS E 61 Q E:ONq (II 000"7 bas) aIdOA1JVOV V"ol 81 Q E :ON cli oooi' 09S) 1NOMAUJNDTI Elz £1 (59:ON 0001, GIi o~s) IdVS1lDoaa 91 (tIZVON 0001, (11 09S) ISHNS6D3I 19Z 9 (otwONq ai (t~z:ON Ci 03s) A-dDvo1D1 101ot UIi 09S) ASdI0GIILA Z91 z

(OE:ON

000'8 GIi OaS) IDO3aHd3V 18 11 aI 000*8 03s) VDAJU~DIs 81 E 01 (091 :0x4 000*6 GIi 09S) Ad~.L)ITIVO E1Z 6 (t,L:ON UH 000'6 das) AYS Vdd VI 0" 8 (OL 1: ON GI 000'6 03s) ADONuIIwo 1t'1 L (uZVON 000*91 GIi O9S) OVOYIldi 8LZ 9 (Eg:ON cli ooo*j7 09S) NNH-fI19CI 6Zt' (LQON CII 000,8t, 09S) AMSId)13S 011" (L01ON 000*09 CII 09S) JVS1D033H V8 (I 6:ON CII 000,08 035S) iciDo&dX99~ 6K" Table XXVI Results of BIMAS H4LA Peptide Binding Prediction Analysis for Binding of Human WTI Peptides to Human HLA B 5101 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 303 VPGVAPTLV (SEQ 3 14.600 ID 2 180 DPMGQQGSL (SEQ 242.000 ID 3 250 VAAGSSSSV (SEQ 157.300 _____IDNO:236) 4 130 NAPYLPSCL (SEQ ID 50.000 144)__ 30 GAAQWAPVL (SEQ 50.000 NO:86) 6 20 GGGGCALPV (SEQ 44.000 ID NO:92) 7 64 PPPPPHSFI (SEQ ID 40.000 NO:157) 8 29 SGAAQWAPV (SEQ 40.000 ID NO:21 1) 9 18 LGGGGGCAL (SEQ 31.460 ID NO: 134) 295 RGIQDVRRV (SEQ 22.000 NO: 179) 11 119 QASSGQARM (SEQ 18.150 NO: 16 1) 12 418 WPSCQKIKFA (SEQ 12.100 13 82 EPHEEQCLS (SEQ ID 12.100 14 110 GPFGPPPPS (SEQ ID 11.000 272 ESDNHT-TPI (SEQ ID 8.000 71) 16 306 VAPTLVRSA (SEQ 7.150 NO:237) 17 280 ILCGAQYRI (SEQ ID 6.92 1 116) 18 219 TPYSSDNLY (SEQ ID 6.600 1) 19 128 FPNAPYLPS (SEQ ID 6.500 204 TPTDSCTGS (SEQ ID 6.050 Table XXVII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WTI Pelptides to Human HLA B 5102 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 295 RGIQDVRRV (SEQ 290.400 NO: 179)__ 2 303 VPGVAPTLV (SEQ 200.000 ID NO:242) 3 180 DPMGQQGSL (SEQ 133.100 NO:59) 4 250 VAAGSSSSV (SEQ 110.000 NO:236) 30 GAAQWAPVL (SEQ 55.000 ID 6 130 NAPYLPSCL (SEQ ID 50.000 NO: 144) 7 20 GGGGCALPV (SEQ 44.000 NO:92) 8 29 SGAAQWAPV (SEQ 44.000 NO:21 1) 9 64 PPPPPHSFI (SEQ ID 40.000 NO: 119 QASSGQARM (SEQ 36.300 ID NO: 16 1) 1 1 110 GPFGPPPPS (SEQ ID 27.500 12 412 KPFSCRWPS (SEQ ID 25.000 NO: 123)__ 13 18 LGGGGGCAL (SEQ 24.200 NO: 134) 14 24 CALPVSGAA (SEQ 16.500 219 TPYSSDNLY (SEQ ID 15.000 16 292 GVFRGIQDV (SEQ 14.641 NO: 103)__ r17 136 SCLESQPAI (SEQ ID 14.520 NO: 198)__ 18 418 WPSCQKKFA (SEQ 12.100 19 269 TGYESDNHT (SEQ 11.000 NO:225) 351 KPYQCDFKD (SEQ 11.000 NO: 124)__ Table XXVIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Bindingz of Human WTI Peptides to Human HLA B 5201 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 191 QQYSVPPPV (SEQ 100.000 NO: 171) 2 32 AQWAPVLDF (SEQ 30.000 ID NO:37) 3 243 LGATLKGVA (SEQ 16.500 NO:133) 4 303 VPGVAPTLV (SEQ 13.500 NO:242) 86 EQCLSAFTV (SEQ ID, 12.000 6 295 RGIQDVRRV (SEQ 10.000 NO: 179) 7 98 GQFTGTAGA (SEQ 8.250 NO:99) 8 292 GVFRGIQDV (SEQ 8.250 NO: 103) 9 29 SGAAQWAPV (SEQ 6.000 NO:21 1) 146 NQGYSTVTF (SEQ 5.500 NO: 150) 11 20 GGGGCALPV (SEQ 5.000 NO:92)__ 12 239 NQMNLGATL (SEQ 4.000 IDNO: 151)__ 13 64 PPPPPHSFI (SEQ ID) 3.600 14 273 SDNHTTPIL (SEQ ID 3.300 286 YRIHTHGVF (SEQ ID 3.000 16 269 TGYESDNHT (SEQ 3.000 NO:225) 17 406 TGKTSEKPF (SEQ ID 2.750 NO:222) 18 327 YPGCNKRYF (SEQ 2.750 IDNO:250) 19 7 DLNALLPAV (SEQ 2.640 104 AGACRYGPF (SEQ 2.500 ID NO:3 1) Table XXIX Results of BIMAS HLA Peptide Binding Prediction Analysis for Bindiny- of Human WTI Peptides to Human HLA B 5801 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 230 TSQLECMTW (SEQ 96.800 NO:234) 2 92 FTVHFSGQF (SEQ ID 60.000 3 120 ASSGQARMF (SEQ 40.000 4 168 AAQFPNI-SF (SEQ 20.000 IDNO:29) 408 KTSEKPFSC (SEQ ID 12.000 NO: 129)__ 6 394 RSDHLKTHT (SEQ 9.900 ID NO: 192) 7 276 HTTPILCGA (SEQ ID 7.200 NO: 115)__ 8 218 RTPYSSDNL (SEQ ID 6.600 NO:194) 9 152 VTFDGTPSY (SEQ ID 6.000 NO 40 FAPPGAS-AY (SEQ 6.000 ID NO:74) 11 213 QALLLRTPY (SEQ ID 4.500 12 347 HTGEKPYQC (SEQ 4.400 NO: 112) 13 252 AGSSSSVKW (SEQ 4.400 NO:32) 14 211 GSQALLLRT (SEQ ID 4.356 102) 174 HSFKHEDPM (SEQ 4.000 ID NO: I 16 317 TSEKRPFMC (SEQ 4.000 NO :233) 17 26 LPVSGAAQw (SEQ 4.000 NO: 138) 18 289 HTHGVFRGI (SEQ ID 3.600 19 222 SSDNLYQMT (SEQ 3.300 NO:2 17) 96 FSGQFTGTA (SEQ ID 3.300 Table XX Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WTrI Peptides to Human HLA CW0301 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 10 ALLPAVPSL (SEQ ID 100.000 2 2 KRYFKLSHL (SEQ 48.000 NO: 127) 3 126 RMFPNAPYL (SEQ 36.000 185)__ 4 3 SDVRDLNAL (SEQ 30.000 NO:206) 239 NQMNLGATL (SEQ 24.000 15 1) 6 225 NLYQMTSQL (SEQ 24.000 NO: 147) 7 180 DPMGQQGSL (SEQ 20.000 8 362 RRFSRSDQL (SEQ ID- 12.000 NO: 187) 9 329 GCNKRYFKL (SEQ 10.000 286 YRIHTHGVF (SEQ ID 10.000 11 301 RRVPGVAPT (SEQ 10.000 NO: 189) 12 24 CALPVSGAA (SEQ 10.000 NO:43)__ 13 136 SCLESQPAI (SEQ ID 7.500 198) 14 437 MHQRNMTKL (SEQ 7.200 ID NO: 143) 390 RKFSRSDHL (SEQ ID 6.000 NO: 183) 16 423 KKFARSDEL (SEQ 6.000 NO: 122) 17 92 FTVHFSGQF (SEQ ID 5.000 18 429 DELVRHHNM (SEQ 5.000 ~~ID 19 130 NAPYLPSCL (SEQ ID) 4.800 30 GAAQWAPVL (SEQ 4.000 ID NO:86) Table XXXI Results of BIMAS HLA Pe~tide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA CW0401 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 356 DFKDCERRF (SEQ 120.000 ID 2 334 YFKLSHLQM (SEQ 100.000 ID 3 180 DPMGQQGSL (SEQ 88.000 NO:59) 4 163 TPSHH.AAQF (SEQ 52.800 327 YPGCNKRYF (SEQ 40.000 NO:250) 6 285 QYRIHTHGV (SEQ 27.500 NO: 175) 7 424 KFARSDELV (SEQ 25.000 NO: 119) 8 326 AYPGCNKRY (SEQ 25.000 NO:42)__ 9 192 QYSVPPPVY (SEQ 25.000 176) 417 RWPSCQKKF (SEQ 22.000 NO: 11 278 TPILCGAQY (SEQ ID 12.000 12 10 ALLPAVPSL (SEQ ID 11.616 13 141 QPAIRNQGY (SEQ 11.000 NO: 170) 14 303 VPGVAPTLV (SEQ 11.000 ~~ID 219 TPYSSDNLY (SEQ ID 10.000 1) 16 39 DFAPPGASA (SEQ 7.920 NO:54) 17 99 QFTGTAGAC (SEQ 6.000 NO: 165) 18 4 DVRDLNALL (SEQ 5.760 NO:62) 19 70 SFIKQEPSW (SEQ ID 5.500 NO:21 0) 63 PPPPPPHSF (SEQ ID 5.280 Table XXXII Results of BIMAS HLA Peptide Binding Prediction Analysis for Bindinp, of Human W71 Peptides to Human HLA CW0602 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 332 KRYFKLSHL (SEQ 9.680 ID NO: 127)__ 2 239 NQMNLGATL (SEQ 6.600 ID NO: 151) 3 130 NAPYLPSCL (SEQ ID 