Patent Publication Number: US-7915393-B2

Title: Compositions and methods for WT1 specific immunotherapy

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 10/427,717, filed Apr. 30, 2003, now issued as U.S. Pat. No. 7,553,494, on Jun. 30, 2009; which is a continuation-in-part of U.S. patent application Ser. No. 10/286,333, filed Oct. 30, 2002, now abandoned; which is a continuation-in-part of U.S. patent application Ser. No. 10/244,830, filed Sep. 16, 2002, now abandoned; which is a continuation-in-part of U.S. patent application Ser. No. 10/195,835, filed Jul. 12, 2002, now issued as U.S. Pat. No. 7,655,249, on Feb. 2, 2010; which is a continuation-in-part of U.S. patent application Ser. No. 10/125,635, filed Apr. 16, 2002, now abandoned; which is a continuation-in-part of U.S. patent application Ser. No. 10/002,603, filed Oct. 30, 2001, now; which is a continuation-in-part of U.S. patent application Ser. No. 09/938,864, filed Aug. 24, 2001, now abandoned; which applications are incorporated herein by reference in their entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made in part with government support under NIH SBIR Phase I grant number IR43CA81752-01A1. The Government may have certain rights in this invention. 
    
    
     STATEMENT REGARDING SEQUENCE LISTING 
     The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 210121 — 610C5_SEQUENCE_LISTING.txt. The text file is 280 KB, was created on May 21, 2009, and is being submitted electronically via EFS-Web. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     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 WT1, and to the use of such compositions for preventing and/or treating malignant diseases. 
     2. Description of the Related Art 
     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 (e.g., 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 exception, 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. 
     BRIEF 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 of the present invention, the polypeptide comprises at least an immunogenic portion of WT1 wherein the immunogenic portion is contained within amino acids 2-281 of WT1. Within certain embodiments, the polypeptide comprises no more than 16 consecutive amino acid residues of a native WT1 polypeptide. Within other embodiments, the polypeptide comprises an immunogenic portion of amino acid residues 1-174 of a native WT1 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 WT1 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 (a) sequences recited in any one or more of Tables II-XLVI, (b) 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 (c) 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 (a) 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), VLDFAPPGA (SEQ ID NO:241), VLDFAPPGAS (SEQ ID NO:411), SEQ ID NOs: 414-450, ALLPAVPSL (SEQ ID NO:451) (b) 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 (c) 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 WT1 protein, wherein the variant differs from the immunogenic portion due to substitutions at between 1 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 not substantially reduced or is enhanced relative to a native WT1 protein. 
     The present invention further provides WT1 polynucleotides that encode a WT1 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 (i) a WT1 polynucleotide; (ii) an antibody or antigen-binding fragment thereof that specifically binds to a WT1 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 (i) a WT1 polynucleotide, (ii) an antigen-presenting 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 WT1 polypeptide are present within a WT1 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 (e.g., acute myeloid, acute lymphocytic and chronic myeloid) and cancers (e.g., 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 WT1 polypeptide, 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 10%, preferably less than 5% and more preferably less than 1%, 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 WT1 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 (e.g., 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 WT1 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 WT1-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. WT1-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 expression 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: (a) incubating a first biological sample with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 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; (b) detecting immunocomplexes formed between the WT1 polypeptide and antibodies in the biological sample that specifically bind to the WT1 polypeptide; (c) repeating steps (a) and (b) 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 (a) incubating the immunocomplexes with a detection reagent that is capable of binding to the immunocomplexes, wherein the detection reagent comprises a reporter group, (b) removing unbound detection reagent, and (c) 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 WT1 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 WT1 expression in a patient may comprise the steps of: (a) incubating a first biological sample with one or more of: (i) a WT1 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; (c) repeating steps (a) and (b) 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 (d) 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: (a) incubating CD4 +  and/or CD8+ T cells isolated from a patient with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide, such that the T cells proliferate; and (b) 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 WT1 expression in a patient, comprising the steps of: (a) incubating CD4 +  and/or CD8+ T cells isolated from a patient with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide, such that the T cells proliferate; (b) cloning one or more cells that proliferated; and (c) 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 WT1 expression in a patient, comprising the steps of: (a) incubating CD4 +  and/or CD8+ T cells isolated from a patient with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide; and (b) detecting the presence or absence of specific activation of the T cells, therefrom determining the presence or absence of a malignant disease associated with WT1 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 WT1 expression in a patient, comprising the steps of: (a) incubating a biological sample obtained from a patient with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide, wherein the incubation is performed under conditions and for a time sufficient to allow immunocomplexes to form; and (b) detecting immunocomplexes formed between the WT1 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 WT1 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 
         FIG. 1  depicts a comparison of the mouse (MO) and human (HU) WT1 protein sequences (SEQ ID NOS: 320 and 319 respectively). 
         FIG. 2  is a Western blot illustrating the detection of WT1 specific antibodies in patients with hematological malignancy (AML). Lane 1 shows molecular weight markers; lane 2 shows a positive control (WT1 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 WT1 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 WT1 specific antibody. 
         FIG. 3  is a Western blot illustrating the detection of a WT1 specific antibody response in B6 mice immunized with TRAMP-C, a WT1 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 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. 
         FIG. 4  is a Western blot illustrating the detection of WT1 specific antibodies in mice immunized with representative WT1 peptides. 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 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. 
         FIGS. 5A to 5C  are graphs illustrating the stimulation of proliferative T cell responses in mice immunized with representative WT1 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 (p1), p117-139 (p2) or p244-262 human (p3). 
         FIGS. 6A and 6B  are histograms illustrating the stimulation of proliferative T cell responses in mice immunized with representative WT1 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), p117-139 (p117), p244-262 (p244) (Vaccine A;  FIG. 6A ) or p287-301 (p287), p299-313 (p299), p421-435 (p421) (Vaccine B;  FIG. 6B ) and spleen cells pulsed with an irrelevant control peptide (irrelevant peptide) at 25 ug/ml and were assayed after 96 hr for proliferation by ( 3 H) thymidine incorporation. Bars represent the stimulation index (SI), which is calculated as the mean of the experimental wells divided by the mean of the control (B6 spleen cells with no antigen). 
         FIGS. 7A-7D  are histograms illustrating the generation of proliferative T-cell lines and clones specific for p117-139 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 p117-139 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 (p6), p117-139 (p117) or an irrelevant control peptide (irrelevant peptide) at 25 ug/ml and were assayed after 96 hr for proliferation by ( 3 H) thymidine incorporation. Bars represent the stimulation index (SI), which is calculated as the mean of the experimental wells divided by the mean of the control (B6 spleen cells with no antigen). 
         FIGS. 8A and 8B  present the results of TSITES Analysis of human WT1 (SEQ ID NO:319) for peptides that have the potential to elicit Th responses. Regions indicated by “A” are AMPHI midpoints of blocks, “R” indicates residues matching the Rothbard/&#39;Taylor motif, “D” indicates residues matching the IAd motif, and ‘d’ indicates residues matching the IEd motif. 
         FIGS. 9A and 9B  are graphs illustrating the elicitation of WT1 peptide-specific CTL in mice immunized with WT1 peptides.  FIG. 9A  illustrates the lysis of target cells by allogeneic cell lines and  FIG. 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. 
         FIGS. 10A-10D  are graphs illustrating the elicitation of WT1 specific CTL, which kill WT1 positive tumor cell lines but do not kill WT1 negative cell lines, following vaccination of B6 mice with WT1 peptide P117.  FIG. 10A  illustrates that T-cells of non-immunized B6 mice do not kill WT1 positive tumor cell lines. 
         FIG. 10B  illustrates the lysis of the target cells by allogeneic cell lines.  FIGS. 10C and 10D  demonstrate the lysis of WT1 positive tumor cell lines, as compared to WT1 negative cell lines in two different experiments. In addition,  FIGS. 10C and 10D  show the lysis of peptide-coated cell lines (WT1 negative cell line E10 coated with the relevant WT1 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. 
         FIGS. 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 WT1 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.  FIG. 11A  shows the cytotoxic activity of the p235-243 specific T cell line against the WT1 negative cell line EL-4 (EL-4, WT1 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 p117-139 (EL-4+p117), p126-134 (EL-4+p126) or p130-138 (EL-4+p130) and the WT1 positive tumor cells BLK-SV40 (BLK-SV40, WT1 positive) and TRAMP-C (TRAMP-C, WT1 positive), as indicated.  FIG. 11B  shows cytotoxic activity of the p117-139 specific T cell line against EL-4; EL-4 pulsed with the relevant peptide P117-139 (EL-4+p117) 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. 
         FIGS. 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.  FIG. 12A  shows the cytotoxic activity of the p117-139 specific T cell line against the WT1 negative cell line EL-4 (EL-4, WT1 negative); the WT1 positive tumor cell line TRAMP-C (TRAMP-C, WT1 positive); TRAMP-C cells incubated with a ten-fold excess (compared to the hot target) of EL-4 cells pulsed with the relevant peptide p117-139 (TRAMP-C+p117 cold target) without  51 Cr labeling and TRAMP-C cells incubated with EL-4 pulsed with an irrelevant peptide without  51 Cr labeling (TRAMP-C+irrelevant cold target), as indicated.  FIG. 12B  shows the cytotoxic activity of the p117-139 specific T cell line against the WT1 negative cell line EL-4 (EL-4, WT1 negative); the WT1 positive tumor cell line BLK-SV40 (BLK-SV40, WT1 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 (BLK-SV40+irrelevant cold target), as indicated. 
         FIGS. 13A-13C  are histograms depicting an evaluation of the 9mer CTL epitope within p117-139. The p117-139 tumor specific CTL line was tested against peptides within aa117-139 containing or lacking an appropriate H-2 b  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.  FIG. 13A  shows the cytotoxic activity of the p117-139 specific T cell line against the WT1 negative cell line EL-4 (EL-4, WT1 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+p126), p128-136 (EL-4+p128), and p130-138 (EL-4+p130).  FIG. 13B  shows the cytotoxic activity of the CTL line after restimulation with p126-134 against the WT1 negative cell line EL-4, EL-4 cells pulsed with p117-139 (EL-4+p117), p126-134 (EL-4+p126) and the WT1 positive tumor cell line TRAMP-C.  FIG. 13C  shows the cytotoxic activity of the CTL line after restimulation with p130-138 against EL-4, EL-4 cells pulsed with p117-139 (EL-4+p117), p130-138 (EL-4+p130) and the WT1 positive tumor cell line TRAMP-C. 
         FIG. 14  depicts serum antibody reactivity to WT1 in 63 patients with AML. Reactivity of serum antibody to WT1/N-terminus protein was evaluated by ELISA in patients with AML. The first and second lanes represent the positive and negative controls, respectively. The first and second lanes represent the positive and negative controls, respectively. Commercially obtained WT1 specific antibody WT180 was used for the positive control. The next 63 lanes represent results using sera from each individual patient. The OD values depicted were from ELISA using a 1:500 serum dilution. The figure includes cumulative data from 3 separate experiments. 
         FIG. 15  depicts serum antibody reactivity to WT1 proteins and control proteins in 2 patients with AML. Reactivity of serum antibody to WT1/full-length, WT1N-terminus, TRX and Ra12 proteins was evaluated by ELISA in 2 patients with AML. The OD values depicted were from ELISA using a 1:500 serum dilution. AML-1 and AML-2 denote serum from 2 of the individual patients in  FIG. 1  with demonstrated antibody reactivity to WT1/full-length. The WT1 full-length protein was expressed as a fusion protein with Ra12. The WT1/N-terminus protein was expressed as a fusion protein with TRX. The control Ra12 and TRX proteins were purified in a similar manner. The results confirm that the serum antibody reactivity against the WT1 fusion proteins is directed against the WT1 portions of the protein. 
         FIG. 16  depicts serum antibody reactivity to WT1 in 81 patients with CML. Reactivity of serum antibody to WT1/full-length protein was evaluated by ELISA in patients with AML. The first and second lanes represent the positive and negative controls, respectively. Commercially obtained WT1 specific antibody WT180 was used for the positive control. The next 81 lanes represent results using sera from each individual patient. The OD values depicted were from ELISA using a 1:500 serum dilution. The figure includes cumulative data from 3 separate experiments. 
         FIG. 17  depicts serum antibody reactivity to WT1 proteins and control proteins in 2 patients with CML. Reactivity of serum antibody to WT1/full-length, WT1/N-terminus, TRX and Ra12 proteins was evaluated by ELISA in 2 patients with CML. The OD values depicted were from ELISA using a 1:500 serum dilution. CML-1 and CML-2 denote serum from 2 of the individual patients in  FIG. 3  with demonstrated antibody reactivity to WT1/full-length. The WT1/full-length protein was expressed as a fusion protein with Ra12. The WT1/N-terminus protein was expressed as a fusion protein with TRX. The control Ra12 and TRX proteins were purified in a similar manner. The results confirm that the serum antibody reactivity against the WT1 fusion proteins is directed against the WT1 portions of the protein. 
         FIG. 18  provides the characteristics of the recombinant WT1 proteins used for serological analysis. 
       FIGS.  19 A 1 - 19 E 3  is a bar graph depicting the antibody responses in mice elicited by vaccination with different doses of WT1 protein. 
         FIGS. 20A  and B is a bar graph of the proliferative T-cell responses in mice immunized with WT1 protein. 
         FIG. 21  is a photograph of human DC, examined by fluorescent microscopy, expressing WT1 following adeno WT1 and Vaccinia WT1 infection. 
         FIG. 22  is a photograph that demonstrates that WT1 expression in human DC is reproducible following adeno WT1 infection and is not induced by a control Adeno infection. 
         FIG. 23  is a graph of an IFN-gamma ELISPOT assay showing that WT1 whole gene in vitro priming elicits WT1 specific T-cell responses. 
         FIG. 24  shows amino acids 2-281 (SEQ ID NO:461) of the WT1 protein and the cDNA encoding these amino acid residues (SEQ ID NO:460). This truncated WT1 protein is referred to as WT1-F. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety. 
     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 WT1 polypeptides, WT1 polynucleotides, antigen-presenting cells (APC, e.g., dendritic cells) that express a WT1 polypeptide, agents such as antibodies that bind to a WT1 polypeptide and/or immune system cells (e.g., T cells) specific for WT1. WT1 Polypeptides of the present invention generally comprise at least a portion of a Wilms Tumor gene product (WT1) 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 (e.g., 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 (e.g., WT1) can provide prophylactic and/or therapeutic benefit for patients afflicted with malignant diseases characterized by increased WT1 gene expression. Such diseases include, but are not limited to, leukemias (e.g., 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 WT1 gene was originally identified and isolated on the basis of a cytogenetic deletion at chromosome 11p13 in patients with Wilms&#39; tumor (see Call et al., U.S. Pat. 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  FIG. 1  and SEQ ID NOs: 319 and 320. 
     WT1 Polypeptides 
     Within the context of the present invention, a WT1 polypeptide is a polypeptide that comprises at least an immunogenic portion of a native WT1 (i.e., a WT1 protein expressed by an organism that is not genetically modified), or a variant thereof, as described herein. A WT1 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 WT1 polypeptide may be an oligopeptide (i.e., consisting of a relatively small number of amino acid residues, such as 8-10 residues, joined by peptide bonds), a full length WT1 protein (e.g., present within a human or non-human animal, such as a mouse) or a polypeptide of intermediate size. Within certain embodiments, the use of WT1 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 WT1 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 (i.e., 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  125 I labeled β2-microglobulin (β2m) into MHC class I/β2m/peptide heterotrimeric complexes (see Parker et al.,  J. Immunol.  152:163, 1994). Alternatively, 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 WT1 residues 117-139; SEQ ID NOs: 2 and 3 respectively), GATLKGVAAGSSSSVKWTE (human WT1 residues 244-262; SEQ ID NO:4), GATLKGVAA (human WT1 residues 244-252; SEQ ID NO:88), CMTWNQMNL (human and mouse WT1 residues 235-243; SEQ ID NOs: 49 and 258 respectively), SCLESQPTI (mouse WT1 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); RMFPNAPYL (human and mouse WT1 residues 126-134; SEQ ID NOs: 185 and 293 respectively), VLDFAPPGA (human WT1 residues 37-45; SEQ ID NO:241), or VLDFAPPGAS (human WT1 residues 37-46; SEQ ID NO:411). Further immunogenic portions are provided in SEQ ID NOs:414-451. 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 T-cell 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 WT1 (e.g., 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. Alternatively, immungenic portions that bind to a particular MHC molecule can be identified by using defined peptide binding motifs such as those described in Rammensee et al., Immunogenetics 41:178-228, 1995. To confirm peptide binding to murine and human class I or class II MHC molecules, peptide binding assays known in the art may be used. To confirm immunogenicity, a peptide may be tested using an HLA A2 or other 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 WT1 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 (i.e., 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. In one embodiment the ability of a variant to react with antigen-specific antisera and/or T-cell lines or clones may be diminished by less than 10%, 5%, 4%, 3%, 2%, 1%, or 0.5%, 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. In one embodiment of the present invention, a variant may be identified by evaluating its ability to bind to a human or a muring HLA molecule. In one preferred embodiment, a variant polypeptide has a modification such that the ability of the variant polypeptide to bind to a class I or class II MHC molecule, for example HLA-A2, HLA-A24, HLA-A1, HLA-A3, HLA-A-68, HLA-A11, HLA-A31, HLA-A33, HLA-B14, HLA-B40, HLA-B60, HLA-B62, HLA-B7, HLA-B8, HLA-B27, HLA-B3501, HLA-B37, HLA-B38, HLA-B39, HLA-B44, HLA-B51, HLA-B52, HLA-B58, HLA-CW03, HLA-CW04, HLA-CW06, or HLA-CW07, is increased relative to that of a wild type (unmodified) WT1 polypeptide. In a further embodiment, the N-terminal portion of WT1 from amino acids 1-281 is modified such that the ability of any number of polypeptides therein that can bind to HLA-A2, HLA-A24, HLA-A1, HLA-A3, HLA-A68, HLA-A11, HLA-A31, HLA-A33, HLA-B14, HLA-B40, HLA-B60, HLA-B62, HLA-B7, HLA-B8, HLA-B27, HLA-B3501, HLA-B37, HLA-B38, HLA-B39, HLA-B44, HLA-B51, HLA-B52, HLA-B58, HLA-CW03, HLA-CW04, HLA-CW06, or HLA-CW07 is increased relative to that of the wild type (unmodified) WT1 polypeptide sequence. In a further embodiment, a polypeptide comprising at least one immunogenic portion (epitope) is modified such that its ability to bind to an MHC molecule, such as HLA-A2, HLA-A24, HLA-A1, HLA-A3, HLA-A68, HLA-A11, HLA-A31, HLA-A33, HLA-B14, HLA-B40, HLA-B60, HLA-B62, HLA-B7, HLA-B8, HLA-B27, HLA-B3501, HLA-B37, HLA-B38, HLA-B39, HLA-B44, HLA-B51, HLA-B52, HLA-B58, HLA-CW03, HLA-CW04, HLA-CW06, or HLA-CW07 is increased. Example 30 describes illustrative variants that can be generated. The skilled artisan would readily recognize that these are illustrative variants and that other variants can be generated in a similar manner for peptides that bind to HLA molecules other than HLA-A2. In a further embodiment, the ability of the variant polypeptide to bind to a HLA molecule is increased by at least 2 fold, preferably at least 3 fold, 4 fold, or 5 fold relative to that of a native WT1 polypeptide. It has been found, within the context of the present invention, that a relatively small number of substitutions (e.g., 1 to 3) within an immunogenic portion of a WT1 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 WT1 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: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. 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. 
     In a preferred embodiment, a variant polypeptide of the WT1 N-terminus (amino acids 1-249) is constructed, wherein the variant polypeptide is capable of binding to an antibody that recognizes full-length WT1 and/or WT1 N-terminus polypeptide. A non-limiting example of an antibody is anti WT1 antibody WT180 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). 
     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 post-translationally 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 (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region. 
     WT1 polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by a WT1 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 been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are  E. coli , yeast or a mammalian cell line such as COS or CHO. Supernatants from suitable 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, Calif.), and may be operated according to the manufacturer&#39;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 WT1 polypeptides. Such mimetics may comprise amino acids linked to one or more amino acid mimetics (i.e., one or more amino acids within the WT1 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 WT1 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 WT1-specific antisera and/or T cell lines or clones is not substantially diminished relative to the ability of a WT1 polypeptide. Such mimetics may be designed based on standard techniques (e.g., 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 WT1 polypeptide. 
     Within other illustrative embodiments, a polypeptide may be a fusion polypeptide that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the polypeptide or to enable the polypeptide to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the polypeptide. 
     Fusion polypeptides may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion polypeptide is expressed as a recombinant polypeptide, allowing the production of increased levels, relative to a non-fused polypeptide, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3′ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides. 
     A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al.,  Gene  40:39-46, 1985; Murphy et al.,  Proc. Natl. Acad. Sci. USA  83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. 
     The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5′ to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3′ to the DNA sequence encoding the second polypeptide. 
     The fusion polypeptide can comprise a polypeptide as described herein together with an unrelated immunogenic protein, such as an immunogenic protein capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al.  New Engl. J. Med.,  336:86-91, 1997). 
     In one preferred embodiment, the immunological fusion partner is derived from a  Mycobacterium  sp., such as a  Mycobacterium tuberculosis -derived Ra12 fragment. Ra12 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences is described in U.S. patent application 60/158,585, now issued as U.S. Pat. No. 7,009,042, the disclosure of which is incorporated herein by reference in its entirety. Briefly, Ra12 refers to a polynucleotide region that is a subsequence of a  Mycobacterium tuberculosis  MTB32A nucleic acid. MTB32A is a serine protease of 32 KD molecular weight encoded by a gene in virulent and avirulent strains of  M. tuberculosis . The nucleotide sequence and amino acid sequence of MTB32A have been described (for example, U.S. patent application 60/158,585, now issued as U.S. Pat. No. 7,009,042; see also, Skeiky et al.,  Infection and Immun . (1999) 67:3998-4007, incorporated herein by reference). C-terminal fragments of the MTB32A coding sequence express at high levels and remain as soluble polypeptides throughout the purification process. Moreover, Ra12 may enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One preferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ra12 polynucleotides generally comprise at least about 15 consecutive nucleotides, at least about 30 nucleotides, at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, or at least about 300 nucleotides that encode a portion of a Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Ra12 polypeptide or a portion thereof) or may comprise a variant of such a sequence. Ra12 polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ra12 polypeptide. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native Ra12 polypeptide or a portion thereof. 
     Within other preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium  Haemophilus influenza  B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in  E. coli  (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenzae virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used. 
     In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from  Streptococcus pneumoniae , which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of  E. coli  C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see  Biotechnology  10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion polypeptide. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305. 
     Within another illustrative embodiment the fusion partner comprises a twin arginine translocation (TAT) signal peptide from the TorA signal peptide in  E. coli  on the N-terminus; see J. Mol. Microbiol. (2000) 2(2): 179-189; Journal of Bacteriology, January 2001 p604-610 Vol 183, No 2; Journal of Biochemistry Vol 276, Mar. 16, 2001 pp 8159-8164), hereby incorporated herein in its entirety. 
     Yet another illustrative embodiment involves fusion polypeptides, and the polynucleotides encoding them, wherein the fusion partner comprises a targeting signal capable of directing a polypeptide to the endosomal/lysosomal compartment, as described in U.S. Pat. No. 5,633,234. An immunogenic polypeptide of the invention, when fused with this targeting signal, will associate more efficiently with MHC class II molecules and thereby provide enhanced in vivo stimulation of CD4 +  T-cells specific for the polypeptide. 
     The invention provides truncated forms of WT1 polypeptides that can be recombinantly expressed in  E. coli  without the addition of a fusion partner. Examples of these truncated forms are shown in SEQ ID NOs:342-346, and are encoded by polynucleotides shown in SEQ ID NOs:337-341. In variations of these truncations, the first 76 amino acids of WT1 can be fused to the C-terminus of the protein, creating a recombinant protein that is easier to express in  E. coli . Other hosts in addition to  E. coli  can also be used, such as, for example,  B. megaterium . The protein can further be prepared without a histidine tag. 
     In other embodiments, different subunits can be made and fused together in an order which differs from that of native WT1. In addition, fusions can be made with, for example, Ra12. Exemplary fusion proteins are shown in SEQ ID NOs: 332-336 and can be encoded by polynucleotides shown in SEQ ID NOs: 327-331. 
     WT1 Polynucleotides 
     Any polynucleotide that encodes a WT1 polypeptide as described herein is a WT1 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. 
     WT1 polynucleotides may encode a native WT1 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 WT1 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 WT1 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 WT1 polypeptide (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×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 WT1 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. 
     Therefore, according to another aspect of the present invention, polynucleotide compositions are provided that comprise some or all of a polynucleotide sequence set forth herein, complements of a polynucleotide sequence set forth herein, and degenerate variants of a polynucleotide sequence set forth herein. In certain preferred embodiments, the polynucleotide sequences set forth herein encode immunogenic polypeptides, as described above. 
     Once an immunogenic portion of WT1 is identified, as described above, a WT1 polynucleotide may be prepared using any of a variety of techniques. For example, a WT1 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—P118: 1434-1414: 5′ GAG AGT CAG ACT TGA AAG CAGT 3′ (SEQ ID NO:5) and P135: 5′ CTG AGC dC 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. WT1 polynucleotides may also be prepared by synthesizing oligonucleotide components, and ligating components together to generate the complete polynucleotide. 
     WT1 polynucleotides may also be synthesized by any method known in the art, including chemical synthesis (e.g., 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 WT1 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 (e.g., by transfecting antigen-presenting cells such as dendritic cells with a cDNA construct encoding a WT1 polypeptide, and administering the transfected cells to the patient). 