6.600 NO: 144) 4 7 DLNALLPAV (SEQ 6.000 ID NO: 58) 441 NMTKLQLAL (SEQ 6.000 ID NO: 149) 6 225 NLYQMTSQL (SEQ 6.000 ID NO: 147) 74 DVRDLN ALL (SEQ 6.000 NO:62) 8 3 SDVRDLNAL (SEQ 4.400 NO:206) 9 10 ALLPAVPSL (SEQ ID 4.000 213 QALLLRTPY (SEQ ID 3.300 NO: 160)__ 11 319 EKRPFMCAY (SEQ 3.000 12 30 GAAQWAPVL (SEQ 2.200 13 242 NLGATLKGV (SEQ 2.200 ID NO: 146) 14 292 GVFRGIQDV (SEQ 2.200 NO: 103) 207 DSCTGSQAL (SEQ 2.200 NO:61)__ 16 362 RRFSRSDQL (SEQ ID 2.200 187)__ 17 439 QRNMTKLQL (SEQ 2.200 NO: 173)__ 18 295 RGIQDVRRV (SEQ 2.200 ID NO: 179) 19 423 KKFARSDEL (SEQ 2.200 ID NO: 122) 180 DPMGQQGSL (SEQ 2.200 Table XXXIII Results of BIMAS HLA Peptide Bindingz Prediction Analysis for Bindinp, of Human WTI Pep~tides to Human HLA CW0702 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 319 EKRPFMCAY (SEQ 26.880 NO:67)__ 2 326 AYPGCNKRY (SEQ 24.000 NO:42)__ 3 40 FAPPGASAY (SEQ 14.784 NO:74) 4 192 QYSVPPPVY (SEQ 12.000 176) 278 TPILCGAQY (SEQ ID 12.000 6 219 TPYSSDNLY (SEQ ID 12.000 NO:23 7 213 QALLLRTPY (SEQ ID 8.800 NO: 160) 8 125 ARMFPNAPY (SEQ 8.000 ~~ID NO:38) 9 327 YPGCNKRYF (SEQ 6.600 NO:250) 152 VTFDGTPSY (SEQ ID 5.600 11 141 QPAIRNQGY (SEQ 4.800 NO: 170) 12 345 RKHTGEKPY (SEQ 4.000 NO: 184) 13 185 QGSLGEQQY (SEQ 4.000 NO: 166) 14 101 TGTAGACRY (SEQ 4.000 NO:224) 375 RRHTGVKPF (SEQ 4.000 ID NO:188) 16 263 GQSNHSTGY (SEQ 4.000 NO: 100) 17 163 TPSHHAAQF (SEQ 3.000 ID NO:228) 18 33 QWAPVLDFA (SEQ 2.688 NO: 19 130 NAPYLPSCL (SEQ ID 2.640 144) 84 HEEQCLSAF (SEQ ID 2.400 Table XXXIV Results of BIMAS HLA Peltide Binding Prediction Analysis for Bindinp, of Human WTI Peptides to Mouse MHC Class I Db Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 235 CMTWNQMNL (SEQ 5255.7 12 _ID NO:49) I RMFPNAPYL (SEQ ID NO: 185) 1990.800 3 221 1YSSDNLYQM (SEQ 930.000 4 228 QMTSQLECM (SEQ 33.70 1 NO: 169) 239 NQMNLGATL (SEQ 2 1.470 NOA 51) 6 441 NMTKLQLAL (SEQ 19.908 NO: 149) 7 437 MHQRNMTKL (SEQ 19.837 NO: 143) 8 136 SCLESQPAI (SEQ ID 11.177 198) 9 174 HSFKHEDPM (SEQ 10.800 NO: I10)__ 302 RVPGVAPTL (SEQ 10.088 NO: 11 130 NAPYLPSCL (SEQ ID 8.400 12 10 ALLPAVPSL (SEQ ID 5.988 13 208 SCTGSQALL (SEQ ID 4.435 14 209 CTGSQALLL (SEQ ID 3.548 NO:52) 238 WNQMNLGAT (SEQ 3.300 ID NO:245) 16 218 RTPYSSDNL (SEQ ID 3.185 194) 17 24 CALPVSGAA (SEQ 2.85 1 ID NO:43) 18 18 LGGGGGCAL (SEQ 2.177 ID NO: 134) 19 142 PAIRNQGYS (SEQ ID 2.160 NO: 152) 30 GAAQWAPVL (SEQ 1.680 1 ID NO:86) Table XXXV Results of BIMAS HLA P'eptide Binding Prediction Analysis for Binding of Human WTI Pentides to Mouse MHC Class I Dd Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 112 FGPPPPSQA (SEQ ID 48.000 NO:76) 2 122 SGQARMFPN (SEQ 36.000 ID NO:212) 3 104 AGACRYGPF (SEQ 30.000 NO:31) 4 218 RTPYSSDNL (SEQ ID 28.800 194) 130 NAPYLPSCL (SEQ ID 20.000 144) 6 302 RVPGVAYTL (SEQ 20.000 NO: 195) 7 18 LGGGGGCAL (SEQ 20.000 NO: 134) 8 81 AEPHEEQCL (SEQ ID 10.000 9 29 SGAAQWAPV (SEQ 7.200 NO:21 1) 423 KKFARSDEL (SEQ 7.200 NO: 122) 11 295 RGIQDVRRV (SEQ 7.200 NO: 179) 12 390 RKFSRSDHL (SEQ ID 6.000 183) 13 332 KRYFKLSHL (SEQ 6.000 NO: 127) 14 362 RR.FSRSDQL (SEQ ID 6.000 417 RWPSCQKKF (SEQ 6.000 NO: 196) 16 160 YGHTPSHHA (SEQ 6.000 NO:249) 17 20 GGGGCALPV (SEQ 6.000 18 329 GCNKRYFKL (SEQ 5.000 NO:90) 19 372 RHQRRHTGV (SEQ 4.500 ID NO: 18 1) 52 GGPAP P NO:93 ID 4.000 APPAP(SE3 I Table XXXVI Results of BIMAS HLA Pentide Binding Prediction Analysis for Bindinp, of Human WTI Peptides to Mouse MHC Class I Kb Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 329 GCNKCRYFKL (SEQ 24.000 ID 2 225 NLYQMTSQL (SEQ 10.000 ID NO: 147) 3 420 SCQKKFARS (SEQ 3.960 NO:200) 4 218 RTPYSSDNL (SEQ ID 3.630 NO: 194) 437 MHQRNMTKL (SEQ 3.600 IDNO: 6 387 TCQRKFSRS, (SEQ ID 3.600 7 302 RVPGVAPTL (SEQ 3.300 NO:]195) 8 130 NAPYLPSCL (SEQ ID 3.000 144) 9 289 HTHGVFRGI (SEQ ID 3.000 113) 43 PGASAYGSL (SEQ 2.400 ID NO: 153) 11 155 DGTPSYGHT (SEQ 2.400 ID NO:56) 12 273 SDNHTTPIL (SEQ ID 2.200 NO :204) 13 126 RMFPNAPYL (SEQ 2.200 ID NO: 185) 14 128 FPNAPYLPS, (SEQ ID 2.000 NO:79) 3 SDVRDLNAL (SEQ 1.584 16 207 DSCTGSQAL (SEQ 1.584 NO:61) 17 332 KRYFKLSI-L (SEQ 1.500 NO: 18 18 LGGGGGCAL (SEQ 1.320 ID NO: 134) 19 233 LECMTWNQM (SEQ 1.320 ID NO: 131) 441 NMTKLQLAL (SEQ 1.200 D NO: 149) Table XXXVII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WTI Pe.1tides to Mouse MHC Class I Kd Score (Estimate of Hlf Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 285 QYRIHTHGV (SEQ 600.000 NOA175)__ 2 424 KFARSDELV (SEQ 288.000 ID NO: 119) 3 334 YFKLSHLQM (SEQ 120.000 ~~ID 4 136 SCLESQPTI (SEQ ID 115.200 199) 239 NQMNLGATL (SEQ 115.200 ID NO: 15 6 10 ALLPAVSSL (SEQ ID 115.200 7 47 AYGSLGGPA (SEQ 86.400 ID NO:4 1) 8 180 DPMGQQGSL (SEQ 80.000 ID NO:59) 9 270 GYESDNHTA (SEQ 72.000 IDNO: 105) 326 AYPGCNKRY (SEQ 60.000 11 192 QYSVPPPVY (SEQ 60.000 ID NO: 176) 12 272 ESDNHTAPI (SEQ ID, 57.600 13 289 HTHGVFRGI (SEQ ID 57.600 113) 14 126 DVRDLNALL (SEQ 57.600 NO:62) 4 CTGSQALLL (SEQ ID 57.600 :52) 16 208 SCTGSQALL (SEQ ID 48.000 17 441 NMTKLQLAL (SEQ 48.000 NO: 149) 18 207 DSCTGSQAL (SEQ 48.000 ID NO:61) 19 130 NAPYLPSCL (SEQ ID 48.000 Table XXXVIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WTI Peptides to Mouse MHC Class I Kk Score (Estimate of Half Time of Subsequence Residue. Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 81 AEPHEEQCL (SEQ ID 40.000 ~~NO:30) 2 85 EEQCLSAFT (SEQ ID 40.000 3 429 DEL VRHHNM (SEQ 20.000 4 315 SETSEKRPF (SEQ ID 20.000 261 TEGQSNHST (SEQ ID 20.000 6 410 SEKPFSCRW (SEQ 10.000 NO:207) 7 272 ESDNHTTPI (SEQ ID 10.000 NO:71) 8 318 SEKRPFMCA (SEQ 10.000 ID NO:208) 9 138 LESQPAIRN (SEQ ID 10.000 NO: 132)__ 233 LECMTWNQM (SEQ 10.000 NO: 13 11 298 QDVRRVPGV (SEQ 10.000 NO: 164) 12 84 HEEQCLSAF (SEQ ID 10.000 107) 13 349 GEKPYQCDF (SEQ 10.000 NO:91) 14 289 HTHGVFRGI (SEQ ID 10.000 113) 179 EDPMGQQGS (SEQ 8.000 16 136 SCLESQPAI (SEQ ID 5.000 198) 17 280 ILCGAQYRI (SEQ ID 5.000 NO: 116) 18 273 SDNHITPIL (SEQ ID 4.000 19 428 SDELVRHHN (SEQ 4.000 IDNO:203) 3 SDVRDLNAL (SEQ 4.000 Table XXXIX Results of BIMAS HLA Peltide-Binding Prediction Analysis for Bindin of Human WTI Peptides to Mouse MHC Class I Ld Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 163 TPSHHAAQF (SEQ 360.000 NO:228) 2 327 YPGCNKRYF (SEQ 300.000 ID NO:250) 3 180 DPMGQQGSL (SEQ 150.000 NO:59) 4 26 LPVSGAAQW (SEQ 93.600 ~~~IDNO: 138) 278 TPILCGAQY (SEQ ID 72.000 6 141 QPAIRNQGY (SEQ 60.000 NO: 170) 7 219 TPYSSDNLY (SEQ ID 60.000 1) 8 303 VPGVAPTLV (SEQ 60.000 ID NO:242) 9 120 ASSGQARMF (SEQ 50.000 NO:40)__ 63 PPPPPPHSF (SEQ ID 45.000 11 113 GPPPPSQAS (SEQ ID 45.000 12 157 TPSYGHTPS (SEQ ID 39.000 13 207 DSCTGSQAL (SEQ 32.500 ID NO:61) 14 110 GPFGPPPPS (SEQ ID 30.000 NO:96) 82 EPHEEQCLS (SEQ ID 30.000 16 412 KPFSCRWPS (SEQ ID 123) 17 418 WPSCQKKFA (SEQ ID NO:246) 18 221 YSSDNLYQM (SEQ 19 204 TPTDSCTGS (SEQ ID 3 0.000 128 FPNAPYLPS (SEQ ID 30.000 Table XL Results of BIMAS HLA Peptide Binding Prediction Analysis for Bindin of Human WTI Peptides to Cattle HLA Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 350 EKPYQCDFK (SEQ 1000.00 NO:66) 2 319 EKRPFMCAY (SEQ 500.000 NO:67) 3 423 KKFARSDEL (SEQ 500.000 ID NO: 122) 4 345 RKHTGEKPY (SEQ 5 00.000 NO: 184) 390 RKFSRSDHL (SEQID 5 00.000 183) 6 137 CLESQPAIR (SEQ ID 120.000 7 380 VKPFQCKTC (SEQ 100.000 NO:239)__ 8 407 GKTSEKPFS (SEQ ID 100.000 9 335 FKLSHLQMH (SEQ 100.000 NO:78) 247 LKGVAAGSS (SEQ 100.000 NO: 13 11 370 LKRJ-QRRI-T (SEQ 100.000 IDNO: 12 258 VKWTEGQSN (SEQ 100.000 13 398 LKTHTRTHT (SEQ 100.000 NO: 137) 14 331 NKRYFKLSH (SEQ 100.000 NO: 145) 357 FKDCERRFS (SEQ ID 100.000 16 385 CKTCQRKFS (SEQ 100.000 NO:46) 17 294 FRGIQDVRR (SEQ ID 80.000 NO:81) 18 368 DQLKR.HQRR (SEQ 80.000 ID 19 432 VRHHNMHQR (SEQ 80.000 1 118 j SQASSGQAR (SEQ 80.000 16) Table XLI Results of BIMAS HLA Pevtide Binding Prediction Analysis for Bindingz of Mouse WTI Peptides to Mouse MHC Class I A 0201 Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence), 1 126 RMFPNAPYL (SEQ 313.968 ID NO:293) 2 187 SLQEQQYSV (SEQ 285.163 NO:299) 3 10 ALLPAVSSL (SEQ ID 181.794 4 225 NLYQMTSQL (SEQ 68.360 NO:284) 292 GVFRGIQDV (SEQ 5 1.790 ID NO:270) 6 93 TLHFSGQFT (SEQ ID 40.986 7 191 QQYSVPPPV (SEQ 22.566 NO:290) 8 280 ILCGAQYRI (SEQ ID 17.736 9 441 NMTKLHVAL (SEQ 15.428 NO:285) 235 CMTWNQMNL (SEQ 15.428 11 7 DLNALLPAV (SEQ-7 11.998 NO:26 1) 12 242 NLGATLKGM (SEQ 11.426 13 227 YQMTSQLEC (SEQ 8.573 NO:307) 14 239 NQMNLGATL (SEQ 8.014 ID NO:286) 309 TLVRSASET (SEQ ID 7.452 NO:303) 16 408 KTSEKPFSC (SEQ ID 5.743 17 340 LQMHSRKHT (SEQ 4.75 2 NO:280) 18 228 QMTSQLECM (SEQ 4.044 IDNO:289) 19 37 VLDFAPPGA (SEQ 3.378 NO:304)__ 302 RVSGVAPTL (SEQ 1.869 IDNO:295) Table XLII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Mouse WTI Peotides to Mouse MHC Class I Db* Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 221 YSSDNLYQM (SEQ 3 12.000 ID NO:308) 2 126 RMFPNAPYL (SEQ 260.000 3 235 CMTWNQM NL (SEQ 260.000 ID NO:258) 4 437 MHQRNMTKL (SEQ 200.000 ID NO:28 1) 238 WNQfvfNLGAT (SEQ 12.000 NO:305) 6 130 NAPYLPSCL (SEQ ID 8.580 7 3 SDVRDLNAL (SEQ 7.920 ID NO:298) 8 136 SCLESQPTI (SEQ ID 7.920 9 81 AEPHEEQCL (SEQ IDI 6.600 10 ALLPAVSSL (SEQ ID 6.600 NO:255) 11 218 RTPYSSDNL (SEQ ID 6.000 12 441 NMTkL HVAL (SEQ 3.432 NO:285) 13 228 QMTSQLECM (SEQ 3.120 NO:289) 14 174 HSFKHEDPM (SEQ 3.120 NO:272) 242 NLGATLKGM (SEQ 2.640 16 261 TEGQSNHGI (SEQ ID 2.640 17 225 NLYQMTSQL (SEQ 2.640 18 207 DSCTGSQAL (SEQ 2.600 NO:263)__ 19 119 QASSGQARM (SEQ 2.600 18 LGGGGGCGL (SEQ 2.600 NO:279)__ Table XLIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Bindiniz of Mouse WTI Pep~tides to Mouse MHC Class 1 Kb Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 329 GCNKRYFKL (SEQ 24.000 2 225 NLYQMTSQL (SEQ 10.000 NO:284)__ 3 420 SCQKKFARS (SEQ 3.960 IDNO:297) 4 218 RTPYSSDNL (SEQ ID 3.630 437 MHQRNMTKL (SEQ 3.600 L 6 387 TCQRKFSRS (SEQ DF 3.600 7 289 HTHGVFRGI (SEQ ID 3.000 ~~NO:273) 8 130 NAPYLPSCL (SEQ ID 3.000 9 43 PGASAYGSL (SEQ 2.400 NO:2 87) 155 DGAPSYGHT (SEQ 2.400 _____IDNO:260) 11 126 RMFPNAPYL (SEQ 2.200 NO:293) 12 128 FPNAPYLPS (SEQ ID 2.000 NO:267) 13 207 DSCTGSQAL (SEQ 1.584 NO:263)) 14 3 SDVRDLNAL (SEQ 1.584 NO:298) 332 KRYFKLSHL (SEQ 1.500 NO:276) 16 233 LECMTWNQM (SEQ 1.320 NO:278) 17 18 LGGGGGCGL (SEQ 1.320 NO:279) 18 242 NLGATLKGM (SEQ 1.200 NO:283) 19 123 GQARMFPN (SEQ ID 1.200 441 NMTKLHVAL (SEQ 1.200 Table XLIV Results of BIMAS HLA Peptide Binding! Prediction Analysis for Bindingz of Mouse WTI Peptides to Mouse MHC Class I Kd Score (Estimate of Half Time of Subsequence Residue Disassociation of a Molecule Rank Start Position Listing Containing This Subsequence) 1 285 QYRIHTHGV (SEQ 600.000 ~~ID NO:291) 2 424 KFARSDELV (SEQ 288.000 _____IDNO:275) 3 334 fYFKLSHLQM (SQ120.000 NO:306)__ 4 136 SCLESQPTI (SEQ ID 115.200 239 NQMNLGATL (SEQ 115.200 NO:286) 6 10 ALLPAVSSL (SEQ ID 115.200 7 47 AYGSLGGPA (SEQ 86.400 NO:256) 8 180 DPMGQQGSL (SEQ 80.000 NO:262) 9 270 GYESDNHTA (SEQ 72.000 ID NO:27 1) 192 QYSVPPPVY (SEQ 60.000 NO:292) 11 326 AYPGCNKRY (SEQ 60.000 12 289 HTHGVFRGI (SEQ ID 57.600 13 4 DVRDLNALL (SEQ 57.600 NO:264)__ 14 126 RMFPNAPYL (SEQ 5 7.600 ID NO:293) 209 CTGSQALLL (SEQ ID 48.000 16 86 EQCLSAFTL (SEQ ID 48.000 17 302 RVSGVAPTL (SEQ 48.000 NO :295) 18 218 RTPYSSDNL (SEQ ID 48.000 19 272 ESDNHTAPI (SEQ ID 48.000 225 NLYQMTSQL (SEQ 48.000 _____IDNO:284) Table XLV Results of TSites Pelptide Binding Prediction Analysis for Human WTI Peptides Capable of Eliciting a Helper T cell Response Peptide fSequence p6-23 RDLNALLPAVPSLGGGG (SEQ ID NO: 1) p30-35 GAAQWA (SEQ ID NO:309) p 4 5 -56 ASAYGSLGGPAP (SEQ ID NO:3 p91-105 IAFTVHFSGQFTGTAG (SEQ ID NO:31 1) p 117-13 9 PSQASSGQARMFPNAPYLPSCLE (SEQ ID NO:2) p1.67-171 HAAQF (SEQ ID NO:312) p202-233 CHTPTDSCTGSQALLLRTPYSSDNLYQMTSQL (SEQ ID NO:3 13) p244-262 GATLKGVAAGSSSSVKWTE (SEQ ID NO:4) p287-318 RIHTHGVFRGIQDVRRVPGVAPTLVRSASETS (SEQ ID NO:3 14) p333-336 RYFK (SEQ ID NO:3 p361-374 ERRFSRSDQLKRHQ (SEQ ID NO:3 16) p389-410 QRKFSRSDHLKTHTRTHTGKTS (SEQ ID NO:3 17) p421-441 ICQKKFARSDELVRHHNMHQRN (SEQ ID NO:3 18) Certain CTL peptides (shown in Table XLVI) were selected for further study. For each peptide in Table XLVI, scores obtained using BIMAS HLA peptide binding prediction analysis are provided.