     Polynucleotides that encode a WT1 polypeptide may generally be used for production of the polypeptide, in vitro or in vivo. WT1 polynucleotides that are complementary to a coding sequence (i.e., antisense polynucleotides) may also be used as a probe or to inhibit WT1 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. In particular, one embodiment of the invention comprises expression vectors which incorporate the nucleic acid molecules of the invention, in operable linkage (i.e., “operably linked”) to an expression control sequence (promoter). Illustrative vectors include, but are not limited, those described herein in Examples 20 and 40. Construction of vaccinia virus or attenuated viral vectors is well within the skill of the art, as is the transformation or transfection of cells, to produce eukaryotic cell lines, or prokaryotic cell strains which encode the molecule of interest. Exemplary of the host cells which can be employed in this fashion are COS cells, CHO cells, yeast cells, insect cells (e.g.,  Spodoptera frugiperda  or Sf-9 cells), NIH 3T3 cells, and so forth. Prokaryotic cells, such as  E. coli  and other bacteria may also be used. 
     Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art. cDNA constructs within such a vector may be used, for example, to transfect human or animal cell lines for use in establishing WT1 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 (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art. 
     Antibody Compositions, Fragments thereof and Other Binding Agents 
     According to another aspect, the present invention further provides binding agents, such as antibodies and antigen-binding fragments thereof, that exhibit immunological binding to a WT1 polypeptide disclosed herein, or to a portion, variant or derivative thereof. An antibody, or antigen-binding fragment thereof, is said to “specifically bind,” “immunogically bind,” and/or is “immunologically reactive” to a WT1 polypeptide of the invention if it reacts at a detectable level (within, for example, an ELISA assay) with the polypeptide, and does not react detectably with unrelated polypeptides under similar conditions. 
     Immunological binding, as used in this context, generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K d ) of the interaction, wherein a smaller K d  represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (K on ) and the “off rate constant” (K off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of K off /K on  enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant K d . See, generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473. 
     An “antigen-binding site,” or “binding portion” of an antibody refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus the term “FR” refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” 
     Binding agents may be further capable of differentiating between patients with and without a WT1-associated cancer, using the representative assays provided herein. For example, antibodies or other binding agents that bind to a tumor protein will preferably generate a signal indicating the presence of a cancer in at least about 20% of patients with the disease, more preferably at least about 30% of patients. Alternatively, or in addition, the antibody will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without the cancer. To determine whether a binding agent satisfies this requirement, biological samples (e.g., blood, sera, sputum, urine and/or tumor biopsies) from patients with and without a cancer (as determined using standard clinical tests) may be assayed as described herein for the presence of polypeptides that bind to the binding agent. Preferably, a statistically significant number of samples with and without the disease will be assayed. Each binding agent should satisfy the above criteria; however, those of ordinary skill in the art will recognize that binding agents may be used in combination to improve sensitivity. 
     Any agent that satisfies the above requirements may be a binding agent. For example, a binding agent may be a ribosome, with or without a peptide component, an RNA molecule or a polypeptide. In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane,  Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support. 
     Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein,  Eur. J. Immunol.  6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 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. 
     A number of therapeutically useful molecules are known in the art which comprise antigen-binding sites that are capable of exhibiting immunological binding properties of an antibody molecule. The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the “F(ab)” fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the “F(ab′) 2 ” fragment which comprises both antigen-binding sites. An “Fv” fragment can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent V H ::V L  heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096. 
     A single chain Fv (“sFv”) polypeptide is a covalently linked V H ::V L  heterodimer which is expressed from a gene fusion including V H - and V L -encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methods have been described to discern chemical structures for converting the naturally aggregated—but chemically separated—light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al. 
     Each of the above-described molecules includes a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain FR set which provide support to the CDRS and define the spatial relationship of the CDRs relative to each other. As used herein, the term “CDR set” refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3” respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a “molecular recognition unit.” Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site. 
     As used herein, the term “FR set” refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRS. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain “canonical” structures—regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains. 
     A number of “humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains (Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J. Immunol. 138:4534-4538; and Brown et al. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a human supporting FR prior to fusion with an appropriate human antibody constant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature 321:522-525), and rodent CDRs supported by recombinantly veneered rodent FRs (European Patent Publication No. 519,596, published Dec. 23, 1992). These “humanized” molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. 
     As used herein, the terms “veneered FRs” and “recombinantly veneered FRs” refer to the selective replacement of FR residues from, e.g., a rodent heavy or light chain V region, with human FR residues in order to provide a xenogeneic molecule comprising an antigen-binding site which retains substantially all of the native FR polypeptide folding structure. Veneering techniques are based on the understanding that the ligand binding characteristics of an antigen-binding site are determined primarily by the structure and relative disposition of the heavy and light chain CDR sets within the antigen-binding surface. Davies et al. (1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificity can be preserved in a humanized antibody only wherein the CDR structures, their interaction with each other, and their interaction with the rest of the V region domains are carefully maintained. By using veneering techniques, exterior (e.g., solvent-accessible) FR residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic, or substantially non-immunogenic veneered surface. 
     The process of veneering makes use of the available sequence data for human antibody variable domains compiled by Kabat et al., in Sequences of Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Health and Human Services, U.S. Government Printing Office, 1987), updates to the Kabat database, and other accessible U.S. and foreign databases (both nucleic acid and protein). Solvent accessibilities of V region amino acids can be deduced from the known three-dimensional structure for human and murine antibody fragments. There are two general steps in veneering a murine antigen-binding site. Initially, the FRs of the variable domains of an antibody molecule of interest are compared with corresponding FR sequences of human variable domains obtained from the above-identified sources. The most homologous human V regions are then compared residue by residue to corresponding murine amino acids. The residues in the murine FR which differ from the human counterpart are replaced by the residues present in the human moiety using recombinant techniques well known in the art. Residue switching is only carried out with moieties which are at least partially exposed (solvent accessible), and care is exercised in the replacement of amino acid residues which may have a significant effect on the tertiary structure of V region domains, such as proline, glycine and charged amino acids. 
     In this manner, the resultant “veneered” murine antigen-binding sites are thus designed to retain the murine CDR residues, the residues substantially adjacent to the CDRs, the residues identified as buried or mostly buried (solvent inaccessible), the residues believed to participate in non-covalent (e.g., electrostatic and hydrophobic) contacts between heavy and light chain domains, and the residues from conserved structural regions of the FRs which are believed to influence the “canonical” tertiary structures of the CDR loops. These design criteria are then used to prepare recombinant nucleotide sequences which combine the CDRs of both the heavy and light chain of a murine antigen-binding site into human-appearing FRs that can be used to transfect mammalian cells for the expression of recombinant human antibodies which exhibit the antigen specificity of the murine antibody molecule. 
     In another embodiment of the invention, monoclonal antibodies of the present invention may be coupled to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include  90 Y,  123 I,  125 I,  131 I,  186 Re,  188 Re,  211 At, and  212 Bi. Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin,  Pseudomonas  exotoxin,  Shigella  toxin, and pokeweed antiviral protein. 
     A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other. 
     Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible. 
     It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, Ill.), may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al. 
     Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group which is cleavable during or upon internalization into a cell. A number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. 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 that provide multiple sites for attachment can be used. Alternatively, a carrier can be used. 
     A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Pat. 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. Pat. No. 4,673,562, to Davison et al. discloses representative chelating compounds and their synthesis. 
     T Cells 
     Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for WT1. 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™ system, available from CellPro Inc., Bothell Wash. (see also U.S. Pat. Nos. 5,240,856; 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 WT1 polypeptide, polynucleotide encoding a WT1 polypeptide and/or an antigen presenting cell (APC) that expresses a WT1 polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the WT1 polypeptide. Preferably, a WT1 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 WT1 polypeptide. For example, T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37° C. with WT1 polypeptide (e.g., 5 to 25 μg/ml) or cells synthesizing a comparable amount of WT1 polypeptide. It may be desirable to incubate a separate aliquot of a T cell sample in the absence of WT1 polypeptide to serve as a control. 
     T cells are considered to be specific for a WT1 polypeptide if the T cells kill target cells coated with a WT1 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 (e.g., 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)-2,5-diphenyl-tetrazolium. Alternatively, synthesis of lymphokines (such as interferon-gamma) can be measured or the relative number of T cells that can respond to a WT1 polypeptide may be quantified. Contact with a WT1 polypeptide (200 ng/ml-100 μg/ml, preferably 100 ng/ml-25 μg/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 (e.g., TNF or IFN-γ) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998). 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 WT1 polypeptide, polynucleotide or WT1-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 (e.g., lymphokines), or the generation of cytolytic activity (i.e., generation of cytotoxic T cells specific for WT1). 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 WT1 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 WT1 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 (e.g., greater than 4×10 7 ) may be placed in flasks with media containing human serum. WT1 polypeptide (e.g., peptide at 10 μg/ml) may be added directly, along with tetanus toxoid (e.g., 5 μg/ml). The flasks may then be incubated (e.g., 37° C. for 7 days). For a second IVS, T cells are then harvested and placed in new flasks with 2-3× 10   7  irradiated peripheral blood mononuclear cells. WT1 polypeptide (e.g., 10 μg/ml) is added directly. The flasks are incubated at 37° 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&#39;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, WT1 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 WT1 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 1×10 6  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 (i.e., sensitized to WT1) 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 WT1 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. 
     T cells specific for WT1 can kill cells that express WT1 protein. Introduction of genes encoding T-cell receptor (TCR) chains for WT1 are used as a means to quantitatively and qualitatively improve responses to WT1 bearing leukemia and cancer cells. Vaccines to increase the number of T cells that can react to WT1 positive cells are one method of targeting WT1 bearing cells. T cell therapy with T cells specific for WT1 is another method. An alternative method is to introduce the TCR chains specific for WT1 into T cells or other cells with lytic potential. In a suitable embodiment, the TCR alpha and beta chains are cloned out from a WT1 specific T cell line and used for adoptive T cell therapy, such as described in WO96/30516, incorporated herein by reference. 
     T Cell Receptor Compositions 
     The T cell receptor (TCR) consists of 2 different, highly variable polypeptide chains, termed the T-cell receptor α and β chains, that are linked by a disulfide bond (Janeway, Travers, Walport. Immunobiology. Fourth Ed., 148-159. Elsevier Science Ltd/Garland Publishing. 1999). The α/β heterodimer complexes with the invariant CD3 chains at the cell membrane. This complex recognizes specific antigenic peptides bound to MHC molecules. The enormous diversity of TCR specificities is generated much like immunoglobulin diversity, through somatic gene rearrangement. The β chain genes contain over 50 variable (V), 2 diversity (D), over 10 joining (J) segments, and 2 constant region segments (C). The α chain genes contain over 70 V segments, and over 60 J segments but no D segments, as well as one C segment. During T cell development in the thymus, the D to J gene rearrangement of the β chain occurs, followed by the V gene segment rearrangement to the DJ. This functional VDJβ exon is transcribed and spliced to join to a Cβ. For the α chain, a Vα gene segment rearranges to a Jα gene segment to create the functional exon that is then transcribed and spliced to the Cα. Diversity is further increased during the recombination process by the random addition of P and N-nucleotides between the V, D, and J segments of the β chain and between the V and J segments in the α chain (Janeway, Travers, Walport. Immunobiology. Fourth Ed., 98 and 150. Elsevier Science Ltd/Garland Publishing. 1999). 
     The present invention, in another aspect, provides TCRs specific for a polypeptide disclosed herein, or for a variant or derivative thereof. In accordance with the present invention, polynucleotide and amino acid sequences are provided for the V-J or V-D-J junctional regions or parts thereof for the alpha and beta chains of the T-cell receptor which recognize tumor polypeptides described herein. In general, this aspect of the invention relates to T-cell receptors which recognize or bind tumor polypeptides presented in the context of MHC. In a preferred embodiment the tumor antigens recognized by the T-cell receptors comprise a polypeptide of the present invention. For example, cDNA encoding a TCR specific for a WT1 peptide can be isolated from T cells specific for a tumor polypeptide using standard molecular biological and recombinant DNA techniques. 
     This invention further includes the T-cell receptors or analogs thereof having substantially the same function or activity as the T-cell receptors of this invention which recognize or bind tumor polypeptides. Such receptors include, but are not limited to, a fragment of the receptor, or a substitution, addition or deletion mutant of a T-cell receptor provided herein. This invention also encompasses polypeptides or peptides that are substantially homologous to the T-cell receptors provided herein or that retain substantially the same activity. The term “analog” includes any protein or polypeptide having an amino acid residue sequence substantially identical to the T-cell receptors provided herein in which one or more residues, preferably no more than 5 residues, more preferably no more than 25 residues have been conservatively substituted with a functionally similar residue and which displays the functional aspects of the T-cell receptor as described herein. 
     The present invention further provides for suitable mammalian host cells, for example, non-specific T cells, that are transfected with a polynucleotide encoding TCRs specific for a polypeptide described herein, thereby rendering the host cell specific for the polypeptide. The α and β chains of the TCR may be contained on separate expression vectors or alternatively, on a single expression vector that also contains an internal ribosome entry site (IRES) for cap-independent translation of the gene downstream of the IRES. Said host cells expressing TCRs specific for the polypeptide may be used, for example, for adoptive immunotherapy of WT1-associated cancer as discussed further below. 
     In further aspects of the present invention, cloned TCRs specific for a polypeptide recited herein may be used in a kit for the diagnosis of WT1-associated cancer. For example, the nucleic acid sequence or portions thereof, of tumor-specific TCRs can be used as probes or primers for the detection of expression of the rearranged genes encoding the specific TCR in a biological sample. Therefore, the present invention further provides for an assay for detecting messenger RNA or DNA encoding the TCR specific for a polypeptide. 
     Peptide-MHC Tetrameric Complexes 
     The present invention, in another aspect, provides peptide-MHC tetrameric complexes (tetramers) specific for T cells that recognize a polypeptide disclosed herein, or for a variant or derivative thereof. In one embodiment, tetramers may be used in the detection of WT1 specific T-cells. Tetramers may be used in monitoring WT1 specific immune responses, early detection of WT1 associated malignancies and for monitoring minimal residual disease. Tetramer staining is typically carried out with flow cytometric analysis and can be used to identify groups within a patient population suffering from a WT1 associated disease at a higher risk for relapse or disease progression. 
     Pharmaceutical Compositions 
     In additional embodiments, the present invention concerns formulation of one or more of the polynucleotide, polypeptide, T-cell, TCR, and/or antibody compositions disclosed herein in pharmaceutically-acceptable carriers for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. 
     It will be understood that, if desired, a composition as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may further comprise substituted or derivatized RNA or DNA compositions. 
     Therefore, in another aspect of the present invention, pharmaceutical compositions are provided comprising one or more of the polynucleotide, polypeptide, antibody, TCR, and/or T-cell compositions described herein in combination with a physiologically acceptable carrier. In certain preferred embodiments, the pharmaceutical compositions of the invention comprise immunogenic polynucleotide and/or polypeptide compositions of the invention for use in prophylactic and therapeutic vaccine applications. Vaccine preparation is generally described in, for example, M. F. Powell and M. J. Newman, eds., “Vaccine Design (the subunit and adjuvant approach),” Plenum Press (NY, 1995). Generally, such compositions will comprise one or more polynucleotide and/or polypeptide compositions of the present invention in combination with one or more immunostimulants. 
     It will be apparent that any of the pharmaceutical compositions described herein can contain pharmaceutically acceptable salts of the polynucleotides and polypeptides of the invention. Such salts can be prepared, for example, from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts). 
     In another embodiment, illustrative immunogenic compositions, e.g., vaccine compositions, of the present invention comprise DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. As noted above, the polynucleotide may be administered within any of a variety of delivery systems known to those of ordinary skill in the art. Indeed, numerous gene delivery techniques are well known in the art, such as those described by Rolland,  Crit. Rev. Therap. Drug Carrier Systems  15:143-198, 1998, and references cited therein. Appropriate polynucleotide expression systems will, of course, contain the necessary regulatory DNA regulatory sequences for expression in a patient (such as a suitable promoter and terminating signal). Alternatively, bacterial delivery systems may involve the administration of a bacterium (such as  Bacillus -Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. 
     Therefore, in certain embodiments, polynucleotides encoding immunogenic polypeptides described herein are introduced into suitable mammalian host cells for expression using any of a number of known viral-based systems. In one illustrative embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. A selected nucleotide sequence encoding a polypeptide of the present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109. 
     In addition, a number of illustrative adenovirus-based systems have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476). 
     Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988)  Molec. Cell. Biol.  8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875. 
     Additional viral vectors useful for delivering the polynucleotides encoding polypeptides of the present invention by gene transfer include those derived from the pox family of viruses, such as vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the novel molecules can be constructed as follows. The DNA encoding a polypeptide is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome. The resulting TK.sup.(−) recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto. 
     A vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression or coexpression of one or more polypeptides described herein in host cells of an organism. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide or polynucleotides of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into polypeptide by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126. 
     Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the coding sequences of interest. Recombinant avipoxviruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an Avipox vector is particularly desirable in human and other mammalian species since members of the Avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant Avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545. A number of pox viruses have been developed as live viral vectors for the expression of heterologous proteins (Cepko et al., Cell 37:1053-1062 (1984); Morin et al., Proc. Natl. Acad. Sci. USA 84:4626-4630 (1987); Lowe et al., Proc. Natl. Acad. Sci. USA, 84:3896-3900 (1987); Panicali &amp; Paoletti, Proc. Natl. Acad. Sci. USA, 79:4927-4931 (1982); Machett et al., Proc. Natl. Acad. Sci. USA, 79:7415-7419 (1982)). Representative fowlpox and swinepox virus are available through the ATCC under accession numbers VR-229 and VR-363, respectively. A recombinant vaccinia—CEA is available through the ATCC under accession number VR2323. Other illustrative viral vectors also include, but are not limited to, those described by Therion Biologics (Cambridge, Mass., USA), for example, in U.S. Pat. Nos. 6,051,410, 5,858,726, 5,656,465, 5,804,196, 5,747,324, 6,319,496, 6,165,460. 
     Any of a number of alphavirus vectors can also be used for delivery of polynucleotide compositions of the present invention, such as those vectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE) can also be used, illustrative examples of which can be found in U.S. Pat. Nos. 5,505,947 and 5,643,576. 
     Moreover, molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery under the invention. 
     Additional illustrative information on these and other known viral-based delivery systems can be found, for example, in Fisher-Hoch et al.,  Proc. Natl. Acad. Sci. USA  86:317-321, 1989; Flexner et al.,  Ann. N.Y. Acad. Sci.  569:86-103, 1989; Flexner et al.,  Vaccine  8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,  Biotechniques  6:616-627, 1988; Rosenfeld et al.,  Science  252:431-434, 1991; Kolls et al.,  Proc. Natl. Acad. Sci. USA  91:215-219, 1994; Kass-Eisler et al.,  Proc. Natl. Acad. Sci. USA  90:11498-11502, 1993; Guzman et al.,  Circulation  88:2838-2848, 1993; and Guzman et al.,  Cir. Res.  73:1202-1207, 1993. 
     As would be readily appreciated by the skilled artisan, any number of additional components may be present in a DNA or retroviral vector expressing a WT1 polypeptide or any portion thereof, as described herein. For example, an expression vector for delivery of a polynucleotide or peptide of the present invention may include any number of a variety of costimulatory molecules, including, but not limited to CD28, B7-1, ICAM-1, and LFA-3. A delivery vector may also include any number of cytokines, for example IFN-γ, GM-CSF, or IL-2. In one illustrative embodiment, a recombinant viral vector, e.g. a vaccinia or fowlpox vector, includes B7-1, ICAM-1, and LFA-3. 
     The present invention also comprises the use of any combination of the DNA and/or viral vectors described herein for use in the treatment of malignancies associated with the expression of WT1. In one illustrative embodiment, a recombinant vaccinia viral vector is administered to an animal or human patient afflicted with a WT1-associated malignancy, followed by administration of a recombinant fowlpox vector. In another embodiment of the present invention, the recombinant fowlpox is adminstered twice following administration of the vaccinia vector (e.g. a prime/boost/boost vaccination regimen). 
     In certain embodiments, a polynucleotide may be integrated into the genome of a target cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the polynucleotide may be stably maintained in the cell as a separate, episomal segment of DNA. Such polynucleotide segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. The manner in which the expression construct is delivered to a cell and where in the cell the polynucleotide remains is dependent on the type of expression construct employed. 
     In another embodiment of the invention, a polynucleotide is administered/delivered as “naked” DNA, for example as described in Ulmer et al.,  Science  259:1745-1749, 1993 and reviewed by Cohen,  Science  259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. 
     In still another embodiment, a composition of the present invention can be delivered via a particle bombardment approach, many of which have been described. In one illustrative example, gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, Wis.), some examples of which are described in U.S. Pat. Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799. This approach offers a needle-free delivery approach wherein a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high speed within a helium gas jet generated by a hand held device, propelling the particles into a target tissue of interest. 
     In a related embodiment, other devices and methods that may be useful for gas-driven needle-less injection of compositions of the present invention include those provided by Bioject, Inc. (Portland, Oreg.), some examples of which are described in U.S. Pat. Nos. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412. 
     According to another embodiment, the pharmaceutical compositions described herein will comprise one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T-cell, TCR, and/or APC compositions of this invention. An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A,  Bortadella pertussis  or  Mycobacterium tuberculosis  derived proteins. Certain adjuvants are commercially available as, for example, Freund&#39;s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants. 
     Within certain embodiments of the invention, the adjuvant composition is preferably one that induces an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Th1- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989. 
     Certain preferred adjuvants for eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A (MPL®), preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL™), together with an aluminum salt. MPL® adjuvants are available from Corixa Corporation (Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al.,  Science  273:352, 1996. Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or  Gypsophila  or  Chenopodium  quinoa saponins. Other preferred formulations include more than one saponin in the adjuvant combinations of the present invention, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, β-escin, or digitonin. 
     Alternatively the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOM®s (saponins, cholesterol and phospholipids). Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM®. The saponins may also be formulated with excipients such as CARBOPOL® to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose. 
     In one preferred embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL® adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation employing QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210. 
     Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 is disclosed in WO 00/09159. Preferably the formulation additionally comprises an oil in water emulsion and tocopherol. 
     Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention include MONTANIDE® ISA 720 (Seppic, France), SAF™ (Chiron, Calif., United States), ISCOM®s (CSL), MF-59™ (Chiron), the SBAS™ series of adjuvants (e.g., SBAS™-2 or SBAS™-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (ENHANZYN®) (Corixa, Hamilton, Mont.), RC-529™ (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in U.S. patent application Ser. No. 08/853,826, now issued as U.S. Pat. No. 6,113,918 and U.S. patent application Ser. No. 09/074,720, now issued as U.S. Pat. No. 6,355,257 and in U.S. Pat. No. 6,303,347 the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1. Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention include isotucerosol. 
     Other preferred adjuvants include adjuvant molecules of the general formula
 