Table XLVI ATM Peptide Sequences and HLA Peptide Binding~ Predictions Peptidle Sequence Comments p329-337 GCNKRYFKL Score 24,000 (SEQ ID NOs: 90 and 268) p225-233 NLYQMTSQL binds also to class II and HLA A2, Kd, (SEQ ID NOs: 147 and score 10,000 284)__ p235-243 CMTWNQMfNL binds also to HLA A2, score 5,255,712 (SEQ ID NOs: 49 and 258) p126-134 RMFPNAPYL binds also to Kd, class 11 and HLA A2, (SEQ ID NOs: 185 and score 1,990,800 293) p221-229 YSSDNLYQM binds also to Ld, score 312,000 (SEQ ID NOs: 253 and 308) p228-236 QMTSQLECM score 3,120 (SEQ ID NOs: 169 and 289) p239-247 NQMNLGATL binds also to HLA A 0201, Kd, score (SEQ ID NOs: 151 and 8,015 286) mouse p 13 6-144 SCLESQPTI binds also to Kd, I mismatch to human ID NO:296) human p136-144 SCLESQPAI score 7,920 (SEQ ID NO:198) mouse p10-18 ALLPAVSSL binds also to Kd, HLA A2, I mismatch (SEQ ID NO:255) to human human pl0-18 ALLPAVPSL score 6,600 (SEQ ID NO:34) Peptide binding to C57B1/6 murine MHC was confirmed using the leukemia cell line RMA-S, as described by Ljunggren et al., Nature 346:476-480, 1990.

In brief, RMA-S cells were cultured for 7 hours at 26 0 C in complete medium supplemented with 1% FCS. A total of 106 RMA-S cells were added into each well of a 24-well plate and incubated either alone or with the designated peptide (25ug/ml) for 16 hours at 26°C and additional 3 hours at 37 0 C in complete medium. Cells were then washed three times and stained with fluorescein isothiocyanate-conjugated anti Db or anti-Kb antibody (PharMingen, San Diego, CA). Labeled cells were washed twice, resuspended and fixed in 500ul of PBS with 1% paraformaldehyde and analyzed for fluorescence intensity in a flow cytometer (Becton-Dickinson FACSCalibur®). The percentage of increase of Db or Kb molecules on the surface of the RMA-S cells was measured by increased mean fluorescent intensity of cells incubated with peptide compared with that of cells incubated in medium alone.

Mice were immunized with the peptides capable of binding to murine class I MHC. Following immunization, spleen cells were stimulated in vitro and tested for the ability to lyse targets incubated with WTI peptides. CTL were evaluated with a standard chromium release assay (Chen et al., Cancer Res. 54:1065-1070, 1994). 10 6 target cells were incubated at 37°C with 1501tCi of sodium "Cr for 90 minutes, in the presence or absence of specific peptides. Cells were washed three times and resuspended in RPMI with 5% fetal bovine serum. For the assay, 104 Cr-labeled target cells were incubated with different concentrations of effector cells in a final volume of 2 00tl in U-bottomed 96-well plates. Supernatants were removed after 4 to 7 hours at 37 0 C, and the percentage specific lysis was determined by the formula: specific lysis 100 x (experimental release spontaneous release)/(maximum release-spontaneous release).

100 The results, presented in Table XLVII, show that some WT1 peptides can bind to class I MHC molecules, which is essential for generating CTL. Moreover, several of the peptides were able to elicit peptide specific CTL (Figures 9A and 9B), as determined using chromium release assays. Following immunization to CTL peptides pl0-18 human, p136-144 human, p136-144 mouse and p235-243, peptide specific CTL lines were generated and clones were established. These results indicate that peptide specific CTL can kill malignant cells expressing WT1.

Table XLVII Binding of WTI CTL Peptides to mouse B6 class I antigens Peptide Binding Affinity to Mouse MHC Class I Positive control 91% negative control 0.5.-1.3% p235-243 33.6% p136-144 mouse 27.9% p136-144 human 52% p10-18: human 2.2% p225-233 5.8% p329-337 1.2% p126-134 0.9% p221-229 0.8% p228-236 1.2% p239-247 1% Example Use ofa WTI Polvoentide to Elicit WT1 Specific CTL in Mice This Example illustrates the ability of a representative WTI polypeptide to elicit CTL immunity capable of killing WTI positive tumor cell lines.

P117-139, a peptide with motifs appropriate for binding to class I and class II MHC, was identified as described above using TSITES and BIMAS HLA peptide binding prediction analyses. Mice were immunized as described in Example 3.