HO(CH 2 CH 2 O) n -A-R,  (I)
 
     wherein, n is 1-50, A is a bond or —C(O)—, R is C 1-50  alkyl or Phenyl C 1-50  alkyl. 
     One embodiment of the present invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C 1-50 , preferably C 4 -C 20  alkyl and most preferably C 12  alkyl, and A is a bond. The concentration of the polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12 th  edition: entry 7717). These adjuvant molecules are described in WO 99/52549. 
     The polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2. 
     According to another embodiment of this invention, an immunogenic composition described herein is delivered to a host via antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells. 
     Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman,  Nature  392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy,  Ann. Rev. Med.  50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naïve T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al.,  Nature Med.  4:594-600, 1998). 
     Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells. 
     Dendritic cells are conveniently categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcγ receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1 BB). 
     APCs may generally be transfected with a polynucleotide of the invention (or portion or other variant thereof) such that the encoded polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a pharmaceutical composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al.,  Immunology and cell Biology  75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the tumor polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide. 
     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 typically 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, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration. 
     Carriers for use within such pharmaceutical compositions are biocompatible, and may also be biodegradable. In certain embodiments, the formulation preferably provides a relatively constant level of active component release. In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). 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. 
     In another illustrative embodiment, biodegradable microspheres (e.g., polylactate polyglycolate) are employed as carriers for the compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems such as described in WO/99 40934, and references cited therein, will also be useful for many applications. Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S. Pat. No. 5,928,647, which are capable of inducing a class 1-restricted cytotoxic T lymphocyte responses in a host. 
     In another illustrative embodiment, calcium phosphate core particles are employed as carriers, vaccine adjuvants, or as controlled release matrices for the compositions of this invention. Exemplary calcium phosphate particles are disclosed, for example, in published patent application No. WO/0046147. 
     The pharmaceutical compositions of the invention will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate. 
     The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are typically sealed in such a way to preserve the sterility and stability of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use. 
     The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation, is well known in the art, some of which are briefly discussed below for general purposes of illustration. 
     In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to an animal. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. 
     The active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et al., Nature 1997 Mar. 27; 386(6623):410-4; Hwang et al., Crit. Rev Ther Drug Carrier Syst 1998; 15(3):243-84; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451). Tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. 
     Typically, these formulations will contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. 
     For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth. 
     In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms. 
     Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No. 5,466,468). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. 
     In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington&#39;s Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards. 
     In another embodiment of the invention, the compositions disclosed herein may be formulated in a neutral or salt form. Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. 
     The carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. 
     In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., J Controlled Release 1998 Mar. 2;52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are also well-known in the pharmaceutical arts. Likewise, illustrative transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045. 
     In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like, are used for the introduction of the compositions of the present invention into suitable host cells/organisms. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Alternatively, compositions of the present invention can be bound, either covalently or non-covalently, to the surface of such carrier vehicles. 
     The formation and use of liposome and liposome-like preparations as potential drug carriers is generally known to those of skill in the art (see for example, Lasic, Trends Biotechnol 1998 July; 16(7):307-21; Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., Indian J Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit. Rev Ther Drug Carrier Syst. 1995; 12(2-3):233-61; U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587, each specifically incorporated herein by reference in its entirety). 
     Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other procedures, including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al., J Biol. Chem. 1990 Sep. 25; 265(27):16337-42; Muller et al., DNA Cell Biol. 1990 April; 9(3):221-9). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors and the like, into a variety of cultured cell lines and animals. Furthermore, he use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery. 
     In certain embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). 
     Alternatively, in other embodiments, the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (see, for example, Quintanar-Guerrero et al., Drug Dev Ind Pharm. 1998 December; 24(12):1113-28). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) may be designed using polymers able to be degraded in vivo. Such particles can be made as described, for example, by Couvreur et al., Crit. Rev Ther Drug Carrier Syst. 1988; 5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March; 45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan. 2;50(1-3):31-40; and U.S. Pat. No. 5,145,684. 
     Therapy of Malignant Diseases 
     Immunologic approaches to cancer therapy are based on the recognition that cancer cells can often evade the body&#39;s defenses against aberrant or foreign cells and molecules, and that these defenses might be therapeutically stimulated to regain the lost ground, e.g. pgs. 623-648 in Klein, Immunology (Wiley-Interscience, New York, 1982). Numerous recent observations that various immune effectors can directly or indirectly inhibit growth of tumors has led to renewed interest in this approach to cancer therapy, e.g. Jager, et al., Oncology 2001; 60(1):1-7; Renner, et al., Ann Hematol 2000 December; 79(12):651-9. 
     Four-basic cell types whose function has been associated with antitumor cell immunity and the elimination of tumor cells from the body are: i) B-lymphocytes which secrete immunoglobulins into the blood plasma for identifying and labeling the nonself invader cells; ii) monocytes which secrete the complement proteins that are responsible for lysing and processing the immunoglobulin-coated target invader cells; iii) natural killer lymphocytes having two mechanisms for the destruction of tumor cells, antibody-dependent cellular cytotoxicity and natural killing; and iv) T-lymphocytes possessing antigen-specific receptors and having the capacity to recognize a tumor cell carrying complementary marker molecules (Schreiber, H., 1989, in Fundamental Immunology (ed). W. E. Paul, pp. 923-955). 
     Cancer immunotherapy generally focuses on inducing humoral immune responses, cellular immune responses, or both. Moreover, it is well established that induction of CD4 +  T helper cells is necessary in order to secondarily induce either antibodies or cytotoxic CD8 +  T cells. Polypeptide antigens that are selective or ideally specific for cancer cells, particularly cancer cells associated with WT1 expression, offer a powerful approach for inducing immune responses against cancer associated with WT1 expression, and are an important aspect of the present invention. 
     In further aspects of the present invention, the compositions and vaccines described herein may be used to inhibit the development of malignant diseases (e.g., 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 WT1 expression. In other words, therapeutic methods provided herein may be used to treat an existing WT1-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 WT1 expression. 
     As used herein, a disease is “associated with WT1 expression” if diseased cells (e.g., tumor cells) at some time during the course of the disease generate detectably higher levels of a WT1 polypeptide than normal cells of the same tissue. Association of WT1 expression with a malignant disease does not require that WT1 be present on a tumor. For example, overexpression of WT1 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 WT1 expression may, at a later time, progress to a disease that is characterized by increased WT1 expression. Accordingly, any malignant disease in which diseased cells formerly expressed, currently express or are expected to subsequently express increased levels of WT1 is considered to be “associated with WT1 expression.” 
     Immunotherapy may be performed using any of a variety of techniques, in which compounds or cells provided herein function to remove WT1-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 WT1 or a cell expressing WT1. Alternatively, WT1-expressing cells may be removed ex vivo (e.g., 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 (i.e., prophylactically) or to treat a patient afflicted with a disease (e.g., to prevent or delay progression and/or metastasis of an existing disease). A patient afflicted with a disease may have a minimal residual disease (e.g., 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 (i.e., prevent or delay the relapse, or decrease the severity of a relapse). Within certain preferred embodiments, the patient is afflicted with a leukemia (e.g., AML, CML, ALL or childhood ALL), a myelodysplastic syndrome (MDS) or a cancer (e.g., gastrointestinal, lung, thyroid or breast cancer or a melanoma), where the cancer or leukemia is WT1 positive (i.e., reacts detectably with an anti-WT1 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 WT1-expressing cells. 
     Other diseases associated with WT1 overexpression include kidney cancer (such as renal cell carcinoma, or Wilms tumor), as described in Satoh F., et al.,  Pathol. Int.  50(6):458-71 (2000), and Campbell C. E. et al.,  Int. J. Cancer  78(2):182-8 (1998); and mesothelioma, as described in Amin, K. M. et al.,  Am. J. Pathol.  146(2):344-56 (1995). Harada et al. ( Mol. Urol.  3(4):357-364 (1999) describe WT1 gene expression in human testicular germ-cell tumors. Nonomura et al.  Hinyokika Kiyo  45(8):593-7 (1999) describe molecular staging of testicular cancer using polymerase chain reaction of the testicular cancer-specific genes. Shimizu et al.,  Int. J. Gynecol. Pathol.  19(2):158-63 (2000) describe the immunohistochemical detection of the Wilms&#39; tumor gene (WT1) in epithelial ovarian tumors. 
     WT1 overexpression was also described in desmoplastic small round cell tumors, by Barnoud, R. et al.,  Am. J. Surg. Pathol.  24(6):830-6 (2000); and  Pathol. Res. Pract.  194(10):693-700 (1998). WT1 overexpression in glioblastoma and other cancer was described by Menssen, H. D. et al.,  J. Cancer Res. Clin. Oncol.  126(4):226-32 (2000), “Wilms&#39; tumor gene (WT1) expression in lung cancer, colon cancer and glioblastoma cell lines compared to freshly isolated tumor specimens.” Other diseases showing WT1 overexpression include EBV associated diseases, such as Burkitt&#39;s lymphoma and nasopharyngeal cancer (Spinsanti P. et al.,  Leuk. Lymphoma  38(5-6):611-9 (2000), “Wilms&#39; tumor gene expression by normal and malignant human B lymphocytes.” 
     In  Leukemia  14(9):1634-4 (2000), Pan et al., describe in vitro IL-12 treatment of peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes, and reported an increase in cytotoxicity and reduction in WT1 gene expression. In  Leukemia  13(6):891-900 (1999), Patmasiriwat et al. reported WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia. In  Leukemia  13(3):393-9 (1999), Tamaki et al. reported that the Wilms&#39; tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Expression of the Wilms&#39; tumor gene WT1 in solid tumors, and its involvement in tumor cell growth, was discussed in relation to gastric cancer, colon cancer, lung cancer, breast cancer cell lines, germ cell tumor cell line, ovarian cancer, the uterine cancer, thyroid cancer cell line, hepatocellular carcinoma, in Oji et al.,  Jpn. J. Cancer Res.  90(2):194-204 (1999). 
     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 WT1-specific T-cells in vitro and/or in vivo. Such WT1-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 (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., 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 (i.e., untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient&#39;s tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent 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 μg 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 (e.g., 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 certain embodiments, immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as polypeptides and polynucleotides as provided herein). 
     Within other embodiments, immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system. Examples of effector cells include T cells as discussed above, T lymphocytes (such as CD8 +  cytotoxic T lymphocytes and CD4 +  T-helper tumor-infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein. T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Pat. No. 4,918,164) for passive immunotherapy. 
     Monoclonal antibodies may be labeled with any of a variety of labels for desired selective usages in detection, diagnostic assays or therapeutic applications (as described in U.S. Pat. Nos. 6,090,365; 6,015,542; 5,843,398; 5,595,721; and 4,708,930, hereby incorporated by reference in their entirety as if each was incorporated individually). In each case, the binding of the labelled monoclonal antibody to the determinant site of the antigen will signal detection or delivery of a particular therapeutic agent to the antigenic determinant on the non-normal cell. A further object of this invention is to provide the specific monoclonal antibody suitably labelled for achieving such desired selective usages thereof. 
     Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As noted above, immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic, macrophage, monocyte, fibroblast and/or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al.,  Immunological Reviews  157:177, 1997). 
     Alternatively, a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient. Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration. 
     Within further aspects, methods for inhibiting the development of a malignant disease associated with WT1 expression involve the administration of autologous T cells that have been activated in response to a WT1 polypeptide or WT1-expressing 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 1×10 9  to 1×10 11  T cells/M 2  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 WT1 polypeptide, a polynucleotide encoding a WT1 polypeptide and/or an APC that expresses a WT1 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 WT1-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 WT1 polypeptide, WT1 polynucleotide and/or APC that expresses a WT1 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 WT1-specific T cells may then be administered to a patient using standard techniques. 
     Within other embodiments, WT1-specific T cells as described herein may be used to remove cells expressing WT1 from autologous bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood (e.g., 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 WT1 expressing cells to less than 10%, preferably less than 5% and more preferably less than 1%, 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. 
     Cancer Detection and Diagnostic Compositions, Methods and Kits 
     In general, a cancer associated with WT1 expression may be detected in a patient based on the presence of one or more WT1 proteins and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, sputum urine and/or tumor biopsies) obtained from the patient. In other words, such WT1 proteins may be used as markers to indicate the presence or absence of a cancer. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. 
     Polynucleotide primers and probes may be used to detect the level of mRNA encoding a WT1 protein, which is also indicative of the presence or absence of a cancer. In general, a WT1 sequence should be present at a level that is at least two-fold, preferably three-fold, and more preferably five-fold or higher in tumor tissue than in normal tissue of the same type from which the tumor arose. Expression levels of WT1 in tissue types different from that in which the tumor arose are irrelevant in certain diagnostic embodiments since the presence of tumor cells can be confirmed by observation of predetermined differential expression levels, e.g., 2-fold, 5-fold, etc, in tumor tissue to expression levels in normal tissue of the same type. 
     Other differential expression patterns can be utilized advantageously for diagnostic purposes. For example, in one aspect of the invention, overexpression of WT1 sequence in tumor tissue and normal tissue of the same type, but not in other normal tissue types, e.g. PBMCs, can be exploited diagnostically. In this case, the presence of metastatic tumor cells, for example in a sample taken from the circulation or some other tissue site different from that in which the tumor arose, can be identified and/or confirmed by detecting expression of the tumor sequence in the sample, for example using RT-PCR analysis. In many instances, it will be desired to enrich for tumor cells in the sample of interest, e.g., PBMCs, using cell capture or other like techniques. 
     There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect WT1 polypeptide markers in a sample. See, e.g., Harlow and Lane,  Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a cancer associated with WT1 in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of WT1 polypeptide that binds to the binding agent; and (c) comparing the level of WT1 polypeptide with a predetermined cut-off value. 
     In a preferred embodiment, the assay involves the use of binding agent immobilized on a solid support to bind to and remove the WT1 polypeptide from the remainder of the sample. The bound WT1 polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/WT1 polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to a WT1 polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a WT1 polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled WT1 polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include full length WT1 proteins and polypeptide portions thereof to which the binding agent binds, as described above. 
     The solid support may be any material known to those of ordinary skill in the art to which the WT1 protein may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). 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 binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of binding agent. 
     Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13). 
     In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that WT1 polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group. 
     More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or TWEEN® 20 (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of WT1 polypeptide within a sample obtained from an individual with a cancer associated with WT1 least about 95% of that achieved at equilibrium between bound and unbound polypeptide. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient. 
     Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% TWEEN® 20. The second antibody, which contains a reporter group, may then be added to the solid support. Preferred reporter groups include those groups recited above. 
     The detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products. 
     To determine the presence or absence of a cancer associated with WT1 expression the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value for the detection of a cancer associated with WT1 is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the cancer. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al.,  Clinical Epidemiology: A Basic Science for Clinical Medicine , Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer. 
     In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. Concentration of second binding agent at the area of immobilized antibody indicates the presence of a cancer. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample. 
     Of course, numerous other assay protocols exist that are suitable for use with the WT1 proteins or binding agents of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use tumor polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such WT1-specific antibodies may correlate with the presence of a cancer associated with WT1 expression. 
     A cancer associated with WT1 expression may also, or alternatively, be detected based on the presence of T cells that specifically react with a tumor protein in a biological sample. Within certain methods, a biological sample comprising CD4 +  and/or CD8 +  T cells isolated from a patient is incubated with a WT1 polypeptide, a polynucleotide encoding such a polypeptide and/or an APC that expresses at least an immunogenic portion of such a polypeptide, and the presence or absence of specific activation of the T cells is detected. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37 C with polypeptide (e.g., 5-25 g/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of WT1 polypeptide to serve as a control. For CD4 +  T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8 +  T cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a cancer associated with WT1 expression in the patient. 
     As noted above, a cancer may also, or alternatively, be detected based on the level of mRNA encoding a WT1 protein in a biological sample. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a WT1 cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the WT1 protein. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. 
     Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding a WT1 protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the WT1 protein in a biological sample. 
     To permit hybridization under assay conditions, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a WT1 protein of the invention that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above. Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length. In a preferred embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule having a sequence as disclosed herein. Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al.,  Cold Spring Harbor Symp. Quant. Biol.,  51:263, 1987; Erlich ed., PCR  Technology , Stockton Press, NY, 1989). 
     One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and is reverse transcribed to produce cDNA molecules. PCR amplification using at least one specific primer generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis. Amplification may be performed on biological samples taken from a test patient and from an individual who is not afflicted with a cancer. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the test patient sample as compared to the same dilutions of the non-cancerous sample is typically considered positive. 
     In another aspect of the present invention, cell capture technologies may be used in conjunction, with, for example, real-time PCR to provide a more sensitive tool for detection of metastatic cells expressing WT1 antigens. Detection of WT1-associated cancer cells in biological samples, e.g., bone marrow samples, peripheral blood, and small needle aspiration samples is desirable for diagnosis and prognosis in patients with cancer associated with WT1 expression. 
     Immunomagnetic beads coated with specific monoclonal antibodies to surface cell markers, or tetrameric antibody complexes, may be used to first enrich or positively select cancer cells in a sample. Various commercially available kits may be used, including DYNABEADS® Epithelial Enrich (Dynal Biotech, Oslo, Norway), STEMSEP® (StemCell Technologies, Inc., Vancouver, BC), and ROSETTESEP® (StemCell Technologies). A skilled artisan will recognize that other methodologies and kits may also be used to enrich or positively select desired cell populations. DYNABEADS® Epithelial Enrich contains magnetic beads coated with mAbs specific for two glycoprotein membrane antigens expressed on normal and neoplastic epithelial tissues. The coated beads may be added to a sample and the sample then applied to a magnet, thereby capturing the cells bound to the beads. The unwanted cells are washed away and the magnetically isolated cells eluted from the beads and used in further analyses. 
     ROSETTESEP® can be used to enrich cells directly from a blood sample and consists of a cocktail of tetrameric antibodies that targets a variety of unwanted cells and cross-links them to glycophorin A on red blood cells (RBC) present in the sample, forming rosettes. When centrifuged over FICOLL®, targeted cells pellet along with the free RBC. The combination of antibodies in the depletion cocktail determines which cells will be removed and consequently which cells will be recovered. Antibodies that are available include, but are not limited to: CD2, CD3, CD4, CD5, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD24, CD25, CD29, CD33, CD34, CD36, CD38, CD41, CD45, CD45RA, CD45RO, CD56, CD66B, CD66e, HLA-DR, IgE, and TCRαβ. 
     In another embodiment, the compositions described herein may be used as markers for the progression of cancer. In this embodiment, assays as described above for the diagnosis of a cancer associated with WT1 expression may be performed over time, and the change in the level of reactive polypeptide(s) or polynucleotide(s) evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a cancer is progressing in those patients in whom the level of WT1 polypeptide or polynucleotide detected increases over time. In contrast, the cancer is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time. 
     Certain in vivo diagnostic assays may be performed directly on a tumor. One such assay involves contacting tumor cells with a binding agent. The bound binding agent may then be detected directly or indirectly via a reporter group. Such binding agents may also be used in histological applications. Alternatively, polynucleotide probes may be used within such applications. 
     The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a WT1 protein. Such antibodies or fragments may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding. 
     Alternatively, a kit may be designed to detect the level of mRNA encoding a WT1 protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a WT1 protein. Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a WT1 protein. 
     The following Examples are offered by way of illustration and not by way of limitation. 
     EXAMPLES 
     Example 1 
     Identification of an Immune Response to WT1 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 WT1 specific antibody responses in patients, sera of patients with acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML) and severe aplastic anemia were analyzed using Western blot analysis. Sera were tested for the ability to immunoprecipitate WT1 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 WT1 antibody WT180 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). 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  FIG. 2 . A 52 kD protein in the immunoprecipitate generated using the patient sera was recognized by the WT1 specific antibody. The 52 kD protein migrated at the same size as the positive control. 
     Additional studies analyzed the sera of patients with AML and CML for the presence of antibodies to full-length and truncated WT1 proteins. cDNA constructs representing the human WT1/full-length (aa 1-449), the N-terminus (aa 1-249) (WT1/N-terminus) and C-terminus (aa 267-449) (WT1/C-terminus) region were subcloned into modified pET28 vectors. The WT1/full-length and WT1/N-terminus proteins were expressed as Ra12 fusion proteins. Ra12 is the C-terminal fragment of a secreted  Mycobacterium tuberculosis  protein, denoted as MTB32B. (Skeiky et al.,  Infect Immun.  67; 3998, 1999). The Ra12-WT1/full-length fusion region was cloned 3′ to a histidine-tag in a histidine-tag modified pET28 vector. The WT1/N-terminus region was subcloned into a modified pET28 vector that has a 5′ histidine-tag followed by the thioredoxin (TRX)-WT1/N-terminus fusion region followed by a 3′ histidine-tag. The WT1/C-terminus coding region was subcloned into a modified pET28 vector without a fusion partner containing only the 5′ and 3′ histidine-tag, followed by a Thrombin and EK site. 
     BL21 pLysS  E. coli  (Stratagene, La Jolla, Calif.) were transformed with the three WT1 expression constructs, grown overnight and induced with isopropyl-β-D-thiogalactoside (IPTG). WT1 proteins were purified as follows: Cells were harvested and lysed by incubation in 10 mM Tris, pH 8.0 with Complete Protease Inhibitor Tablets (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) at 37° C. followed by repeated rounds of sonication. Inclusion bodies were washed twice with 10 mM Tris, pH 8.0. Proteins were then purified by metal chelate affinity chromatography over nickel-nitrilotriacetic acid resin (QIAGEN Inc., Valencia, Calif.; Hochuli et al.,  Biologically Active Molecules: 217, 1989) followed by chromatography on a Source Q anion exchange resin (Amersham Pharmacia Biotech, Upsala, Sweden). The identity of the WT1 proteins was confirmed by N-terminal sequencing. 
     Sera from adult patients with de nova AML or CML were studied for the presence of WT1 specific Ab. Recombinant proteins were adsorbed to TC microwell plates (Nunc, Roskilde, Denmark). Plates were washed with PBS/0.5% TWEEN® 20 and blocked with 1% BSA/PBS/0.1% TWEEN® 20. After washing, serum dilutions were added and incubated overnight at 4° C. Plates were washed and Donkey anti-human IgG-HRP secondary antibody was added (Jackson-Immunochem, West Grove, Pa.) and incubated for 2 h at room temperature. Plates were washed, incubated with TMB Peroxidase substrate solution (Kirkegaard and Perry Laboratories, Mass.), quenched with 1N H 2 SO 4 , and immediately read (Cyto-Fluor 2350; Millipore, Bedford, Mass.). 
     For the serological survey, human sera were tested by ELISA over a range of serial dilutions from 1:50 to 1:20,000. A positive reaction was defined as an OD value of a 1:500 diluted serum that exceeded the mean OD value of sera from normal donors (n=96) by three (WT1/full-length, WT1C-terminus) standard deviations. Due to a higher background in normal donors to the WT1/N-terminus protein a positive reaction to WT1/N-terminus was defined as an OD value of 1:500 diluted serum that exceeded the mean OD value of sera from normal donors by four standard deviations. To verify that the patient Ab response was directed against WT1 and not to the Ra12 or TRX fusion part of the protein or possible  E. coli  contaminant proteins, controls included the Ra12 and TRX protein alone purified in a similar manner. Samples that showed reactivity against the Ra12 and/or TRX proteins were excluded from the analysis. 
     To evaluate for the presence of immunity to WT1, Ab to recombinant full-length and truncated WT1 proteins in the sera of normal individuals and patients with leukemia were determined. Antibody reactivity was analyzed by ELISA reactivity to WT1/full-length protein, WT1/N-terminus protein and WT1/C-terminus protein. 
     Only 2 of 96 normal donors had serum antibodies reactive with WT1/full-length protein ( FIG. 18 ). One of those individuals had antibody to WT1/N-terminus protein and one had antibody to WT1/C-terminus protein. In contrast, 16 of 63 patients (25%) with AML had serum antibodies reactive with WT1/full-length protein. By marked contrast, only 2 of 63 patients (3%) had reactivity to WT1/C-terminus protein. Fifteen of 81 patients (19%) with CML had serum antibodies reactive with WT1/full-length protein and 12 of 81 patients (15%) had serum antibodies reactive with WT1/N-terminus. Only 3 of 81 patients (3%) had reactivity to WT1/C-terminus protein. ( FIGS. 16 and 17 .) 
     These data demonstrate that Ab responses to WT1 are detectable in some patients with AML and CML. The greater incidence of antibody in leukemia patients provides strong evidence that immunization to the WT1 protein occurred as a result of patients bearing malignancy that expresses or at some time expressed WT1. Without being limited to a specific theory, it is believed that the observed antibody responses to WT1 most probably result from patients becoming immune to WT1 on their own leukemia cells and provide direct evidence that WT1 can be immunogenic despite being a “self” protein. 
     The presence of antibody to WT1 strongly implies that concurrent helper T cell responses are also present in the same patients. WT1 is an internal protein. Thus, CTL responses are likely to be the most effective in terms of leukemia therapy and the most toxic arm of immunity. Thus, these data provide evidence that therapeutic vaccines directed against WT1 will be able to elicit an immune response to WT1. 
     The majority of the antibodies detected were reactive with epitopes within the N-terminus while only a small subgroup of patients showed a weak antibody response to the C-terminus. This is consistent with observations in the animal model, where immunization with peptides derived from the N-terminus elicited antibody, helper T cell and CTL responses, whereas none of the peptides tested from the C-terminus elicited antibody or T cell responses (Gaiger et al.,  Blood  96:1334, 2000). 
     Example 2 
     Induction of Antibodies to WT1 in Mice Immunized with Cell Lines Expressing WT1 
     This Example illustrates the use of cells expressing WT1 to induce a WT1 specific antibody response in vivo. 
     Detection of existent antibodies to WT1 in patients with leukemia strongly implied that it is possible to immunize to WT1 protein to elicit immunity to WT1. To test whether immunity to WT1 can be generated by vaccination, mice were injected with TRAMP-C, a WT1 positive tumor cell line of B6 origin. Briefly, male B6 mice were immunized with 5×10 6  TRAMP-C cells subcutaneously and boosted twice with 5×10 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 25μM β-2-mercaptoethanol, 200 units of penicillin per ml, 10 mM L-glutamine, and 10% fetal bovine serum. 
     Following immunization to TRAMP-C, a WT1 specific antibody response in the immunized animals was detectable. A representative Western blot is shown in  FIG. 3 . These results show that immunization to WT1 protein can elicit an immune response to WT1 protein. 
     Example 3 
     Induction of Th and Antibody Responses in Mice Immunized with WT1 Peptides 
     This Example illustrates the ability of immunization with WT1 peptides to elicit an immune response specific for WT1. 
     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 
               