Following immunization, spleen cells were stimulated in vitro and tested for the ability to lyse targets incubated with WTI peptides, as well as WTI positive and negative tumor cells. CTL were evaluated with a standard chromium release assay. The results, presented in Figures 10A-10D, show that P117 can elicit WTI specific CTL capable of killing WTI positive tumor cells, whereas no killing of WTI negative cells was observed. These results demonstrate that peptide specific CTL in fact kill malignant cells expressing WT1 and that vaccine and T cell therapy are effective against malignancies that express WTI.

Similar immunizations were performed using the 9-mer class I MHC binding peptides p136-144, p225-233, p 2 3 5 2 4 3 as well as the 23-mer peptide p 1 1 7- 139. Following immunization, spleen cells were stimulated in vitro with each of the 4 peptides and tested for ability to lyse targets incubated with WTI peptides. CTL were generated specific for p136-144, p235-243 and p117-139, but not for p225-233. CTL data for p235-243 and p117-139 are presented in Figures 11A and 11B. Data for peptides p136-144 and p225-233 are not depicted.

CTL lysis demands that the target WTI peptides are endogenously processed and presented in association with tumor cell class I MHC molecules. The above WTI peptide specific CTL were tested for ability to lyse WTI positive versus negative tumor cell lines. CTL specific for p235-243 lysed targets incubated with the p235-243 peptides, but failed to lyse cell lines that expressed WTI proteins (Figure S11A). By marked contrast, CTL specific for p 17-139 lysed targets incubated with pl 17-139 peptides and also lysed malignant cells expressing WT1 (Figure 11B). As a negative control, CTL specific for p 1 17-139 did not lyse WTI negative EL-4 (also referred to herein as Specificity of WT specific lysis was confirmed by cold target inhibition (Figures 12A-12B). Effector cells were plated for various effector: target ratios in 96well U-bottom plates. A ten-fold excess (compared to hot target) of the indicated peptide-coated target without 5 Cr labeling was added. Finally, 104 "'Cr-labeled target cells per well were added and the plates incubated at 37 0 C for 4 hours. The total volume per well was 200pl.

Lysis of TRAMP-C by p 17-139 specific CTL was blocked from 58% to 36% by EL-4 incubated with the relevant peptide p117-139, but not with EL-4 incubated with an irrelevant peptide (Figure 12A). Similarly, lysis of BLK-SV40 was blocked from 18% to 0% by EL-4 incubated with the relevant peptide p117-139 (Figure 12B). Results validate that WTI peptide specific CTL specifically kill malignant cells by recognition of processed WT1.

Several segments with putative CTL motifs are contained within p 1 17- 139. To determine the precise sequence of the CTL epitope all potential 9-mer peptides within p117-139 were synthesized (Table XLVIII). Two of these peptides (p126-134 and p130-138) were shown to bind to H-2b class I molecules (Table XLVIII). CTL generated by immunization with p 17-139 lysed targets incubated with p126-134 and p130-138, but not the other 9-mer peptides within p117-139 (Figure 13A).

The p117-139 specific CTL line was restimulated with either p126-134 or p130-138. Following restimulation with p126-134 or p130-138, both T cell lines demonstrated:peptide specific lysis, but only p130-138 specific CTL shwed lysis of a WTI positive tumor cell line (Figures 13B and 13C). Thus, p130-138 appears to be the naturally processed epitope.

Table XLVIII Binding of WT1 CTL 9mer Peptides within pl 17-139 to mouse B6 class I antigens Peptide Binding Affinity to Mouse MHC Class I P117-125 PSQASSGQA (SEQ ID 2% NO:221) P118-126 SQASSGQAR (SEQ ID 2% NO:216) P119-127 QASSGQARM (SEQ ID 2% NOs: 161 and 288) P120-128 ASSGQARMF (SEQ ID 1% P121-129 SSGQARMFP (SEQ ID 1% NO:222) P122-130 SGQARMFPN (SEQ ID 1% NO:212) P123-131 GQARMFPNA (SEQ ID 1% NOs: 98 and 269) P124-132 QARMFPNAP (SEQ ID 1% NO:223) P125-133 ARMFPNAPY (SEQ ID 1% NO:38) P126-134 RMFPNAPYL (SEQ ID 79% NOs: 185 and 293) P127-135 MFPNAPYLP (SEQ ID 2% NO:224) P128-136 FPNAPYLPS (SEQ ID 1% NOs: 79 and 267) P129-137 PNAPYLPSC (SEQ ID 1% NO:225) P130-138 NAPYLPSCL (SEQ ID 79% NOs: 144 and 282) P131-139 APYLPSCLE (SEQ ID 1% NO:226) Example 6 Identification of WTI Specific mRNA in Mouse Tumor Cell Lines This Example illustrates the use of RT-PCR to detect WTI specific mRNA in cells and cell lines.

Mononuclear cells were isolated by density gradient centrifugation, and were immediately frozen and stored at -800C until analyzed by RT-PCR for the presence of WTI specific mRNA. RT-PCR was generally performed as described by Fraizer et al., Blood 86:4704-4706, 1995. Total RNA was extracted from 107 cells according to standard procedures. RNA pellets were resuspended in 25 gL diethylpyrocarbonate treated water and used directly for reverse transcription. The zincfinger region (exons 7 to 10) was amplified by PCR as a 330 bp mouse cDNA.

Amplification was performed in a thermocycler during one or, when necessary, two sequential rounds of PCR. AmpliTaq DNA Polymerase (Perkin Elmer Cetus, Norwalk, CT), 2.5 mM MgCl, and 20 pmol of each primer in a total reaction volume of 501tl were used. Twenty JtL aliquots of the PCR products were electrophoresed on 2% agarose gels stained with ethidium bromide. The gels were photographed with Polaroid film (Polaroid 667, Polaroid Ltd., Hertfordshire, England). Precautions against cross contamination were taken following the recommendations of Kwok and Higuchi, Nature 339:237-238, 1989. Negative controls included the cDNA- and PCR-reagent mixes with water instead of cDNA in each experiment. To avoid false negatives, the presence of intact RNA and adequate cDNA generation was evaluated for each sample by a control PCR using p-actin primers. Samples that did not amplify with these primers were excluded from analysis.

Primers for amplification of WT1 in mouse cell lines were: P115: 1458- 1478: 5' CCC AGG CTG CAA TAA GAG ATA 3' (forward primer; SEQ ID NO:21); and P116: 1767-1787: 5' ATG TTG TGA TGG CGG ACC AAT 3' (reverse primer; SEQ ID NO:22) (see Inoue et al, Blood 88:2267-2278, 1996; Fraizer et al., Blood 86:4704-4706, 1995).

Beta Actin primers used in the control reactions were: 5' GTG GGG CGC CCC AGG CAC CA 3' (sense primer; SEQ ID NO:23); and 5' GTC CTT AAT GTC ACG CAC GAT TTC 3' (antisense primer; SEQ ID NO:24) Primers for use in amplifying human WTI include: P117: 954-974: GGC ATC TGA GAC CAG TGA GAA 3' (SEQ ID NO:25); and P118: 1434-1414: GAG AGT CAG ACT TGA AAG CAGT 3' (SEQ ID NO:5). For nested RT-PCR, primers may be: P119: 1023-1043: 5' GCT GTC CCA CTT ACA GAT GCA 3' (SEQ ID NO:26); and P120: 1345-1365: 5' TCA AAG CGC CAG CTG GAG TTT 3' (SEQ ID NO:27).