             
            
               
                   
               
               
                 WT1 Peptides 
               
            
           
           
               
               
               
            
               
                 Peptide 
                 Sequence 
                 Comments 
               
               
                   
               
               
                 Mouse: p6-22 
                 RDLNALLPAVSSLGGGG 
                 1 mismatch 
               
               
                   
                 (SEQ ID NO: 13) 
                 relative to 
               
               
                   
                   
                 human WT1 
               
               
                   
                   
                 sequence 
               
               
                   
               
               
                 Human: p6-22 
                 RDLNALLPAVPSLGGGG 
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
               
               
                 Human/mouse: 
                 PSQASSGQARMFPNAPYLPSCLE 
               
               
                 p117-139 
                 (SEQ ID NOs: 2 and 3) 
               
               
                   
               
               
                 Mouse: p244- 
                 GATLKGMAAGSSSSVKWTE 
                 1 mismatch 
               
               
                 262 
                 (SEQ ID NO: 14) 
                 relative to 
               
               
                   
                   
                 human WT1 
               
               
                   
                   
                 sequence 
               
               
                   
               
               
                 Human: p244- 
                 GATLKGVAAGSSSSVKWTE 
               
               
                 262 
                 (SEQ ID NO: 4) 
               
               
                   
               
               
                 Human/mouse: 
                 RIHTHGVFRGIQDVR 
               
               
                 p287-301 
                 (SEQ ID NOs: 15 and 16) 
               
               
                   
               
               
                 Mouse: p299- 
                 VRRVSGVAPTLVRS 
                 1 mismatch 
               
               
                 313 
                 (SEQ ID NO: 17) 
                 relative to 
               
               
                   
                   
                 human WT1 
               
               
                   
                   
                 sequence 
               
               
                   
               
               
                 Human/mouse: 
                 CQKKFARSDELVRHH 
               
               
                 p421-435 
                 (SEQ ID NOs: 19 and 20) 
               
               
                   
               
            
           
         
       
     
     For immunization, peptides were grouped as follows:
         Group A: p6-22 human: 10.9 mg in 1 ml (10 μl=100 μg)
           p117-139 human/mouse: 7.6 mg in 1 ml (14 μl=100 μg)   p244-262 human: 4.6 mg in 1 ml (22 μl=100 μg)   
           Group B: p287-301 human/mouse: 7.2 mg in 1 ml (14 μl=100 μg)
           mouse p299-313: 6.6.mg in 1 ml (15 μl=100 μg)   p421-435 human/mouse: 3.3 mg in 1 ml (30 μl=100 μg)   
           Control: (FBL peptide 100 μg)+CFA/IFA   Control: (CD45 peptide 100 μg)+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 p299-313 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 WT1 peptides or with a control peptide. Peptides were dissolved in 1 ml 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 MONTANIDE®. 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 2×10 5  cells per well with 4×10 5  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 WT1 ( FIG. 4 ). 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. P117-139 elicited proliferative T cell responses ( FIGS. 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). 
       FIGS. 6A and 6B  show the proliferative response observed for each of the three peptides within vaccine A ( FIG. 6A ) and vaccine B ( FIG. 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 ( FIG. 6A ). 
     Subsequent in vitro stimulations were carried out as single peptide stimulations using only p6-22 and p117-139. Stimulation of the Vaccine A specific T cell line with p117-139 resulted in proliferation to p117-139 with no response to p6-22 ( FIG. 7A ). Clones derived from the line were specific for p117-139 ( FIG. 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 ( FIG. 7C ). Clones derived from the line were specific for p6-22 ( FIG. 7D ). 
     These results show that vaccination with WT1 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 WT1 Peptides 
     This Example illustrates the ability of WT1 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. Also indicated in the tables are the defined peptide binding motifs as described by Rammensee, et al., Immunogenetics 41:178-228, 1995, incorporated herein in its entirety. The peptide binding motif for HLA-B14 is described in DiBrino et al., J. Biol. Chem. 269:23426-23434, 1994, also incorporated herein it its entirety. Peptide positions are abbreviated as P1, P2, P3, P4, P5, P6, P7, P8, and P9. 
     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  FIGS. 8A and 8B , and Table XLV. 
     
       
         
           
               
             
               
                 TABLE II 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA A1 (HLA-A1 peptide binding motif 
               
               
                 anchor residues are D or E at position 3 (P3); Y 
               
               
                 at P9; with auxiliary anchors being P at P4 and 
               
               
                 L at P7) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 137 
                 CLESQPAIR 
                 18.000 
               
               
                   
                   
                 (SEQ ID NO: 47) 
               
               
                   
               
               
                 2 
                 80 
                 GAEPHEEQC 
                 9.000 
               
               
                   
                   
                 (SEQ ID NO: 87) 
               
               
                   
               
               
                 3 
                 40 
                 FAPPGASAY 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 74) 
               
               
                   
               
               
                 4 
                 354 
                 QCDFKDCER 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 162) 
               
               
                   
               
               
                 5 
                 2 
                 GSDVRDLNA 
                 3.750 
               
               
                   
                   
                 (SEQ ID NO: 101) 
               
               
                   
               
               
                 6 
                 152 
                 VTFDGTPSY 
                 2.500 
               
               
                   
                   
                 (SEQ ID NO: 244) 
               
               
                   
               
               
                 7 
                 260 
                 WTEGQSNHS 
                 2.250 
               
               
                   
                   
                 (SEQ ID NO: 247) 
               
               
                   
               
               
                 8 
                 409 
                 TSEKPFSCR 
                 1.350 
               
               
                   
                   
                 (SEQ ID NO: 232) 
               
               
                   
               
               
                 9 
                 73 
                 KQEPSWGGA 
                 1.350 
               
               
                   
                   
                 (SEQ ID NO: 125) 
               
               
                   
               
               
                 10 
                 386 
                 KTCQRKFSR 
                 1.250 
               
               
                   
                   
                 (SEQ ID NO: 128) 
               
               
                   
               
               
                 11 
                 37 
                 VLDFAPPGA 
                 1.000 
               
               
                   
                   
                 (SEQ ID NO: 241) 
               
               
                   
               
               
                 12 
                 325 
                 CAYPGCNKR 
                 1.000 
               
               
                   
                   
                 (SEQ ID NO: 44) 
               
               
                   
               
               
                 13 
                 232 
                 QLECMTWNQ 
                 0.900 
               
               
                   
                   
                 (SEQ ID NO: 167) 
               
               
                   
               
               
                 14 
                 272 
                 ESDNHTTPI 
                 0.750 
               
               
                   
                   
                 (SEQ ID NO: 71) 
               
               
                   
               
               
                 15 
                 366 
                 RSDQLKRHQ 
                 0.750 
               
               
                   
                   
                 (SEQ ID NO: 193) 
               
               
                   
               
               
                 16 
                 222 
                 SSDNLYQMT 
                 0.750 
               
               
                   
                   
                 (SEQ ID NO: 217) 
               
               
                   
               
               
                 17 
                 427 
                 RSDELVRHH 
                 0.750 
               
               
                   
                   
                 (SEQ ID NO: 191) 
               
               
                   
               
               
                 18 
                 394 
                 RSDHLKTHT 
                 0.750 
               
               
                   
                   
                 (SEQ ID NO: 192) 
               
               
                   
               
               
                 19 
                 317 
                 TSEKRPFMC 
                 0.675 
               
               
                   
                   
                 (SEQ ID NO: 233) 
               
               
                   
               
               
                 20 
                 213 
                 QALLLRTPY 
                 0.500 
               
               
                   
                   
                 (SEQ ID NO: 160) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE III 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA A 0201 (HLA-A0201 peptide binding motif 
               
               
                 anchor residues are L or M at P2; V or L at P9; 
               
               
                 with an auxiliary anchor being V at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 126 
                 RMFPNAPYL 
                 313.968 
               
               
                   
                   
                 (SEQ ID NO: 185) 
               
               
                   
               
               
                 2 
                 187 
                 SLGEQQYSV 
                 285.163 
               
               
                   
                   
                 (SEQ ID NO: 214) 
               
               
                   
               
               
                 3 
                 10 
                 ALLPAVPSL 
                 181.794 
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
               
                 4 
                 242 
                 NLGATLKGV 
                 159.970 
               
               
                   
                   
                 (SEQ ID NO: 146) 
               
               
                   
               
               
                 5 
                 225 
                 NLYQMTSQL 
                 68.360 
               
               
                   
                   
                 (SEQ ID NO: 147) 
               
               
                   
               
               
                 6 
                 292 
                 GVFRGIQDV 
                 51.790 
               
               
                   
                   
                 (SEQ ID NO: 103) 
               
               
                   
               
               
                 7 
                 191 
                 QQYSVPPPV 
                 22.566 
               
               
                   
                   
                 (SEQ ID NO: 171) 
               
               
                   
               
               
                 8 
                 280 
                 ILCGAQYRI 
                 17.736 
               
               
                   
                   
                 (SEQ ID NO: 116) 
               
               
                   
               
               
                 9 
                 235 
                 CMTWNQMNL 
                 15.428 
               
               
                   
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                 10 
                 441 
                 NMTKLQLAL 
                 15.428 
               
               
                   
                   
                 (SEQ ID NO: 149) 
               
               
                   
               
               
                 11 
                 7 
                 DLNALLPAV 
                 11.998 
               
               
                   
                   
                 (SEQ ID NO: 58) 
               
               
                   
               
               
                 12 
                 227 
                 YQMTSQLEC 
                 8.573 
               
               
                   
                   
                 (SEQ ID NO: 251) 
               
               
                   
               
               
                 13 
                 239 
                 NQMNLGATL 
                 8.014 
               
               
                   
                   
                 (SEQ ID NO: 151) 
               
               
                   
               
               
                 14 
                 309 
                 TLVRSASET 
                 7.452 
               
               
                   
                   
                 (SEQ ID NO: 226) 
               
               
                   
               
               
                 15 
                 408 
                 KTSEKPFSC 
                 5.743 
               
               
                   
                   
                 (SEQ ID NO: 129) 
               
               
                   
               
               
                 16 
                 340 
                 LQMHSRKHT 
                 4.752 
               
               
                   
                   
                 (SEQ ID NO: 139) 
               
               
                   
               
               
                 17 
                 228 
                 QMTSQLECM 
                 4.044 
               
               
                   
                   
                 (SEQ ID NO: 169) 
               
               
                   
               
               
                 18 
                 93 
                 TVHFSGQFT 
                 3.586 
               
               
                   
                   
                 (SEQ ID NO: 235) 
               
               
                   
               
               
                 19 
                 37 
                 VLDFAPPGA 
                 3.378 
               
               
                   
                   
                 (SEQ ID NO: 241) 
               
               
                   
               
               
                 20 
                 86 
                 EQCLSAFTV 
                 3.068 
               
               
                   
                   
                 (SEQ ID NO: 69) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE IV 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA A 0205 (HLA-A0205 peptide binding motif 
               
               
                 anchor residues are L at P9 with auxiliary anchors 
               
               
                 being V, L, I, M at P2 and I, V, L, A, at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 10 
                 ALLPAVPSL 
                 42.000 
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
               
                 2 
                 292 
                 GVFRGIQDV 
                 24.000 
               
               
                   
                   
                 (SEQ ID NO: 103) 
               
               
                   
               
               
                 3 
                 126 
                 RMFPNAPYL 
                 21.000 
               
               
                   
                   
                 (SEQ ID NO: 185) 
               
               
                   
               
               
                 4 
                 225 
                 NLYQMTSQL 
                 21.000 
               
               
                   
                   
                 (SEQ ID NO: 147) 
               
               
                   
               
               
                 5 
                 239 
                 NQMNLGATL 
                 16.800 
               
               
                   
                   
                 (SEQ ID NO: 151) 
               
               
                   
               
               
                 6 
                 302 
                 RVPGVAPTL 
                 14.000 
               
               
                   
                   
                 (SEQ ID NO: 195) 
               
               
                   
               
               
                 7 
                 441 
                 NMTKLQLAL 
                 7.000 
               
               
                   
                   
                 (SEQ ID NO: 149) 
               
               
                   
               
               
                 8 
                 235 
                 CMTWNQMNL 
                 7.000 
               
               
                   
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                 9 
                 187 
                 SLGEQQYSV 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 214) 
               
               
                   
               
               
                 10 
                 191 
                 QQYSVPPPV 
                 4.800 
               
               
                   
                   
                 (SEQ ID NO: 171) 
               
               
                   
               
               
                 11 
                 340 
                 LQMHSRKHT 
                 4.080 
               
               
                   
                   
                 (SEQ ID NO: 139) 
               
               
                   
               
               
                 12 
                 242 
                 NLGATLKGV 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 146) 
               
               
                   
               
               
                 13 
                 227 
                 YQMTSQLEC 
                 3.600 
               
               
                   
                   
                 (SEQ ID NO: 251) 
               
               
                   
               
               
                 14 
                 194 
                 SVPPPVYGC 
                 2.000 
               
               
                   
                   
                 (SEQ ID NO: 218) 
               
               
                   
               
               
                 15 
                 93 
                 TVHFSGQFT 
                 2.000 
               
               
                   
                   
                 (SEQ ID NO: 235) 
               
               
                   
               
               
                 16 
                 280 
                 ILCGAQYRI 
                 1.700 
               
               
                   
                   
                 (SEQ ID NO: 116) 
               
               
                   
               
               
                 17 
                 98 
                 GQFTGTAGA 
                 1.200 
               
               
                   
                   
                 (SEQ ID NO: 99) 
               
               
                   
               
               
                 18 
                 309 
                 TLVRSASET 
                 1.000 
               
               
                   
                   
                 (SEQ ID NO: 226) 
               
               
                   
               
               
                 19 
                 81 
                 AEPHEEQCL 
                 0.980 
               
               
                   
                   
                 (SEQ ID NO: 30) 
               
               
                   
               
               
                 20 
                 73 
                 KQEPSWGGA 
                 0.960 
               
               
                   
                   
                 (SEQ ID NO: 125) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE V 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA A24 (HLA-A24 peptide binding motif 
               
               
                 anchor residues are Y at P2; I, L, or F at P9; 
               
               
                 with auxiliary anchors I or V at P5 and F at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 302 
                 RVPGVAPTL 
                 16.800 
               
               
                   
                   
                 (SEQ ID NO: 195) 
               
               
                   
               
               
                 2 
                 218 
                 RTPYSSDNL 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 194) 
               
               
                   
               
               
                 3 
                 356 
                 DFKDCERRF 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 55) 
               
               
                   
               
               
                 4 
                 126 
                 RMFPNAPYL 
                 9.600 
               
               
                   
                   
                 (SEQ ID NO: 185) 
               
               
                   
               
               
                 5 
                 326 
                 AYPGCNKRY 
                 7.500 
               
               
                   
                   
                 (SEQ ID NO: 42) 
               
               
                   
               
               
                 6 
                 270 
                 GYESDNHT 
                 7.500 
               
               
                   
                   
                 (SEQ ID NO: 106) 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 7 
                 239 
                 NQMNLGATL 
                 7.200 
               
               
                   
                   
                 (SEQ ID NO: 151) 
               
               
                   
               
               
                 8 
                 10 
                 ALLPAVPSL 
                 7.200 
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
               
                 9 
                 130 
                 NAPYLPSCL 
                 7.200 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 10 
                 329 
                 GCNKRYFKL 
                 6.600 
               
               
                   
                   
                 (SEQ ID NO: 90) 
               
               
                   
               
               
                 11 
                 417 
                 RWPSCQKKF 
                 6.600 
               
               
                   
                   
                 (SEQ ID NO: 196) 
               
               
                   
               
               
                 12 
                 47 
                 AYGSLGGPA 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 41) 
               
               
                   
               
               
                 13 
                 180 
                 DPMGQQGSL 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 59) 
               
               
                   
               
               
                 14 
                 4 
                 DVRDLNALL 
                 5.760 
               
               
                   
                   
                 (SEQ ID NO: 62) 
               
               
                   
               
               
                 15 
                 285 
                 QYRIHTHGV 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 175) 
               
               
                   
               
               
                 16 
                 192 
                 QYSVPPPVY 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 176) 
               
               
                   
               
               
                 17 
                 207 
                 DSCTGSQAL 
                 4.800 
               
               
                   
                   
                 (SEQ ID NO: 61) 
               
               
                   
               
               
                 18 
                 441 
                 NMTKLQLAL 
                 4.800 
               
               
                   
                   
                 (SEQ ID NO: 149) 
               
               
                   
               
               
                 19 
                 225 
                 NLYQMTSQL 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 147) 
               
               
                   
               
               
                 20 
                 235 
                 CMTWNQMNL 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE VI 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA A3 (HLA-A3 peptide binding motif 
               
               
                 anchor residues are L, V, or M at P2; K, Y, or 
               
               
                 F at P9; auxiliary anchors are F or Y at P3, I, 
               
               
                 M, F, V, or L at P6; I, L, M, F at P7) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 436 
                 NMHQRNMTK 
                 40.000 
               
               
                   
                   
                 (SEQ ID NO: 148) 
               
               
                   
               
               
                 2 
                 240 
                 QMNLGATLK 
                 20.000 
               
               
                   
                   
                 (SEQ ID NO: 168) 
               
               
                   
               
               
                 3 
                 88 
                 CLSAFTVHF 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 48) 
               
               
                   
               
               
                 4 
                 126 
                 RMFPNAPYL 
                 4.500 
               
               
                   
                   
                 (SEQ ID NO: 185) 
               
               
                   
               
               
                 5 
                 169 
                 AQFPNHSFK 
                 4.500 
               
               
                   
                   
                 (SEQ ID NO: 36) 
               
               
                   
               
               
                 6 
                 10 
                 ALLPAVPSL 
                 4.050 
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
               
                 7 
                 137 
                 CLESQPAIR 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 47) 
               
               
                   
               
               
                 8 
                 225 
                 NLYQMTSQL 
                 3.000 
               
               
                   
                   
                 (SEQ ID NO: 147) 
               
               
                   
               
               
                 9 
                 32 
                 AQWAPVLDF 
                 2.700 
               
               
                   
                   
                 (SEQ ID NO: 37) 
               
               
                   
               
               
                 10 
                 280 
                 ILCGAQYRI 
                 2.700 
               
               
                   
                   
                 (SEQ ID NO: 116) 
               
               
                   
               
               
                 11 
                 386 
                 KTCQRKFSR 
                 1.800 
               
               
                   
                   
                 (SEQ ID NO: 128) 
               
               
                   
               
               
                 12 
                 235 
                 CMTWNQMNL 
                 1.200 
               
               
                   
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                 13 
                 441 
                 NMTKLQLAL 
                 1.200 
               
               
                   
                   
                 (SEQ ID NO: 149) 
               
               
                   
               
               
                 14 
                 152 
                 VTFDGTPSY 
                 1.000 
               
               
                   
                   
                 (SEQ ID NO: 244) 
               
               
                   
               
               
                 15 
                 187 
                 SLGEQQYSV 
                 0.900 
               
               
                   
                   
                 (SEQ ID NO: 214) 
               
               
                   
               
               
                 16 
                 383 
                 FQCKTCQRK 
                 0.600 
               
               
                   
                   
                 (SEQ ID NO: 80) 
               
               
                   
               
               
                 17 
                 292 
                 GVFRGIQDV 
                 0.450 
               
               
                   
                   
                 (SEQ ID NO: 103) 
               
               
                   
               
               
                 18 
                 194 
                 SVPPPVYGC 
                 0.405 
               
               
                   
                   
                 (SEQ ID NO: 218) 
               
               
                   
               
               
                 19 
                 287 
                 RIHTHGVFR 
                 0.400 
               
               
                   
                   
                 (SEQ ID NO: 182) 
               
               
                   
               
               
                 20 
                 263 
                 GQSNHSTGY 
                 0.360 
               
               
                   
                   
                 (SEQ ID NO: 100) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE VII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA A68.1 (HLA-A68.1 peptide binding motif 
               
               
                 anchor residues are V or T at P2; R or K at P9 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 100 
                 FTGTAGACR 
                 100.000 
               
               
                   
                   
                 (SEQ ID NO: 84) 
               
               
                   
               
               
                 2 
                 386 
                 KTCQRKFSR 
                 50.000 
               
               
                   
                   
                 (SEQ ID NO: 128) 
               
               
                   
               
               
                 3 
                 368 
                 DQLKRHQRR 
                 30.000 
               
               
                   
                   
                 (SEQ ID NO: 60) 
               
               
                   
               
               
                 4 
                 312 
                 RSASETSEK 
                 18.000 
               
               
                   
                   
                 (SEQ ID NO: 190) 
               
               
                   
               
               
                 5 
                 337 
                 LSHLQMHSR 
                 15.000 
               
               
                   
                   
                 (SEQ ID NO: 141) 
               
               
                   
               
               
                 6 
                 364 
                 FSRSDQLKR 
                 15.000 
               
               
                   
                   
                 (SEQ ID NO: 83) 
               
               
                   
               
               
                 7 
                 409 
                 TSEKPFSCR 
                 15.000 
               
               
                   
                   
                 (SEQ ID NO: 232) 
               
               
                   
               
               
                 8 
                 299 
                 DVRRVPGVA 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 63) 
               
               
                   
               
               
                 9 
                 4 
                 DVRDLNALL 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 62) 
               
               
                   
               
               
                 10 
                 118 
                 SQASSGQAR 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 216) 
               
               
                   
               
               
                 11 
                 343 
                 HSRKHTGEK 
                 9.000 
               
               
                   
                   
                 (SEQ ID NO: 111) 
               
               
                   
               
               
                 12 
                 169 
                 AQFPNHSFK 
                 9.000 
               
               
                   
                   
                 (SEQ ID NO: 36) 
               
               
                   
               
               
                 13 
                 292 
                 GVFRGIQDV 
                 8.000 
               
               
                   
                   
                 (SEQ ID NO: 103) 
               
               
                   
               
               
                 14 
                 325 
                 CAYPGCNKR 
                 7.500 
               
               
                   
                   
                 (SEQ ID NO: 44) 
               
               
                   
               
               
                 15 
                 425 
                 FARSDELVR 
                 7.500 
               
               
                   
                   
                 (SEQ ID NO: 75) 
               
               
                   
               
               
                 16 
                 354 
                 QCDFKDCER 
                 7.500 
               
               
                   
                   
                 (SEQ ID NO: 162) 
               
               
                   
               
               
                 17 
                 324 
                 MCAYPGCNK 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 142) 
               
               
                   
               
               
                 18 
                 251 
                 AAGSSSSVK 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 28) 
               
               
                   
               
               
                 19 
                 379 
                 GVKPFQCKT 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 104) 
               
               
                   
               
               
                 20 
                 137 
                 CLESQPAIR 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 47) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE VIII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA A 1101 (HLA-A1101 peptide binding motif 
               
               
                 anchor residues are K at P9; auxiliary anchor 
               
               
                 residues are V, I, F, Y at P2, M, L, F, Y, I, A, 
               
               
                 at P3, L, I, Y, V, or F at P7) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 386 
                 KTCQRKFSR 
                 1.800 
               
               
                   
                   
                 (SEQ ID NO: 128) 
               
               
                   
               
               
                 2 
                 169 
                 AQFPNHSFK 
                 1.200 
               
               
                   
                   
                 (SEQ ID NO: 36) 
               
               
                   
               
               
                 3 
                 436 
                 NMHQRNMTK 
                 0.800 
               
               
                   
                   
                 (SEQ ID NO: 148) 
               
               
                   
               
               
                 4 
                 391 
                 KFSRSDHLK 
                 0.600 
               
               
                   
                   
                 (SEQ ID NO: 120) 
               
               
                   
               
               
                 5 
                 373 
                 HQRRHTGVK 
                 0.600 
               
               
                   
                   
                 (SEQ ID NO: 109) 
               
               
                   
               
               
                 6 
                 383 
                 FQCKTCQRK 
                 0.600 
               
               
                   
                   
                 (SEQ ID NO: 80) 
               
               
                   
               
               
                 7 
                 363 
                 RFSRSDQLK 
                 0.600 
               
               
                   
                   
                 (SEQ ID NO: 178) 
               
               
                   
               
               
                 8 
                 240 
                 QMNLGATLK 
                 0.400 
               
               
                   
                   
                 (SEQ ID NO: 168) 
               
               
                   
               
               
                 9 
                 287 
                 RIHTHGVFR 
                 0.240 
               
               
                   
                   
                 (SEQ ID NO: 182) 
               
               
                   
               
               
                 10 
                 100 
                 FTGTAGACR 
                 0.200 
               
               
                   
                   
                 (SEQ ID NO: 84) 
               
               
                   
               
               
                 11 
                 324 
                 MCAYPGCNK 
                 0.200 
               
               
                   
                   
                 (SEQ ID NO: 142) 
               
               
                   
               
               
                 12 
                 251 
                 AAGSSSSVK 
                 0.200 
               
               
                   
                   
                 (SEQ ID NO: 28) 
               
               
                   
               
               
                 13 
                 415 
                 SCRWPSCQK 
                 0.200 
               
               
                   
                   
                 (SEQ ID NO: 201) 
               
               
                   
               
               
                 14 
                 118 
                 SQASSGQAR 
                 0.120 
               
               
                   
                   
                 (SEQ ID NO: 216) 
               
               
                   
               
               
                 15 
                 292 
                 GVFRGIQDV 
                 0.120 
               
               
                   
                   
                 (SEQ ID NO: 103) 
               
               
                   
               
               
                 16 
                 137 
                 CLESQPAIR 
                 0.080 
               
               
                   
                   
                 (SEQ ID NO: 47) 
               
               
                   
               
               
                 17 
                 425 
                 FARSDELVR 
                 0.080 
               
               
                   
                   
                 (SEQ ID NO: 75) 
               
               
                   
               
               
                 18 
                 325 
                 CAYPGCNKR 
                 0.080 
               
               
                   
                   
                 (SEQ ID NO: 44) 
               
               
                   
               
               
                 19 
                 312 
                 RSASETSEK 
                 0.060 
               
               
                   
                   
                 (SEQ ID NO: 190) 
               
               
                   
               
               
                 20 
                 65 
                 PPPPHSFIK 
                 0.060 
               
               
                   
                   
                 (SEQ ID NO: 156) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE IX 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA A 3101 
               
               
                 (HLA-A3101 peptide binding motif anchor residues 
               
               
                 are R at P9; auxiliary anchors L, V, Y, or F at 
               
               
                 P2; F, L, Y, W at P3, L, F, V, I, at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 386 
                 KTCQRKFSR (SEQ ID NO: 128) 
                 9.000 
               
               
                   
               
               
                 2 
                 287 
                 RIHTHGVFR (SEQ ID NO: 182) 
                 6.000 
               
               
                   
               
               
                 3 
                 137 
                 CLESQPAIR (SEQ ID NO: 47) 
                 2.000 
               
               
                   
               
               
                 4 
                 118 
                 SQASSGQAR (SEQ ID NO: 216) 
                 2.000 
               
               
                   
               
               
                 5 
                 368 
                 DQLKRHQRR (SEQ ID NO: 60) 
                 1.200 
               
               
                   
               
               
                 6 
                 100 
                 FTGTAGACR (SEQ ID NO: 84) 
                 1.000 
               
               
                   
               
               
                 7 
                 293 
                 VFRGIQDVR (SEQ ID NO: 238) 
                 0.600 
               
               
                   
               
               
                 8 
                 325 
                 CAYPGCNKR (SEQ ID NO: 44) 
                 0.600 
               
               
                   
               
               
                 9 
                 169 
                 AQFPNHSFK (SEQ ID NO: 36) 
                 0.600 
               
               
                   
               
               
                 10 
                 279 
                 PILCGAQYR (SEQ ID NO: 155) 
                 0.400 
               
               
                   
               
               
                 11 
                 436 
                 NMHQRNMTK (SEQ ID NO: 148) 
                 0.400 
               
               
                   
               
               
                 12 
                 425 
                 FARSDELVR (SEQ ID NO: 75) 
                 0.400 
               
               
                   
               
               
                 13 
                 32 
                 AQWAPVLDF (SEQ ID NO: 37) 
                 0.240 
               
               
                   
               
               
                 14 
                 240 
                 QMNLGATLK (SEQ ID NO: 168) 
                 0.200 
               
               
                   
               
               
                 15 
                 354 
                 QCDFKDCER (SEQ ID NO: 162) 
                 0.200 
               
               
                   
               
               
                 16 
                 373 
                 HQRRHTGVK (SEQ ID NO: 109) 
                 0.200 
               
               
                   
               
               
                 17 
                 383 
                 FQCKTCQRK (SEQ ID NO: 80) 
                 0.200 
               
               
                   
               
               
                 18 
                 313 
                 SASETSEKR (SEQ ID NO: 197) 
                 0.200 
               
               
                   
               
               
                 19 
                 358 
                 KDCERRFSR (SEQ ID NO: 118) 
                 0.180 
               
               
                   
               
               
                 20 
                 391 
                 KFSRSDHLK (SEQ ID NO: 120) 
                 0.180 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE X 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA A 3302 
               
               
                 (HLA-A3302 peptide binding motif anchor residues 
               
               
                 are R at P9; auxiliary anchors A, I, L, F, Y, 
               
               
                 or V at P2) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 337 
                 LSHLQMHSR (SEQ ID NO: 141) 
                 15.000 
               
               
                   
               
               
                 2 
                 409 
                 TSEKPFSCR (SEQ ID NO: 232) 
                 15.000 
               
               
                   
               
               
                 3 
                 364 
                 FSRSDQLKR (SEQ ID NO: 83) 
                 15.000 
               
               
                   
               
               
                 4 
                 137 
                 CLESQPAIR (SEQ ID NO: 47) 
                 9.000 
               
               
                   
               
               
                 5 
                 368 
                 DQLKRHQRR (SEQ ID NO: 60) 
                 9.000 
               
               
                   
               
               
                 6 
                 287 
                 RIHTHGVFR (SEQ ID NO: 182) 
                 4.500 
               
               
                   
               
               
                 7 
                 210 
                 TGSQALLLR (SEQ ID NO: 223) 
                 3.000 
               
               
                   
               
               
                 8 
                 425 
                 FARSDELVR (SEQ ID NO: 75) 
                 3.000 
               
               
                   
               
               
                 9 
                 313 
                 SASETSEKR (SEQ ID NO: 197) 
                 3.000 
               
               
                   
               
               
                 10 
                 293 
                 VFRGIQDVR (SEQ ID NO: 238) 
                 3.000 
               
               
                   
               
               
                 11 
                 354 
                 QCDFKDCER (SEQ ID NO: 162) 
                 3.000 
               
               
                   
               
               
                 12 
                 100 
                 FTGTAGACR (SEQ ID NO: 84) 
                 3.000 
               
               
                   
               
               
                 13 
                 118 
                 SQASSGQAR (SEQ ID NO: 216) 
                 3.000 
               
               
                   
               
               
                 14 
                 325 
                 CAYPGCNKR (SEQ ID NO: 44) 
                 3.000 
               
               
                   
               
               
                 15 
                 207 
                 DSCTGSQAL (SEQ ID NO: 61) 
                 1.500 
               
               
                   
               
               
                 16 
                 139 
                 ESQPAIRNQ (SEQ ID NO: 72) 
                 1.500 
               
               
                   
               
               
                 17 
                 299 
                 DVRRVPGVA (SEQ ID NO: 63) 
                 1.500 
               
               
                   
               
               
                 18 
                 419 
                 PSCQKKFAR (SEQ ID NO: 159) 
                 1.500 
               
               
                   
               
               
                 19 
                 272 
                 ESDNHTTPI (SEQ ID NO: 71) 
                 1.500 
               
               
                   
               
               
                 20 
                 4 
                 DVRDLNALL (SEQ ID NO: 62) 
                 1.500 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XI 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B14 
               
               
                 (HLA-B14 peptide binding motif anchor residues 
               
               
                 are R at P2, Y at P3, R at P5, L at P9. 
               