Table XLVIII shows the results of WTI PCR analysis of mouse tumor cell lines. Within Table IV, indicates a strong WT I PCR amplification product in the first step RT PCR, indicates a WT1 amplification product that is detectable by first step WTI RT PCR, indicates a product that is detectable only in the second step of WTI RT PCR, and indicates WTI PCR negative.

Table XLIX Detection of WTI mRNA in Mouse Tumor Cell Lines Cell Line WT1 mRNA K562 (human leukemia; ATCC): Positive control; (Lozzio and Lozzio, Blood 45:321-334, 1975) TRAMPC (SV40 transformed prostate, B6); Foster et al., Cancer Res. 57:3325-3330, 1997 HD2 (SV40-transf. fibroblast, B6; ATCC); Nature 276:510-511, 1978 CTLL (T-cell, B6; ATCC); Gillis, Nature 268:154-156, 1977) FM (FBL-3 subline, leukemia, B6); Glynn and Fefer, Cancer Res. 28:434-439, 1968 BALB 3T3 (ATCC); Aaroston and Todaro, J. Cell. Physiol. 72:141-148, 1968 S49.1 (Lymphoma, T-cell like, B/C; ATCC); Horibata and Harris, Exp. Cell. Res. 60:61, 1970 BNL CL.2 (embryonic liver, B/C; ATCC); Nature 276:510-511, 1978 MethA (sarcoma, Old et al., Ann. NY Acad Sci. 101:80- 106, 1962 P3.6.2.8.1 (myeloma, B/C; ATCC); Proc. Natl. Acad Sci. USA 66:344. 1970 P2N (leukemia, DBA/2; ATCC); Melling et al., J Immunol.

117:1267-1274, 1976 BCL1 (lymphoma, B/C; ATCC); Slavin and Strober, Nature 272:624-626, 1977 LSTRA (lymphoma, Glynn et al., Cancer Res. 28:434- 439, 1968 E1I/EL-4 (lymphoma, B6); Glynn et al., Cancer Res. 28:434- 439, 1968 From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as. an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Claims (10)

1. A polypeptide wherein the polypeptide is an immunogenic portion of a native WT1 polypeptide and consists essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144.

2. A polypeptide according to claim 1, wherein the polypeptide consists of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144.

3. A pharmaceutical composition comprising a polypeptide according to claim 1, in combination with a pharmaceutically acceptable carrier or excipient.

4. A vaccine comprising a polypeptide according to claim 1, in combination with a non-specific immune response enhancer. A vaccine according to claim 4, wherein the immune response enhancer is an adjuvant.

6. A vaccine comprising: a WT1 polypeptide, wherein the polypeptide is an immunogenic portion of a native WTI polypeptide and consists essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and a non-specific immune response enhancer that preferentially enhances a T- cell response in a patient.

7. A vaccine according to claim 6, wherein the immune response enhancer is selected from the group consisting of Montanide ISA50, Seppic MONTANIDE ISA 720, cytokines GM-CSF, Flat3-ligand), microspheres, dimethyl dioctadecyl ammonium bromide (DDA) based adjuvants, AS-1, AS-2, Ribi Adjuvant system based adjuvants, QS21, saponin based adjuvants, Syntex adjuvant in its microfluidized form, MV, ddMV, immune stimulating complex (iscom) based adjuvants and inactivated toxins. P QOPERHPMCom- Corp-1-n1' I2 I QO(cded cioms doc.2A12IM7

107- 8. A polynucleotide encoding a polypeptide according to claim 1. 9. A pharmaceutical composition comprising a polynucleotide encoding a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144. A pharmaceutical composition comprising: a T-cell that specifically reacts with a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and a pharmaceutically acceptable carrier or excipient. 11. A pharmaceutical composition comprising: an antigen presenting cell that expresses a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and a pharmaceutically acceptable carrier or excipient. 12. A vaccine comprising a polynucleotide encoding a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144. 13. A vaccine comprising: an antigen presenting cell that expresses a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and a pharmaceutically acceptable carrier or excipient. 14. A vaccine according to any one of claims 12 to 13, wherein the immune response enhancer preferentially enhances a T-cell response in a patient. A vaccine according to any one of claims 12 to 13, wherein the immune response enhancer preferentially enhances a T-cell response in a patient. 16. A method for enhancing or inducing an immune response in a human patient, P 1OPER\HPM\Comxa Corp oauo.,\12162190' doe cla- d.2LRn/2(X)7

108- comprising administering to a patient a pharmaceutical composition comprising: a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and a physiologically acceptable carrier or excipient. 17. A method for enhancing or inducing an immune response in a human patient, comprising administering to a patient a pharmaceutical composition according to any one of claims 3, 9, 10 or 11. 18. A method for enhancing or inducing an immune response in a human patient, comprising administering to a patient a vaccine comprising: a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and a non-specific immune response enhancer. 19. A method for enhancing or inducing an immune response in a human patient, comprising administering to a patient a vaccine according to any one of claims 4, 6, 12 or 13, and thereby enhancing or inducing an immune response specific for WTI or a cell expressing WT1 in a patient. A method for inhibiting the development of malignant disease associated with WTI expression in a human patient, comprising administering to a human patient a pharmaceutical composition comprising: a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO: 144; and a physiologically acceptable carrier or excipient; and thereby inhibiting the development of a malignant disease associated with WTI expression in the human patient. 21. A method for inhibiting the development of malignant disease associated with WTI expression in a human patient, comprising administering to a human patient a P %OPER'IIPAWCom.l C I ,,,cded dau,, doc.21/207