               
                 Three of the 4 anchor residues is sufficient) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 362 
                 RRFSRSDQL (SEQ ID NO: 187) 
                 1000.000 
               
               
                   
               
               
                 2 
                 332 
                 KRYFKLSHL (SEQ ID NO: 127) 
                 300.000 
               
               
                   
               
               
                 3 
                 423 
                 KKFARSDEL (SEQ ID NO: 122) 
                 150.000 
               
               
                   
               
               
                 4 
                 390 
                 RKFSRSDHL (SEQ ID NO: 183) 
                 150.000 
               
               
                   
               
               
                 5 
                 439 
                 QRNMTKLQL (SEQ ID NO: 173) 
                 20.000 
               
               
                   
               
               
                 6 
                 329 
                 GCNKRYFKL (SEQ ID NO: 90) 
                 10.000 
               
               
                   
               
               
                 7 
                 10 
                 ALLPAVPSL (SEQ ID NO: 34) 
                 10.000 
               
               
                   
               
               
                 8 
                 180 
                 DPMGQQGSL (SEQ ID NO: 59) 
                 9.000 
               
               
                   
               
               
                 9 
                 301 
                 RRVPGVAPT (SEQ ID NO: 189) 
                 6.000 
               
               
                   
               
               
                 10 
                 126 
                 RMFPNAPYL (SEQ ID NO: 185) 
                 5.000 
               
               
                   
               
               
                 11 
                 371 
                 KRHQRRHTG (SEQ ID NO: 126) 
                 5.000 
               
               
                   
               
               
                 12 
                 225 
                 NLYQMTSQL (SEQ ID NO: 147) 
                 5.000 
               
               
                   
               
               
                 13 
                 144 
                 IRNQGYSTV (SEQ ID NO: 117) 
                 4.000 
               
               
                   
               
               
                 14 
                 429 
                 DELVRHHNM (SEQ ID NO: 53) 
                 3.000 
               
               
                   
               
               
                 15 
                 437 
                 MHQRNMTKL (SEQ ID NO: 143) 
                 3.000 
               
               
                   
               
               
                 16 
                 125 
                 ARMFPNAPY (SEQ ID NO: 38) 
                 3.000 
               
               
                   
               
               
                 17 
                 239 
                 NQMNLGATL (SEQ ID NO: 151) 
                 3.000 
               
               
                   
               
               
                 18 
                 286 
                 YRIHTHGVF (SEQ ID NO: 252) 
                 3.000 
               
               
                   
               
               
                 19 
                 174 
                 HSFKHEDPM (SEQ ID NO: 110) 
                 3.000 
               
               
                   
               
               
                 20 
                 372 
                 RHQRRHTGV (SEQ ID NO: 181) 
                 3.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B40 
               
               
                 (HLA-B40 peptide binding motif anchor residues 
               
               
                 are E at P2, L, W, M, or A at P9; 
               
               
                 auxiliary anchors are F, I, or V at P3) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 81 
                 AEPHEEQCL (SEQ ID NO: 30) 
                 40.000 
               
               
                   
               
               
                 2 
                 429 
                 DELVRHHNM (SEQ ID NO: 53) 
                 24.000 
               
               
                   
               
               
                 3 
                 410 
                 SEKPFSCRW (SEQ ID NO: 207) 
                 20.000 
               
               
                   
               
               
                 4 
                 318 
                 SEKRPFMCA (SEQ ID NO: 208) 
                 15.000 
               
               
                   
               
               
                 5 
                 233 
                 LECMTWNQM (SEQ ID NO: 131) 
                 12.000 
               
               
                   
               
               
                 6 
                 3 
                 SDVRDLNAL (SEQ ID NO: 206) 
                 10.000 
               
               
                   
               
               
                 7 
                 349 
                 GEKPYQCDF (SEQ ID NO: 91) 
                 8.000 
               
               
                   
               
               
                 8 
                 6 
                 RDLNALLPA (SEQ ID NO: 177) 
                 5.000 
               
               
                   
               
               
                 9 
                 85 
                 EEQCLSAFT (SEQ ID NO: 65) 
                 4.000 
               
               
                   
               
               
                 10 
                 315 
                 SETSEKRPF (SEQ ID NO: 209) 
                 4.000 
               
               
                   
               
               
                 11 
                 261 
                 TEGQSNHST (SEQ ID NO: 221) 
                 4.000 
               
               
                   
               
               
                 12 
                 23 
                 GCALPVSGA (SEQ ID NO: 89) 
                 3.000 
               
               
                   
               
               
                 13 
                 38 
                 LDFAPPGAS (SEQ ID NO: 130) 
                 3.000 
               
               
                   
               
               
                 14 
                 273 
                 SDNHTTPIL (SEQ ID NO: 204) 
                 2.500 
               
               
                   
               
               
                 15 
                 206 
                 TDSCTGSQA (SEQ ID NO: 220) 
                 2.500 
               
               
                   
               
               
                 16 
                 24 
                 CALPVSGAA (SEQ ID NO: 43) 
                 2.000 
               
               
                   
               
               
                 17 
                 98 
                 GQFTGTAGA (SEQ ID NO: 99) 
                 2.000 
               
               
                   
               
               
                 18 
                 30 
                 GAAQWAPVL (SEQ ID NO: 86) 
                 2.000 
               
               
                   
               
               
                 19 
                 84 
                 HEEQCLSAF (SEQ ID NO: 107) 
                 2.000 
               
               
                   
               
               
                 20 
                 26 
                 LPVSGAAQW (SEQ ID NO: 138) 
                 2.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XIII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B60 
               
               
                 (HLA-B60 peptide binding motif anchor residues 
               
               
                 are E at P2, L at P9; auxiliary anchors 
               
               
                 are I or V at P7) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 81 
                 AEPHEEQCL (SEQ ID NO: 30) 
                 160.000 
               
               
                   
               
               
                 2 
                 3 
                 SDVRDLNAL (SEQ ID NO: 206) 
                 40.000 
               
               
                   
               
               
                 3 
                 429 
                 DELVRHHNM (SEQ ID NO: 53) 
                 40.000 
               
               
                   
               
               
                 4 
                 233 
                 LECMTWNQM (SEQ ID NO: 131) 
                 22.000 
               
               
                   
               
               
                 5 
                 273 
                 SDNHTTPIL (SEQ ID NO: 204) 
                 20.000 
               
               
                   
               
               
                 6 
                 209 
                 CTGSQALLL (SEQ ID NO: 52) 
                 8.000 
               
               
                   
               
               
                 7 
                 30 
                 GAAQWAPVL (SEQ ID NO: 86) 
                 8.000 
               
               
                   
               
               
                 8 
                 318 
                 SEKRPFMCA (SEQ ID NO: 208) 
                 8.000 
               
               
                   
               
               
                 9 
                 180 
                 DPMGQQGSL (SEQ ID NO: 59) 
                 8.000 
               
               
                   
               
               
                 10 
                 138 
                 LESQPAIRN (SEQ ID NO: 132) 
                 5.280 
               
               
                   
               
               
                 11 
                 239 
                 NQMNLGATL (SEQ ID NO: 151) 
                 4.400 
               
               
                   
               
               
                 12 
                 329 
                 GCNKRYFKL (SEQ ID NO: 90) 
                 4.400 
               
               
                   
               
               
                 13 
                 130 
                 NAPYLPSCL (SEQ ID NO: 144) 
                 4.400 
               
               
                   
               
               
                 14 
                 85 
                 EEQCLSAFT (SEQ ID NO: 65) 
                 4.400 
               
               
                   
               
               
                 15 
                 208 
                 SCTGSQALL (SEQ ID NO: 202) 
                 4.000 
               
               
                   
               
               
                 16 
                 207 
                 DSCTGSQAL (SEQ ID NO: 61) 
                 4.000 
               
               
                   
               
               
                 17 
                 218 
                 RTPYSSDNL (SEQ ID NO: 194) 
                 4.000 
               
               
                   
               
               
                 18 
                 261 
                 TEGQSNHST (SEQ ID NO: 221) 
                 4.000 
               
               
                   
               
               
                 19 
                 18 
                 LGGGGGCAL (SEQ ID NO: 134) 
                 4.000 
               
               
                   
               
               
                 20 
                 221 
                 YSSDNLYQM (SEQ ID NO: 253) 
                 2.200 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XIV 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA B61 
               
               
                 (HLA-B61 peptide binding motif anchor residues 
               
               
                 are E at P2, V at P9; auxiliary anchors 
               
               
                 are F, I, L, V, Y, W at P3, I at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 318 
                 SEKRPFMCA (SEQ ID NO: 208) 
                 20.000 
               
               
                   
               
               
                 2 
                 429 
                 DELVRHHNM (SEQ ID NO: 53) 
                 16.000 
               
               
                   
               
               
                 3 
                 298 
                 QDVRRVPGV (SEQ ID NO: 164) 
                 10.000 
               
               
                   
               
               
                 4 
                 81 
                 AEPHEEQCL (SEQ ID NO: 30) 
                 8.000 
               
               
                   
               
               
                 5 
                 233 
                 LECMTWNQM (SEQ ID NO: 131) 
                 8.000 
               
               
                   
               
               
                 6 
                 6 
                 RDLNALLPA (SEQ ID NO: 177) 
                 5.500 
               
               
                   
               
               
                 7 
                 85 
                 EEQCLSAFT (SEQ ID NO: 65) 
                 4.000 
               
               
                   
               
               
                 8 
                 261 
                 TEGQSNHST (SEQ ID NO: 221) 
                 4.000 
               
               
                   
               
               
                 9 
                 206 
                 TDSCTGSQA (SEQ ID NO: 220) 
                 2.500 
               
               
                   
               
               
                 10 
                 295 
                 RGIQDVRRV (SEQ ID NO: 179) 
                 2.200 
               
               
                   
               
               
                 11 
                 3 
                 SDVRDLNAL (SEQ ID NO: 206) 
                 2.000 
               
               
                   
               
               
                 12 
                 250 
                 VAAGSSSSV (SEQ ID NO: 236) 
                 2.000 
               
               
                   
               
               
                 13 
                 29 
                 SGAAQWAPV (SEQ ID NO: 211) 
                 2.000 
               
               
                   
               
               
                 14 
                 315 
                 SETSEKRPF (SEQ ID NO: 209) 
                 1.600 
               
               
                   
               
               
                 15 
                 138 
                 LESQPAIRN (SEQ ID NO: 132) 
                 1.200 
               
               
                   
               
               
                 16 
                 244 
                 GATLKGVAA (SEQ ID NO: 88) 
                 1.100 
               
               
                   
               
               
                 17 
                 20 
                 GGGGCALPV (SEQ ID NO: 92) 
                 1.100 
               
               
                   
               
               
                 18 
                 440 
                 RNMTKLQLA (SEQ ID NO: 186) 
                 1.100 
               
               
                   
               
               
                 19 
                 23 
                 GCALPVSGA (SEQ ID NO: 89) 
                 1.100 
               
               
                   
               
               
                 20 
                 191 
                 QQYSVPPPV (SEQ ID NO: 171) 
                 1.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XV 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA B62 
               
               
                 (HLA-B62 peptide binding motif anchor residues 
               
               
                 are Q or L at P2, F or Y at P9; auxiliary 
               
               
                 anchors are I or V at P5) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 146 
                 NQGYSTVTF (SEQ ID NO: 150) 
                 211.200 
               
               
                   
               
               
                 2 
                 32 
                 AQWAPVLDF (SEQ ID NO: 37) 
                 96.000 
               
               
                   
               
               
                 3 
                 263 
                 GQSNHSTGY (SEQ ID NO: 100) 
                 96.000 
               
               
                   
               
               
                 4 
                 88 
                 CLSAFTVHF (SEQ ID NO: 48) 
                 96.000 
               
               
                   
               
               
                 5 
                 17 
                 SLGGGGGCA (SEQ ID NO: 215) 
                 9.600 
               
               
                   
               
               
                 6 
                 239 
                 NQMNLGATL (SEQ ID NO: 151) 
                 8.800 
               
               
                   
               
               
                 7 
                 191 
                 QQYSVPPPV (SEQ ID NO: 171) 
                 8.000 
               
               
                   
               
               
                 8 
                 98 
                 GQFTGTAGA (SEQ ID NO: 99) 
                 8.000 
               
               
                   
               
               
                 9 
                 384 
                 QCKTCQRKF (SEQ ID NO: 163) 
                 6.000 
               
               
                   
               
               
                 10 
                 40 
                 FAPPGASAY (SEQ ID NO: 74) 
                 4.800 
               
               
                   
               
               
                 11 
                 227 
                 YQMTSQLEC (SEQ ID NO: 251) 
                 4.800 
               
               
                   
               
               
                 12 
                 187 
                 SLGEQQYSV (SEQ ID NO: 214) 
                 4.400 
               
               
                   
               
               
                 13 
                 86 
                 EQCLSAFTV (SEQ ID NO: 69) 
                 4.400 
               
               
                   
               
               
                 14 
                 152 
                 VTFDGTPSY (SEQ ID NO: 244) 
                 4.400 
               
               
                   
               
               
                 15 
                 101 
                 TGTAGACRY (SEQ ID NO: 224) 
                 4.000 
               
               
                   
               
               
                 16 
                 242 
                 NLGATLKGV (SEQ ID NO: 146) 
                 4.000 
               
               
                   
               
               
                 17 
                 92 
                 FTVHFSGQF (SEQ ID NO: 85) 
                 4.000 
               
               
                   
               
               
                 18 
                 7 
                 DLNALLPAV (SEQ ID NO: 58) 
                 4.000 
               
               
                   
               
               
                 19 
                 123 
                 GQARMFPNA (SEQ ID NO: 98) 
                 4.000 
               
               
                   
               
               
                 20 
                 280 
                 ILCGAQYRI (SEQ ID NO: 116) 
                 3.120 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XVI 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA B7 
               
               
                 (HLA-B7 peptide binding motif anchor residues 
               
               
                 are P at P2, L or F at P9; auxiliary 
               
               
                 anchor is R at P2) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 180 
                 DPMGQQGSL (SEQ ID NO: 59) 
                 240.000 
               
               
                   
               
               
                 2 
                 4 
                 DVRDLNALL (SEQ ID NO: 62) 
                 200.000 
               
               
                   
               
               
                 3 
                 302 
                 RVPGVAPTL (SEQ ID NO: 195) 
                 20.000 
               
               
                   
               
               
                 4 
                 30 
                 GAAQWAPVL (SEQ ID NO: 86) 
                 12.000 
               
               
                   
               
               
                 5 
                 239 
                 NQMNLGATL (SEQ ID NO: 151) 
                 12.000 
               
               
                   
               
               
                 6 
                 130 
                 NAPYLPSCL (SEQ ID NO: 144) 
                 12.000 
               
               
                   
               
               
                 7 
                 10 
                 ALLPAVPSL (SEQ ID NO: 34) 
                 12.000 
               
               
                   
               
               
                 8 
                 299 
                 DVRRVPGVA (SEQ ID NO: 63) 
                 5.000 
               
               
                   
               
               
                 9 
                 208 
                 SCTGSQALL (SEQ ID NO: 202) 
                 4.000 
               
               
                   
               
               
                 10 
                 303 
                 VPGVAPTLV (SEQ ID NO: 242) 
                 4.000 
               
               
                   
               
               
                 11 
                 18 
                 LGGGGGCAL (SEQ ID NO: 134) 
                 4.000 
               
               
                   
               
               
                 12 
                 218 
                 RTPYSSDNL (SEQ ID NO: 194) 
                 4.000 
               
               
                   
               
               
                 13 
                 207 
                 DSCTGSQAL (SEQ ID NO: 61) 
                 4.000 
               
               
                   
               
               
                 14 
                 209 
                 CTGSQALLL (SEQ ID NO: 52) 
                 4.000 
               
               
                   
               
               
                 15 
                 329 
                 GCNKRYFKL (SEQ ID NO: 90) 
                 4.000 
               
               
                   
               
               
                 16 
                 235 
                 CMTWNQMNL (SEQ ID NO: 49) 
                 4.000 
               
               
                   
               
               
                 17 
                 441 
                 NMTKLQLAL (SEQ ID NO: 149) 
                 4.000 
               
               
                   
               
               
                 18 
                 126 
                 RMFPNAPYL (SEQ ID NO: 185) 
                 4.000 
               
               
                   
               
               
                 19 
                 225 
                 NLYQMTSQL (SEQ ID NO: 147) 
                 4.000 
               
               
                   
               
               
                 20 
                 143 
                 AIRNQGYST (SEQ ID NO: 33) 
                 3.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XVII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA B8 
               
               
                 (HLA-B8 peptide binding motif anchor residues 
               
               
                 are K at P3, K or R at P5, L at P9) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 329 
                 GCNKRYFKL (SEQ ID NO: 90) 
                 16.000 
               
               
                   
               
               
                 2 
                 4 
                 DVRDLNALL (SEQ ID NO: 62) 
                 12.000 
               
               
                   
               
               
                 3 
                 316 
                 ETSEKRPFM (SEQ ID NO: 73) 
                 3.000 
               
               
                   
               
               
                 4 
                 180 
                 DPMGQQGSL (SEQ ID NO: 59) 
                 1.600 
               
               
                   
               
               
                 5 
                 208 
                 SCTGSQALL (SEQ ID NO: 202) 
                 0.800 
               
               
                   
               
               
                 6 
                 130 
                 NAPYLPSCL (SEQ ID NO: 144) 
                 0.800 
               
               
                   
               
               
                 7 
                 244 
                 GATLKGVAA (SEQ ID NO: 88) 
                 0.800 
               
               
                   
               
               
                 8 
                 30 
                 GAAQWAPVL (SEQ ID NO: 86) 
                 0.800 
               
               
                   
               
               
                 9 
                 299 
                 DVRRVPGVA (SEQ ID NO: 63) 
                 0.400 
               
               
                   
               
               
                 10 
                 420 
                 SCQKKFARS (SEQ ID NO: 200) 
                 0.400 
               
               
                   
               
               
                 11 
                 387 
                 TCQRKFSRS (SEQ ID NO: 219) 
                 0.400 
               
               
                   
               
               
                 12 
                 225 
                 NLYQMTSQL (SEQ ID NO: 147) 
                 0.400 
               
               
                   
               
               
                 13 
                 141 
                 QPAIRNQGY (SEQ ID NO: 170) 
                 0.400 
               
               
                   
               
               
                 14 
                 10 
                 ALLPAVPSL (SEQ ID NO: 34) 
                 0.400 
               
               
                   
               
               
                 15 
                 207 
                 DSCTGSQAL (SEQ ID NO: 61) 
                 0.400 
               
               
                   
               
               
                 16 
                 384 
                 QCKTCQRKF (SEQ ID NO: 163) 
                 0.400 
               
               
                   
               
               
                 17 
                 136 
                 SCLESQPAI (SEQ ID NO: 198) 
                 0.300 
               
               
                   
               
               
                 18 
                 347 
                 HTGEKPYQC (SEQ ID NO: 112) 
                 0.300 
               
               
                   
               
               
                 19 
                 401 
                 HTRTHTGKT (SEQ ID NO: 114) 
                 0.200 
               
               
                   
               
               
                 20 
                 332 
                 KRYFKLSHL (SEQ ID NO: 127) 
                 0.200 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XVIII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA B 2702 
               
               
                 (HLA-B2702 peptide binding motif anchor 
               
               
                 residues are R at P2; F, Y, I, L, or W at P9) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 332 
                 KRYFKLSHL (SEQ ID NO: 127) 
                 900.000 
               
               
                   
               
               
                 2 
                 362 
                 RRFSRSDQL (SEQ ID NO: 187) 
                 900.000 
               
               
                   
               
               
                 3 
                 286 
                 YRIHTHGVF (SEQ ID NO: 252) 
                 200.000 
               
               
                   
               
               
                 4 
                 125 
                 ARMFPNAPY (SEQ ID NO: 38) 
                 200.000 
               
               
                   
               
               
                 5 
                 375 
                 RRHTGVKPF (SEQ ID NO: 188) 
                 180.000 
               
               
                   
               
               
                 6 
                 32 
                 AQWAPVLDF (SEQ ID NO: 37) 
                 100.000 
               
               
                   
               
               
                 7 
                 301 
                 RRVPGVAPT (SEQ ID NO: 189) 
                 60.000 
               
               
                   
               
               
                 8 
                 439 
                 QRNMTKLQL (SEQ ID NO: 173) 
                 60.000 
               
               
                   
               
               
                 9 
                 126 
                 RMFPNAPYL (SEQ ID NO: 185) 
                 22.500 
               
               
                   
               
               
                 10 
                 426 
                 ARSDELVRH (SEQ ID NO: 39) 
                 20.000 
               
               
                   
               
               
                 11 
                 146 
                 NQGYSTVTF (SEQ ID NO: 150) 
                 20.000 
               
               
                   
               
               
                 12 
                 144 
                 IRNQGYSTV (SEQ ID NO: 117) 
                 20.000 
               
               
                   
               
               
                 13 
                 389 
                 QRKFSRSDH (SEQ ID NO: 172) 
                 20.000 
               
               
                   
               
               
                 14 
                 263 
                 GQSNHSTGY (SEQ ID NO: 100) 
                 20.000 
               
               
                   
               
               
                 15 
                 416 
                 CRWPSCQKK (SEQ ID NO: 50) 
                 20.000 
               
               
                   
               
               
                 16 
                 191 
                 QQYSVPPPV (SEQ ID NO: 171) 
                 10.000 
               
               
                   
               
               
                 17 
                 217 
                 LRTPYSSDN (SEQ ID NO: 140) 
                 10.000 
               
               
                   
               
               
                 18 
                 107 
                 CRYGPFGPP (SEQ ID NO: 51) 
                 10.000 
               
               
                   
               
               
                 19 
                 98 
                 GQFTGTAGA (SEQ ID NO: 99) 
                 10.000 
               
               
                   
               
               
                 20 
                 239 
                 NQMNLGATL (SEQ ID NO: 151) 
                 6.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XIX 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA B 2705 
               
               
                 (HLA-B2705 peptide binding motif anchor 
               
               
                 residues are R at P2; L or F at P9; Y, R, 
               
               
                 H, and K, have also been found at P9 
               
               
                 for naturally processed epitopes) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 332 
                 KRYFKLSHL (SEQ ID NO: 127) 
                 30000.000 
               
               
                   
               
               
                 2 
                 362 
                 RRFSRSDQL (SEQ ID NO: 187) 
                 30000.000 
               
               
                   
               
               
                 3 
                 416 
                 CRWPSCQKK (SEQ ID NO: 50) 
                 10000.000 
               
               
                   
               
               
                 4 
                 439 
                 QRNMTKLQL (SEQ ID NO: 173) 
                 2000.000 
               
               
                   
               
               
                 5 
                 286 
                 YRIHTHGVF (SEQ ID NO: 252) 
                 1000.000 
               
               
                   
               
               
                 6 
                 125 
                 ARMFPNAPY (SEQ ID NO: 38) 
                 1000.000 
               
               
                   
               
               
                 7 
                 294 
                 FRGIQDVRR (SEQ ID NO: 81) 
                 1000.000 
               
               
                   
               
               
                 8 
                 432 
                 VRHHNMHQR (SEQ ID NO: 243) 
                 1000.000 
               
               
                   
               
               
                 9 
                 169 
                 AQFPNHSFK (SEQ ID NO: 36) 
                 1000.000 
               
               
                   
               
               
                 10 
                 375 
                 RRHTGVKPF (SEQ ID NO: 188) 
                 900.000 
               
               
                   
               
               
                 11 
                 126 
                 RMFPNAPYL (SEQ ID NO: 185) 
                 750.000 
               
               
                   
               
               
                 12 
                 144 
                 IRNQGYSTV (SEQ ID NO: 117) 
                 600.000 
               
               
                   
               
               
                 13 
                 301 
                 RRVPGVAPT (SEQ ID NO: 189) 
                 600.000 
               
               
                   
               
               
                 14 
                 32 
                 AQWAPVLDF (SEQ ID NO: 37) 
                 500.000 
               
               
                   
               
               
                 15 
                 191 
                 QQYSVPPPV (SEQ ID NO: 171) 
                 300.000 
               
               
                   
               
               
                 16 
                 373 
                 HQRRHTGVK (SEQ ID NO: 109) 
                 200.000 
               
               
                   
               
               
                 17 
                 426 
                 ARSDELVRH (SEQ ID NO: 39) 
                 200.000 
               
               
                   
               
               
                 18 
                 383 
                 FQCKTCQRK (SEQ ID NO: 80) 
                 200.000 
               
               
                   
               
               
                 19 
                 239 
                 NQMNLGATL (SEQ ID NO: 151) 
                 200.000 
               
               
                   
               
               
                 20 
                 389 
                 QRKFSRSDH (SEQ ID NO: 172) 
                 200.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XX 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA B 3501 
               
               
                 (HLA-B3501 peptide binding motif anchor 
               
               
                 residues are P at P2; Y, F, M, L, or I at P9) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 278 
                 TPILCGAQY (SEQ ID NO: 227) 
                 40.000 
               
               
                   
               
               
                 2 
                 141 
                 QPAIRNQGY (SEQ ID NO: 170) 
                 40.000 
               
               
                   
               
               
                 3 
                 219 
                 TPYSSDNLY (SEQ ID NO: 231) 
                 40.000 
               
               
                   
               
               
                 4 
                 327 
                 YPGCNKRYF (SEQ ID NO: 250) 
                 20.000 
               
               
                   
               
               
                 5 
                 163 
                 TPSHHAAQF (SEQ ID NO: 228) 
                 20.000 
               
               
                   
               
               
                 6 
                 180 
                 DPMGQQGSL (SEQ ID NO: 59) 
                 20.000 
               
               
                   
               
               
                 7 
                 221 
                 YSSDNLYQM (SEQ ID NO: 253) 
                 20.000 
               
               
                   
               
               
                 8 
                 26 
                 LPVSGAAQW (SEQ ID NO: 138) 
                 10.000 
               
               
                   
               
               
                 9 
                 174 
                 HSFKHEDPM (SEQ ID NO: 110) 
                 10.000 
               
               
                   
               
               
                 10 
                 82 
                 EPHEEQCLS (SEQ ID NO: 68) 
                 6.000 
               
               
                   
               
               
                 11 
                 213 
                 QALLLRTPY (SEQ ID NO: 160) 
                 6.000 
               
               
                   
               
               
                 12 
                 119 
                 QASSGQARM (SEQ ID NO: 161) 
                 6.000 
               
               
                   
               
               
                 13 
                 4 
                 DVRDLNALL (SEQ ID NO: 62) 
                 6.000 
               
               
                   
               
               
                 14 
                 40 
                 FAPPGASAY (SEQ ID NO: 74) 
                 6.000 
               
               
                   
               
               
                 15 
                 120 
                 ASSGQARMF (SEQ ID NO: 40) 
                 5.000 
               
               
                   
               
               
                 16 
                 207 
                 DSCTGSQAL (SEQ ID NO: 61) 
                 5.000 
               
               
                   
               
               
                 17 
                 303 
                 VPGVAPTLV (SEQ ID NO: 242) 
                 4.000 
               
               
                   
               
               
                 18 
                 316 
                 ETSEKRPFM (SEQ ID NO: 73) 
                 4.000 
               
               
                   
               
               
                 19 
                 152 
                 VTFDGTPSY (SEQ ID NO: 244) 
                 4.000 
               
               
                   
               
               
                 20 
                 412 
                 KPFSCRWPS (SEQ ID NO: 123) 
                 4.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXI 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA B 3701 
               
               
                 (HLA-B3701 peptide binding motif anchor residues 
               
               
                 are F, M, or L at P8; I or L at P9; auxiliary 
               
               
                 anchors are D, E at P2 and V, I at P5) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 3 
                 SDVRDLNAL (SEQ ID NO: 206) 
                 40.000 
               
               
                   
               
               
                 2 
                 273 
                 SDNHTTPIL (SEQ ID NO: 204) 
                 40.000 
               
               
                   
               
               
                 3 
                 81 
                 AEPHEEQCL (SEQ ID NO: 30) 
                 10.000 
               
               
                   
               
               
                 4 
                 298 
                 QDVRRVPGV (SEQ ID NO: 164) 
                 8.000 
               
               
                   
               
               
                 5 
                 428 
                 SDELVRHHN (SEQ ID NO: 203) 
                 6.000 
               
               
                   
               
               
                 6 
                 85 
                 EEQCLSAFT (SEQ ID NO: 65) 
                 5.000 
               
               
                   
               
               
                 7 
                 208 
                 SCTGSQALL (SEQ ID NO: 202) 
                 5.000 
               
               
                   
               
               
                 8 
                 4 
                 DVRDLNALL (SEQ ID NO: 62) 
                 5.000 
               
               
                   
               
               
                 9 
                 209 
                 CTGSQALLL (SEQ ID NO: 52) 
                 5.000 
               
               
                   
               
               
                 10 
                 38 
                 LDFAPPGAS (SEQ ID NO: 130) 
                 4.000 
               
               
                   
               
               
                 11 
                 223 
                 SDNLYQMTS (SEQ ID NO: 205) 
                 4.000 
               
               
                   
               
               
                 12 
                 179 
                 EDPMGQQGS (SEQ ID NO: 64) 
                 4.000 
               
               
                   
               
               
                 13 
                 206 
                 TDSCTGSQA (SEQ ID NO: 220) 
                 4.000 
               
               
                   
               
               
                 14 
                 6 
                 RDLNALLPA (SEQ ID NO: 177) 
                 4.000 
               
               
                   
               
               
                 15 
                 84 
                 HEEQCLSAF (SEQ ID NO: 107) 
                 2.000 
               
               
                   
               
               
                 16 
                 233 
                 LECMTWNQM (SEQ ID NO: 131) 
                 2.000 
               
               
                   
               
               
                 17 
                 429 
                 DELVRHHNM (SEQ ID NO: 53) 
                 2.000 
               
               
                   
               
               
                 18 
                 315 
                 SETSEKRPF (SEQ ID NO: 209) 
                 2.000 
               
               
                   
               
               
                 19 
                 349 
                 GEKPYQCDF (SEQ ID NO: 91) 
                 2.000 
               
               
                   
               
               
                 20 
                 302 
                 RVPGVAPTL (SEQ ID NO: 195) 
                 1.500 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Human HLA B 3801 
               
               
                 (HLA-B3801 peptide binding motif anchor 
               
               
                 residues are F or L at P9; Auxiliary anchors 
               
               
                 are H at P2 and D or E at P3) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 437 
                 MHQRNMTKL (SEQ ID NO: 143) 
                 36.000 
               
               
                   
               
               
                 2 
                 434 
                 HHNMHQRNM (SEQ ID NO: 108) 
                 6.000 
               
               
                   
               
               
                 3 
                 372 
                 RHQRRHTGV (SEQ ID NO: 181) 
                 6.000 
               
               
                   
               
               
                 4 
                 180 
                 DPMGQQGSL (SEQ ID NO: 59) 
                 4.000 
               
               
                   
               
               
                 5 
                 433 
                 RHHNMHQRN (SEQ ID NO: 180) 
                 3.900 
               
               
                   
               
               
                 6 
                 165 
                 SHHAAQFPN (SEQ ID NO: 213) 
                 3.900 
               
               
                   
               
               
                 7 
                 202 
                 CHTPTDSCT (SEQ ID NO: 45) 
                 3.000 
               
               
                   
               
               
                 8 
                 396 
                 DHLKTHTRT (SEQ ID NO: 57) 
                 3.000 
               
               
                   
               
               
                 9 
                 161 
                 GHTPSHHAA (SEQ ID NO: 94) 
                 3.000 
               
               
                   
               
               
                 10 
                 302 
                 RVPGVAPTL (SEQ ID NO: 195) 
                 2.600 
               
               
                   
               
               
                 11 
                 417 
                 RWPSCQKKF (SEQ ID NO: 196) 
                 2.400 
               
               
                   
               
               
                 12 
                 327 
                 YPGCNKRYF (SEQ ID NO: 250) 
                 2.400 
               
               
                   
               
               
                 13 
                 208 
                 SCTGSQALL (SEQ ID NO: 202) 
                 2.000 
               
               
                   
               
               
                 14 
                 163 
                 TPSHHAAQF (SEQ ID NO: 228) 
                 2.000 
               
               
                   
               
               
                 15 
                 120 
                 ASSGQARMF (SEQ ID NO: 40) 
                 2.000 
               
               
                   
               
               
                 16 
                 18 
                 LGGGGGCAL (SEQ ID NO: 134) 
                 2.000 
               
               
                   
               
               
                 17 
                 177 
                 KHEDPMGQQ (SEQ ID NO: 121) 
                 1.800 
               
               
                   
               
               
                 18 
                 83 
                 PHEEQCLSA (SEQ ID NO: 154) 
                 1.800 
               
               
                   
               
               
                 19 
                 10 
                 ALLPAVPSL (SEQ ID NO: 34) 
                 1.300 
               
               
                   
               
               
                 20 
                 225 
                 NLYQMTSQL (SEQ ID NO: 147) 
                 1.300 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXIII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B 3901 
               
               
                 (HLA-B3901 peptide binding motif anchor 
               
               
                 residues are R or H at P2; L at P9; 
               
               
                 auxiliary anchors are I, V, or L at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of 
               
               
                   
                   
                   
                 a Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This Sub- 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 sequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 437 
                 MHQRNMTKL (SEQ ID NO: 143) 
                 135.000 
               
               
                   
               
               
                 2 
                 332 
                 KRYFKLSHL (SEQ ID NO: 127) 
                 45.000 
               
               
                   
               
               
                 3 
                 434 
                 HHNMHQRNM (SEQ ID NO: 108) 
                 30.000 
               
               
                   
               
               
                 4 
                 362 
                 RRFSRSDQL (SEQ ID NO: 187) 
                 30.000 
               
               
                   
               
               
                 5 
                 372 
                 RHQRRHTGV (SEQ ID NO: 181) 
                 30.000 
               
               
                   
               
               
                 6 
                 10 
                 ALLPAVPSL (SEQ ID NO: 34) 
                 9.000 
               
               
                   
               
               
                 7 
                 439 
                 QRNMTKLQL (SEQ ID NO: 173) 
                 7.500 
               
               
                   
               
               
                 8 
                 390 
                 RKFSRSDHL (SEQ ID NO: 183) 
                 6.000 
               
               
                   
               
               
                 9 
                 396 
                 DHLKTHTRT (SEQ ID NO: 57) 
                 6.000 
               
               
                   
               
               
                 10 
                 239 
                 NQMNLGATL (SEQ ID NO: 151) 
                 6.000 
               
               
                   
               
               
                 11 
                 423 
                 KKFARSDEL (SEQ ID NO: 122) 
                 6.000 
               
               
                   
               
               
                 12 
                 126 
                 RMFPNAPYL (SEQ ID NO: 185) 
                 6.000 
               
               
                   
               
               
                 13 
                 225 
                 NLYQMTSQL (SEQ ID NO: 147) 
                 6.000 
               
               
                   
               
               
                 14 
                 180 
                 DPMGQQGSL (SEQ ID NO: 59) 
                 6.000 
               
               
                   
               
               
                 15 
                 144 
                 IRNQGYSTV (SEQ ID NO: 117) 
                 5.000 
               
               
                   
               
               
                 16 
                 136 
                 SCLESQPAI (SEQ ID NO: 198) 
                 4.000 
               
               
                   
               
               
                 17 
                 292 
                 GVFRGIQDV (SEQ ID NO: 103) 
                 3.000 
               
               
                   
               
               
                 18 
                 302 
                 RVPGVAPTL (SEQ ID NO: 195) 
                 3.000 
               
               
                   
               
               
                 19 
                 208 
                 SCTGSQALL (SEQ ID NO: 202) 
                 3.000 
               
               
                   
               
               
                 20 
                 207 
                 DSCTGSQAL (SEQ ID NO: 61) 
                 3.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXIV 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B 3902 (HLA-B3902 peptide binding motif 
               
               
                 anchor residues are K or Q at P2; L at P9; 
               
               
                 auxiliary anchors are I, L, F, or V at P5) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of 
               
               
                   
                   
                   
                 Disassociation 
               
               
                   
                   
                   
                 of a 
               
               
                   
                   
                   
                 Molecule 
               
               
                   
                   
                   
                 Containing 
               
               
                   
                 Start 
                 Subsequence 
                 This 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 239 
                 NQMNLGATL (SEQ ID NO: 
                 24.000 
               
               
                   
                   
                 151) 
               
               
                   
               
               
                 2 
                 390 
                 RKFSRSDHL (SEQ ID NO: 
                 20.000 
               
               
                   
                   
                 183) 
               
               
                   
               
               
                 3 
                 423 
                 KKFARSDEL (SEQ ID NO: 
                 20.000 
               
               
                   
                   
                 122) 
               
               
                   
               
               
                 4 
                 32 
                 AQWAPVLDF (SEQ ID NO: 
                 5.000 
               
               
                   
                   
                 37) 
               
               
                   
               
               
                 5 
                 146 
                 NQGYSTVTF (SEQ ID NO: 
                 5.000 
               
               
                   
                   
                 150) 
               
               
                   
               
               
                 6 
                 130 
                 NAPYLPSCL (SEQ ID NO: 
                 2.400 
               
               
                   
                   
                 144) 
               
               
                   
               
               
                 7 
                 225 
                 NLYQMTSQL (SEQ ID NO: 
                 2.400 
               
               
                   
                   
                 147) 
               
               
                   
               
               
                 8 
                 30 
                 GAAQWAPVL (SEQ ID NO: 
                 2.400 
               
               
                   
                   
                 86) 
               
               
                   
               
               
                 9 
                 441 
                 NMTKLQLAL (SEQ ID NO: 
                 2.400 
               
               
                   
                   
                 149) 
               
               
                   
               
               
                 10 
                 302 
                 RVPGVAPTL (SEQ ID NO: 
                 2.400 
               
               
                   
                   
                 195) 
               
               
                   
               
               
                 11 
                 126 
                 RMFPNAPYL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 185) 
               
               
                   
               
               
                 12 
                 218 
                 RTPYSSDNL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 194) 
               
               
                   
               
               
                 13 
                 209 
                 CTGSQALLL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 52) 
               
               
                   
               
               
                 14 
                 332 
                 KRYFKLSHL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 127) 
               
               
                   
               
               
                 15 
                 180 
                 DPMGQQGSL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 59) 
               
               
                   
               
               
                 16 
                 437 
                 MHQRNMTKL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 143) 
               
               
                   
               
               
                 17 
                 207 
                 DSCTGSQAL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 61) 
               
               
                   
               
               
                 18 
                 208 
                 SCTGSQALL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 202) 
               
               
                   
               
               
                 19 
                 329 
                 GCNKRYFKL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 90) 
               
               
                   
               
               
                 20 
                 10 
                 ALLPAVPSL (SEQ ID NO: 
                 2.000 
               
               
                   
                   
                 34) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXV 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B 4403 (HLA-B4403 peptide binding motif 
               
               
                 anchor residues are E at P2; Y or F at P9) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 315 
                 SETSEKRPF 
                 80.000 
               
               
                   
                   
                 (SEQ ID NO: 209) 
               
               
                   
               
               
                 2 
                 349 
                 GEKPYQCDF 
                 80.000 
               
               
                   
                   
                 (SEQ ID NO: 91) 
               
               
                   
               
               
                 3 
                 84 
                 HEEQCLSAF 
                 60.000 
               
               
                   
                   
                 (SEQ ID NO: 107) 
               
               
                   
               
               
                 4 
                 410 
                 SEKPFSCRW 
                 48.000 
               
               
                   
                   
                 (SEQ ID NO: 207) 
               
               
                   
               
               
                 5 
                 429 
                 DELVRHHNM 
                 24.000 
               
               
                   
                   
                 (SEQ ID NO: 53) 
               
               
                   
               
               
                 6 
                 278 
                 TPILCGAQY 
                 15.000 
               
               
                   
                   
                 (SEQ ID NO: 227) 
               
               
                   
               
               
                 7 
                 141 
                 QPAIRNQGY 
                 9.000 
               
               
                   
                   
                 (SEQ ID NO: 170) 
               
               
                   
               
               
                 8 
                 40 
                 FAPPGASAY 
                 9.000 
               
               
                   
                   
                 (SEQ ID NO: 74) 
               
               
                   
               
               
                 9 
                 213 
                 QALLLRTPY 
                 9.000 
               
               
                   
                   
                 (SEQ ID NO: 160) 
               
               
                   
               
               
                 10 
                 318 
                 SEKRPFMCA 
                 8.000 
               
               
                   
                   
                 (SEQ ID NO: 208) 
               
               
                   
               
               
                 11 
                 81 
                 AEPHEEQCL 
                 8.000 
               
               
                   
                   
                 (SEQ ID NO: 30) 
               
               
                   
               
               
                 12 
                 152 
                 VTFDGTPSY 
                 4.500 
               
               
                   
                   
                 (SEQ ID NO: 244) 
               
               
                   
               
               
                 13 
                 101 
                 TGTAGACRY 
                 4.500 
               
               
                   
                   
                 (SEQ ID NO: 224) 
               
               
                   
               
               
                 14 
                 120 
                 ASSGQARMF 
                 4.500 
               
               
                   
                   
                 (SEQ ID NO: 40) 
               
               
                   
               
               
                 15 
                 261 
                 TEGQSNHST 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 221) 
               
               
                   
               
               
                 16 
                 85 
                 EEQCLSAFT 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 65) 
               
               
                   
               
               
                 17 
                 233 
                 LECMTWNQM 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 131) 
               
               
                   
               
               
                 18 
                 104 
                 AGACRYGPF 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 31) 
               
               
                   
               
               
                 19 
                 3 
                 SDVRDLNAL 
                 3.000 
               
               
                   
                   
                 (SEQ ID NO: 206) 
               
               
                   
               
               
                 20 
                 185 
                 QGSLGEQQY 
                 3.000 
               
               
                   
                   
                 (SEQ ID NO: 166) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXVI 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B 5101 (HLA-B5101 peptide binding motif 
               
               
                 anchor residues are A, P, or G at P2; F or I  
               
               
                 at P9) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 303 
                 VPGVAPTLV 
                 314.600 
               
               
                   
                   
                 (SEQ ID NO: 242) 
               
               
                   
               
               
                 2 
                 180 
                 DPMGQQGSL 
                 242.000 
               
               
                   
                   
                 (SEQ ID NO: 59) 
               
               
                   
               
               
                 3 
                 250 
                 VAAGSSSSV 
                 157.300 
               
               
                   
                   
                 (SEQ ID NO: 236) 
               
               
                   
               
               
                 4 
                 130 
                 NAPYLPSCL 
                 50.000 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 5 
                 30 
                 GAAQWAPVL 
                 50.000 
               
               
                   
                   
                 (SEQ ID NO: 86) 
               
               
                   
               
               
                 6 
                 20 
                 GGGGCALPV 
                 44.000 
               
               
                   
                   
                 (SEQ ID NO: 92) 
               
               
                   
               
               
                 7 
                 64 
                 PPPPPHSFI 
                 40.000 
               
               
                   
                   
                 (SEQ ID NO: 157) 
               
               
                   
               
               
                 8 
                 29 
                 SGAAQWAPV 
                 40.000 
               
               
                   
                   
                 (SEQ ID NO: 211) 
               
               
                   
               
               
                 9 
                 18 
                 LGGGGGCAL 
                 31.460 
               
               
                   
                   
                 (SEQ ID NO: 134) 
               
               
                   
               
               
                 10 
                 295 
                 RGIQDVRRV 
                 22.000 
               
               
                   