109- pharmaceutical composition according to any one of claims 3, 9, 10 or 11, and thereby inhibiting the development of a malignant disease in the patient. 22. A method for inhibiting the development of malignant disease associated with WT1 expression in a human patient, comprising administering to a human patient a vaccine comprising: a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO: 144; and a non-specific immune response enhancer; and thereby inhibiting the development of a malignant disease in the patient. 23. A method for inhibiting the development of a malignant disease associated with WTI expression in a patient, comprising administering to a patient a vaccine according to any one of claims 4, 6, 12 or 13, and thereby inhibiting the development of a malignant disease in the patient. 24. A method according to claim 20 or 22, wherein the malignant disease is a leukemia. A method according to claim 24, wherein the leukaemia is acute myeloid leukemia, acute lymphocytic leukemia, or chronic myeloid leukemia. 26. A method according to claim 20 or 22, wherein the malignant disease is a cancer. 27. A method according to claim 26, wherein the cancer is breast, lung, thyroid or gastrointestinal cancer. 28. A method according to claim 21, wherein the malignant disease is a leukemia. 29. A method according to claim 28, wherein the leukemia is acute myeloid leukemia, acute lymphocytic leukemia, or chronic myeloid leukemia. P iOPERHPMWCo-rx CopaioW2 362904 1 mtdc clans dom.2A112(007 -110- A method according to claim 21, wherein the malignant disease is a cancer. 31. A method according to claim 30, wherein the cancer is breast, lung, thyroid or gastrointestinal cancer. 32. A method according to claim 23, wherein the malignant disease is a leukemia. 33. A method according to claim 32, wherein the leukemia is acute myeloid leukemia, acute lymphocytic leukemia, or chronic myeloid leukemia. 34. A method according to claim 23, wherein the malignant disease is a cancer. A method according to claim 34, wherein the cancer is breast, lung, thyroid or gastrointestinal cancer. 36. A method for removing cells expressing WTI from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood, comprising contacting bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood with T-cells that specifically react with a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144, wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of WT1 positive cells to less than of the number of myeloid or lymphatic cells in the bone marrow, peripheral blood, or fraction of bone marrow or peripheral blood. 37. A method for inhibiting the development of a malignant disease associated with WTI expression in a patient, comprising administering to a patient bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood prepared according to the method of claim 36. 38. A method according to claim 37, wherein the bone marrow, peripheral blood or P %OPERU{PMC- Copm.at.MI 2312 I 362Y ded .1n. doc-2A)i2M7 111 fraction is autologous. 39. A method according to claim 37, wherein the bone marrow, peripheral blood or fraction is syngeneic or allogenic. A method for stimulating or expanding T-cells, comprising contacting T-cells with: a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; a polynucleotide encoding a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; or an antigen presenting cell that expresses a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144, under conditions and for a time sufficient to permit the stimulation and/or expansion of T-cells. 41. A method according to claim 40, wherein the T-cells are present within bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood. 42. A method according to claim 40, wherein the bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood is obtained from a patient afflicted with a malignant disease associated with WTI expression. 43. A method according to claim 40, wherein the bone marrow, peripheral blood or a fraction of a bone marrow or peripheral blood is obtained from a mammal that is not afflicted with a malignant disease associated with WT1 expression. 44. A method according to claim 40, wherein the T-cells are cloned prior to expansion. A method for stimulating and/or expanding T-cells in a mammal, comprising administering to a mammal a pharmaceutical composition comprising: one or more of: P 1OPER~I4PM\Con' Corprl 2f,2 i20Xn ded clims dm-2A)1/2('7 -112- a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (ii) a polynucleotide encoding a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and (iii) an antigen presenting cell that expresses a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and a physiologically acceptable carrier or excipient, and thereby stimulating and/or expanding T-cells in a mammal. 46. A method for stimulating and/or expanding T-cells in a mammal, comprising administering to a mammal a vaccine comprising: one or more of: a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (ii) a polynucleotide encoding as WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (ii) an antigen presenting cell that expresses a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and a non-specific immune response enhancer, and thereby stimulating and/or expanding T-cells in a mammal. 47. A method for inhibiting the development of a malignant disease associated with WTI expression in a patient, comprising administering to a patient T-cells prepared according to the method of claim 48. A method according to claim 47, wherein the bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood is obtained from a patient afflicted with a malignant disease associated with WTI expression. 49. A method according to claim 47, wherein the bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood is obtained from a mammal that is not P 1OPERHPMWCona. Co p ooo 2VIMM 219(roodd claims doc.2A)3/2(7

113- afflicted with a malignant disease associate with WTI expression. A method for inhibiting the development of a malignant disease associated with WTI expression in a patient, comprising the steps of: incubating CD4+ T-cells isolated from a patient with one or more of: a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (ii) a polynucleotide encoding a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (iii) an antigen presenting cell that expresses a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; such that T-cells proliferate, and administering to the patient an effective amount of the proliferated T-cells, and therefrom inhibiting the development of a malignant disease in the patient. 51. A method according to claim 50, wherein the malignant disease is a cancer or a leukemia. 52. A method according to claim 50, wherein the step of incubating the T-cells is repeated one or more times. 53. A method for inhibiting the development of a malignant disease associated with WTI expression in a patient, comprising the steps of: incubating CD4+ T-cells isolated from a patient with one or more of: a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO: 144; (ii) a polynucleotide encoding a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (iii) an antigen presenting cell that expresses a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; P %OPER\HPM'Co-,x CovrmoaliM11162190amedod cI.ms d.2flIRfbX7 -114- such that T-cells proliferate, and cloning one or more cells that proliferated in the presence of WT1 polypeptide; and administering to the patient an effective amount of the cloned T-cells. 54. A method according to claim 53, wherein the malignant disease is a cancer or a leukemia. A method for inhibiting the development of a malignant disease associated with WTI expression in a patient, comprising the steps of: incubating CD8+ T-cells isolated from a patient with one or more of: a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (ii) a polynucleotide encoding a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (iii) an antigen presenting cell that expresses a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; such that T-cells proliferate, and administering to the patient an effective amount of the proliferated T-cells, and therefrom inhibiting the development of a malignant disease in a patient. 56. A method according to claim 55, wherein the malignant disease is a cancer or a leukemia. 57. A method according to claim 55, wherein the step of incubating the T-cells is repeated on or more times. 58. A method for inhibiting the development of a malignant disease associated with WTI expression in a patient, comprising the steps of: incubating CD8+ T-cells isolated from a patient with one or more of: P \OPEMPMNIC C0pWallMon~.\I62l9M odu clims doe.2A)3tX)7 -115- a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (ii) a polynucleotide encoding a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (iii) an antigen presenting cell that expresses a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; such that T-cells proliferate, and cloning one or more cells that proliferated in the presence of WTI polypeptide; and administering to the patient an effective amount of the cloned T-cells. 59. A method according to claim 58, wherein the malignant disease is a cancer or a leukemia. A method for determining the presence or absence of a malignant disease associated with WT1 expression in a patient, comprising the steps of: incubating CD4+ T-cells isolated from a patient with one or more of: a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (ii) a polynucleotide encoding as WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and (iii) an antigen presenting cell that expresses a WT1 polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and detecting the presence or absence of specific activation of T-cells, therefrom determining the presence or absence of a malignant disease associated with WTI expression. 61. A method according to claim 60, wherein the malignant disease is a cancer or a leukemia. P %OPERkHPMCwx. Cv o.tv.'I 2 I6219Mvdcd c1I.sd-Oc2A)3t2(1E7 -116- 62. A method according to claim 60, wherein the step of detecting comprises detecting the presence or absence of proliferation of the T-cells. 63. A method for determining the presence or absence of a malignant disease associated with WTI expression in a patient, comprising the steps of: incubating CD8+ T-cells isolated from a patient with one or more of: a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (ii) a polynucleotide encoding a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; (iii) an antigen presenting cell that expresses a WTI polypeptide consisting essentially of a sequence selected from SEQ ID NO:2 or SEQ ID NO:144; and detecting the presence or absence of specific activation of T-cells, therefrom determining the presence or absence of a malignant disease associated with WT1 expression. 64. A method according to claim 61, wherein the malignant disease is a cancer or a leukemia. A method according to claim 61, wherein the step of detecting comprises detecting the presence or absence of generation of cytolytic activity. 66. A polypeptide according to any one of claims 1 and 2, for use as an active therapeutic substance. 67. A polypeptide according to any one of claims 1 and 2, for use in the manufacture of a medicament for enhancing or inducing an immune response in a patient. 68. An isolated peptide according to any one of claims 1 and 2, or a pharmaceutical composition according to any one of claims 3, 9 and 11, or a vaccine according to any one P \OPER\iPMCoI a CoqpoialionlI 21621 niended claims doc.-2V2f 07 -117- of claims 4, 5 to 7, 12 and 15, or a polynucleotide according to claim 8, or a method according to any one of claims 16 to 35, 36 to 49, and 50 to 65 substantially as hereinbefore described with reference to the accompanying Examples and Figures.