                   
                 (SEQ ID NO: 179) 
               
               
                   
               
               
                 11 
                 119 
                 QASSGQARM 
                 18.150 
               
               
                   
                   
                 (SEQ ID NO: 161) 
               
               
                   
               
               
                 12 
                 418 
                 WPSCQKKFA 
                 12.100 
               
               
                   
                   
                 (SEQ ID NO: 246) 
               
               
                   
               
               
                 13 
                 82 
                 EPHEEQCLS 
                 12.100 
               
               
                   
                   
                 (SEQ ID NO: 68) 
               
               
                   
               
               
                 14 
                 110 
                 GPFGPPPPS 
                 11.000 
               
               
                   
                   
                 (SEQ ID NO: 96) 
               
               
                   
               
               
                 15 
                 272 
                 ESDNHTTPI 
                 8.000 
               
               
                   
                   
                 (SEQ ID NO: 71) 
               
               
                   
               
               
                 16 
                 306 
                 VAPTLVRSA 
                 7.150 
               
               
                   
                   
                 (SEQ ID NO: 237) 
               
               
                   
               
               
                 17 
                 280 
                 ILCGAQYRI 
                 6.921 
               
               
                   
                   
                 (SEQ ID NO: 116) 
               
               
                   
               
               
                 18 
                 219 
                 TPYSSDNLY 
                 6.600 
               
               
                   
                   
                 (SEQ ID NO: 231) 
               
               
                   
               
               
                 19 
                 128 
                 FPNAPYLPS 
                 6.500 
               
               
                   
                   
                 (SEQ ID NO: 79) 
               
               
                   
               
               
                 20 
                 204 
                 TPTDSCTGS 
                 6.050 
               
               
                   
                   
                 (SEQ ID NO: 230) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXVII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B 5102 (HLA-B5102 peptide binding motif 
               
               
                 anchor residues are P, A, or G at P2; I or V at 
               
               
                 P9; auxiliary anchor is Y at P3) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 295 
                 RGIQDVRRV 
                 290.400 
               
               
                   
                   
                 (SEQ ID NO: 179) 
               
               
                   
               
               
                 2 
                 303 
                 VPGVAPTLV 
                 200.000 
               
               
                   
                   
                 (SEQ ID NO: 242) 
               
               
                   
               
               
                 3 
                 180 
                 DPMGQQGSL 
                 133.100 
               
               
                   
                   
                 (SEQ ID NO: 59) 
               
               
                   
               
               
                 4 
                 250 
                 VAAGSSSSV 
                 110.000 
               
               
                   
                   
                 (SEQ ID NO: 236) 
               
               
                   
               
               
                 5 
                 30 
                 GAAQWAPVL 
                 55.000 
               
               
                   
                   
                 (SEQ ID NO: 86) 
               
               
                   
               
               
                 6 
                 130 
                 NAPYLPSCL 
                 50.000 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 7 
                 20 
                 GGGGCALPV 
                 44.000 
               
               
                   
                   
                 (SEQ ID NO: 92) 
               
               
                   
               
               
                 8 
                 29 
                 SGAAQWAPV 
                 44.000 
               
               
                   
                   
                 (SEQ ID NO: 211) 
               
               
                   
               
               
                 9 
                 64 
                 PPPPPHSFI 
                 40.000 
               
               
                   
                   
                 (SEQ ID NO: 157) 
               
               
                   
               
               
                 10 
                 119 
                 QASSGQARM 
                 36.300 
               
               
                   
                   
                 (SEQ ID NO: 161) 
               
               
                   
               
               
                 11 
                 110 
                 GPFGPPPPS 
                 27.500 
               
               
                   
                   
                 (SEQ ID NO: 96) 
               
               
                   
               
               
                 12 
                 412 
                 KPFSCRWPS 
                 25.000 
               
               
                   
                   
                 (SEQ ID NO: 123) 
               
               
                   
               
               
                 13 
                 18 
                 LGGGGGCAL 
                 24.200 
               
               
                   
                   
                 (SEQ ID NO: 134) 
               
               
                   
               
               
                 14 
                 24 
                 CALPVSGAA 
                 16.500 
               
               
                   
                   
                 (SEQ ID NO: 43) 
               
               
                   
               
               
                 15 
                 219 
                 TPYSSDNLY 
                 15.000 
               
               
                   
                   
                 (SEQ ID NO: 231) 
               
               
                   
               
               
                 16 
                 292 
                 GVFRGIQDV 
                 14.641 
               
               
                   
                   
                 (SEQ ID NO: 103) 
               
               
                   
               
               
                 17 
                 136 
                 SCLESQPAI 
                 14.520 
               
               
                   
                   
                 (SEQ ID NO: 198) 
               
               
                   
               
               
                 18 
                 418 
                 WPSCQKKFA 
                 12.100 
               
               
                   
                   
                 (SEQ ID NO: 246) 
               
               
                   
               
               
                 19 
                 269 
                 TGYESDNHT 
                 11.000 
               
               
                   
                   
                 (SEQ ID NO: 225) 
               
               
                   
               
               
                 20 
                 351 
                 KPYQCDFKD 
                 11.000 
               
               
                   
                   
                 (SEQ ID NO: 124) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXVIII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B 5201 (HLA-B5201 peptide binding motif 
               
               
                 anchor residues are I or V at P8; I or V at P9; 
               
               
                 auxiliary anchors are Q at P2, F, Y, or W at P3, 
               
               
                 L, I, or V at P5) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 191 
                 QQYSVPPPV 
                 100.000 
               
               
                   
                   
                 (SEQ ID NO: 171) 
               
               
                   
               
               
                 2 
                 32 
                 AQWAPVLDF 
                 30.000 
               
               
                   
                   
                 (SEQ ID NO: 37) 
               
               
                   
               
               
                 3 
                 243 
                 LGATLKGVA 
                 16.500 
               
               
                   
                   
                 (SEQ ID NO: 133) 
               
               
                   
               
               
                 4 
                 303 
                 VPGVAPTLV 
                 13.500 
               
               
                   
                   
                 (SEQ ID NO: 242) 
               
               
                   
               
               
                 5 
                 86 
                 EQCLSAFTV 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 69) 
               
               
                   
               
               
                 6 
                 295 
                 RGIQDVRRV 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 179) 
               
               
                   
               
               
                 7 
                 98 
                 GQFTGTAGA 
                 8.250 
               
               
                   
                   
                 (SEQ ID NO: 99) 
               
               
                   
               
               
                 8 
                 292 
                 GVFRGIQDV 
                 8.250 
               
               
                   
                   
                 (SEQ ID NO: 103) 
               
               
                   
               
               
                 9 
                 29 
                 SGAAQWAPV 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 211) 
               
               
                   
               
               
                 10 
                 146 
                 NQGYSTVTF 
                 5.500 
               
               
                   
                   
                 (SEQ ID NO: 150) 
               
               
                   
               
               
                 11 
                 20 
                 GGGGCALPV 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 92) 
               
               
                   
               
               
                 12 
                 239 
                 NQMNLGATL 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 151) 
               
               
                   
               
               
                 13 
                 64 
                 PPPPPHSFI 
                 3.600 
               
               
                   
                   
                 (SEQ ID NO: 157) 
               
               
                   
               
               
                 14 
                 273 
                 SDNHTTPIL 
                 3.300 
               
               
                   
                   
                 (SEQ ID NO: 204) 
               
               
                   
               
               
                 15 
                 286 
                 YRIHTHGVF 
                 3.000 
               
               
                   
                   
                 (SEQ ID NO: 252) 
               
               
                   
               
               
                 16 
                 269 
                 TGYESDNHT 
                 3.000 
               
               
                   
                   
                 (SEQ ID NO: 225) 
               
               
                   
               
               
                 17 
                 406 
                 TGKTSEKPF 
                 2.750 
               
               
                   
                   
                 (SEQ ID NO: 222) 
               
               
                   
               
               
                 18 
                 327 
                 YPGCNKRYF 
                 2.750 
               
               
                   
                   
                 (SEQ ID NO: 250) 
               
               
                   
               
               
                 19 
                 7 
                 DLNALLPAV 
                 2.640 
               
               
                   
                   
                 (SEQ ID NO: 58) 
               
               
                   
               
               
                 20 
                 104 
                 AGACRYGPF 
                 2.500 
               
               
                   
                   
                 (SEQ ID NO: 31) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXIX 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA B 5801 (HLA-B5801 peptide binding motif 
               
               
                 anchor residues are A, S, or T at P2; F or W at 
               
               
                 P9; auxiliary anchors are P, E, or K at P4, V, 
               
               
                 I, L, M, of F, at P5) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 230 
                 TSQLECMTW 
                 96.800 
               
               
                   
                   
                 (SEQ ID NO: 234) 
               
               
                   
               
               
                 2 
                 92 
                 FTVHFSGQF 
                 60.000 
               
               
                   
                   
                 (SEQ ID NO: 85) 
               
               
                   
               
               
                 3 
                 120 
                 ASSGQARMF 
                 40.000 
               
               
                   
                   
                 (SEQ ID NO: 40) 
               
               
                   
               
               
                 4 
                 168 
                 AAQFPNHSF 
                 20.000 
               
               
                   
                   
                 (SEQ ID NO: 29) 
               
               
                   
               
               
                 5 
                 408 
                 KTSEKPFSC 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 129) 
               
               
                   
               
               
                 6 
                 394 
                 RSDHLKTHT 
                 9.900 
               
               
                   
                   
                 (SEQ ID NO: 192) 
               
               
                   
               
               
                 7 
                 276 
                 HTTPILCGA 
                 7.200 
               
               
                   
                   
                 (SEQ ID NO: 115) 
               
               
                   
               
               
                 8 
                 218 
                 RTPYSSDNL 
                 6.600 
               
               
                   
                   
                 (SEQ ID NO: 194) 
               
               
                   
               
               
                 9 
                 152 
                 VTFDGTPSY 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 244) 
               
               
                   
               
               
                 10 
                 40 
                 FAPPGASAY 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 74) 
               
               
                   
               
               
                 11 
                 213 
                 QALLLRTPY 
                 4.500 
               
               
                   
                   
                 (SEQ ID NO: 160) 
               
               
                   
               
               
                 12 
                 347 
                 HTGEKPYQC 
                 4.400 
               
               
                   
                   
                 (SEQ ID NO: 112) 
               
               
                   
               
               
                 13 
                 252 
                 AGSSSSVKW 
                 4.400 
               
               
                   
                   
                 (SEQ ID NO: 32) 
               
               
                   
               
               
                 14 
                 211 
                 GSQALLLRT 
                 4.356 
               
               
                   
                   
                 (SEQ ID NO: 102) 
               
               
                   
               
               
                 15 
                 174 
                 HSFKHEDPM 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 110) 
               
               
                   
               
               
                 16 
                 317 
                 TSEKRPFMC 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 233) 
               
               
                   
               
               
                 17 
                 26 
                 LPVSGAAQW 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 138) 
               
               
                   
               
               
                 18 
                 289 
                 HTHGVFRGI 
                 3.600 
               
               
                   
                   
                 (SEQ ID NO: 113) 
               
               
                   
               
               
                 19 
                 222 
                 SSDNLYQMT 
                 3.300 
               
               
                   
                   
                 (SEQ ID NO: 217) 
               
               
                   
               
               
                 20 
                 96 
                 FSGQFTGTA 
                 3.300 
               
               
                   
                   
                 (SEQ ID NO: 82) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXX 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA CW0301 (HLA-CW0301 peptide binding motif 
               
               
                 anchor residues are L, F, M, or I at P9; auxiliary 
               
               
                 anchors are V, I, Y, L, or M, at P3, P at P4, F or 
               
               
                 Y at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 10 
                 ALLPAVPSL 
                 100.000 
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
               
                 2 
                 332 
                 KRYFKLSHL 
                 48.000 
               
               
                   
                   
                 (SEQ ID NO: 127) 
               
               
                   
               
               
                 3 
                 126 
                 RMFPNAPYL 
                 36.000 
               
               
                   
                   
                 (SEQ ID NO: 185) 
               
               
                   
               
               
                 4 
                 3 
                 SDVRDLNAL 
                 30.000 
               
               
                   
                   
                 (SEQ ID NO: 206) 
               
               
                   
               
               
                 5 
                 239 
                 NQMNLGATL 
                 24.000 
               
               
                   
                   
                 (SEQ ID NO: 151) 
               
               
                   
               
               
                 6 
                 225 
                 NLYQMTSQL 
                 24.000 
               
               
                   
                   
                 (SEQ ID NO: 147) 
               
               
                   
               
               
                 7 
                 180 
                 DPMGQQGSL 
                 20.000 
               
               
                   
                   
                 (SEQ ID NO: 59) 
               
               
                   
               
               
                 8 
                 362 
                 RRFSRSDQL 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 187) 
               
               
                   
               
               
                 9 
                 329 
                 GCNKRYFKL 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 90) 
               
               
                   
               
               
                 10 
                 286 
                 YRIHTHGVF 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 252) 
               
               
                   
               
               
                 11 
                 301 
                 RRVPGVAPT 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 189) 
               
               
                   
               
               
                 12 
                 24 
                 CALPVSGAA 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 43) 
               
               
                   
               
               
                 13 
                 136 
                 SCLESQPAI 
                 7.500 
               
               
                   
                   
                 (SEQ ID NO: 198) 
               
               
                   
               
               
                 14 
                 437 
                 MHQRNMTKL 
                 7.200 
               
               
                   
                   
                 (SEQ ID NO: 143) 
               
               
                   
               
               
                 15 
                 390 
                 RKFSRSDHL 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 183) 
               
               
                   
               
               
                 16 
                 423 
                 KKFARSDEL 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 122) 
               
               
                   
               
               
                 17 
                 92 
                 FTVHFSGQF 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 85) 
               
               
                   
               
               
                 18 
                 429 
                 DELVRHHNM 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 53) 
               
               
                   
               
               
                 19 
                 130 
                 NAPYLPSCL 
                 4.800 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 20 
                 30 
                 GAAQWAPVL 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 86) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXXI 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA CW0401 (HLA-CW0401 peptide binding motif 
               
               
                 anchor residues are Y, P, or F at P2; L, F, or 
               
               
                 M at P9; auxiliary anchors are V, I, or L at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 356 
                 DFKDCERRF 
                 120.000 
               
               
                   
                   
                 (SEQ ID NO: 55) 
               
               
                   
               
               
                 2 
                 334 
                 YFKLSHLQM 
                 100.000 
               
               
                   
                   
                 (SEQ ID NO: 248) 
               
               
                   
               
               
                 3 
                 180 
                 DPMGQQGSL 
                 88.000 
               
               
                   
                   
                 (SEQ ID NO: 59) 
               
               
                   
               
               
                 4 
                 163 
                 TPSHHAAQF 
                 52.800 
               
               
                   
                   
                 (SEQ ID NO: 228) 
               
               
                   
               
               
                 5 
                 327 
                 YPGCNKRYF 
                 40.000 
               
               
                   
                   
                 (SEQ ID NO: 250) 
               
               
                   
               
               
                 6 
                 285 
                 QYRIHTHGV 
                 27.500 
               
               
                   
                   
                 (SEQ ID NO: 175) 
               
               
                   
               
               
                 7 
                 424 
                 KFARSDELV 
                 25.000 
               
               
                   
                   
                 (SEQ ID NO: 119) 
               
               
                   
               
               
                 8 
                 326 
                 AYPGCNKRY 
                 25.000 
               
               
                   
                   
                 (SEQ ID NO: 42) 
               
               
                   
               
               
                 9 
                 192 
                 QYSVPPPVY 
                 25.000 
               
               
                   
                   
                 (SEQ ID NO: 176) 
               
               
                   
               
               
                 10 
                 417 
                 RWPSCQKKF 
                 22.000 
               
               
                   
                   
                 (SEQ ID NO: 196) 
               
               
                   
               
               
                 11 
                 278 
                 TPILCGAQY 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 227) 
               
               
                   
               
               
                 12 
                 10 
                 ALLPAVPSL 
                 11.616 
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
               
                 13 
                 141 
                 QPAIRNQGY 
                 11.000 
               
               
                   
                   
                 (SEQ ID NO: 170) 
               
               
                   
               
               
                 14 
                 303 
                 VPGVAPTLV 
                 11.000 
               
               
                   
                   
                 (SEQ ID NO: 242) 
               
               
                   
               
               
                 15 
                 219 
                 TPYSSDNLY 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 231) 
               
               
                   
               
               
                 16 
                 39 
                 DFAPPGASA 
                 7.920 
               
               
                   
                   
                 (SEQ ID NO: 54) 
               
               
                   
               
               
                 17 
                 99 
                 QFTGTAGAC 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 165) 
               
               
                   
               
               
                 18 
                 4 
                 DVRDLNALL 
                 5.760 
               
               
                   
                   
                 (SEQ ID NO: 62) 
               
               
                   
               
               
                 19 
                 70 
                 SFIKQEPSW 
                 5.500 
               
               
                   
                   
                 (SEQ ID NO: 210) 
               
               
                   
               
               
                 20 
                 63 
                 PPPPPPHSF 
                 5.280 
               
               
                   
                   
                 (SEQ ID NO: 158) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXXII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA CW0602 (HLA-CW0602 peptide binding motif 
               
               
                 anchor residues are L, I, V, or Y at P9; auxiliary 
               
               
                 anchors are I, L, F, or M at P5, V, I, 
               
               
                 or L at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 332 
                 KRYFKLSHL 
                 9.680 
               
               
                   
                   
                 (SEQ ID NO: 127) 
               
               
                   
               
               
                 2 
                 239 
                 NQMNLGATL 
                 6.600 
               
               
                   
                   
                 (SEQ ID NO: 151) 
               
               
                   
               
               
                 3 
                 130 
                 NAPYLPSCL 
                 6.600 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 4 
                 7 
                 DLNALLPAV 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 58) 
               
               
                   
               
               
                 5 
                 441 
                 NMTKLQLAL 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 149) 
               
               
                   
               
               
                 6 
                 225 
                 NLYQMTSQL 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 147) 
               
               
                   
               
               
                 7 
                 4 
                 DVRDLNALL 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 62) 
               
               
                   
               
               
                 8 
                 3 
                 SDVRDLNAL 
                 4.400 
               
               
                   
                   
                 (SEQ ID NO: 206) 
               
               
                   
               
               
                 9 
                 10 
                 ALLPAVPSL 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
               
                 10 
                 213 
                 QALLLRTPY 
                 3.300 
               
               
                   
                   
                 (SEQ ID NO: 160) 
               
               
                   
               
               
                 11 
                 319 
                 EKRPFMCAY 
                 3.000 
               
               
                   
                   
                 (SEQ ID NO: 67) 
               
               
                   
               
               
                 12 
                 30 
                 GAAQWAPVL 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 86) 
               
               
                   
               
               
                 13 
                 242 
                 NLGATLKGV 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 146) 
               
               
                   
               
               
                 14 
                 292 
                 GVFRGIQDV 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 103) 
               
               
                   
               
               
                 15 
                 207 
                 DSCTGSQAL 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 61) 
               
               
                   
               
               
                 16 
                 362 
                 RRFSRSDQL 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 187) 
               
               
                   
               
               
                 17 
                 439 
                 QRNMTKLQL 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 173) 
               
               
                   
               
               
                 18 
                 295 
                 RGIQDVRRV 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 179) 
               
               
                   
               
               
                 19 
                 423 
                 KKFARSDEL 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 122) 
               
               
                   
               
               
                 20 
                 180 
                 DPMGQQGSL 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 59) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXXIII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Human HLA CW0702 (HLA-CW0702 peptide binding motif 
               
               
                 anchor residues are Y, F, or L at P9; auxiliary 
               
               
                 anchors are Y or P at P2, V, Y, I, L, F, or M at 
               
               
                 P5, V, I, L, or M at P6) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 319 
                 EKRPFMCAY 
                 26.880 
               
               
                   
                   
                 (SEQ ID NO: 67) 
               
               
                   
               
               
                 2 
                 326 
                 AYPGCNKRY 
                 24.000 
               
               
                   
                   
                 (SEQ ID NO: 42) 
               
               
                   
               
               
                 3 
                 40 
                 FAPPGASAY 
                 14.784 
               
               
                   
                   
                 (SEQ ID NO: 74) 
               
               
                   
               
               
                 4 
                 192 
                 QYSVPPPVY 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 176) 
               
               
                   
               
               
                 5 
                 278 
                 TPILCGAQY 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 227) 
               
               
                   
               
               
                 6 
                 219 
                 TPYSSDNLY 
                 12.000 
               
               
                   
                   
                 (SEQ ID NO: 231) 
               
               
                   
               
               
                 7 
                 213 
                 QALLLRTPY 
                 8.800 
               
               
                   
                   
                 (SEQ ID NO: 160) 
               
               
                   
               
               
                 8 
                 125 
                 ARMFPNAPY 
                 8.000 
               
               
                   
                   
                 (SEQ ID NO: 38) 
               
               
                   
               
               
                 9 
                 327 
                 YPGCNKRYF 
                 6.600 
               
               
                   
                   
                 (SEQ ID NO: 250) 
               
               
                   
               
               
                 10 
                 152 
                 VTFDGTPSY 
                 5.600 
               
               
                   
                   
                 (SEQ ID NO: 244) 
               
               
                   
               
               
                 11 
                 141 
                 QPAIRNQGY 
                 4.800 
               
               
                   
                   
                 (SEQ ID NO: 170) 
               
               
                   
               
               
                 12 
                 345 
                 RKHTGEKPY 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 184) 
               
               
                   
               
               
                 13 
                 185 
                 QGSLGEQQY 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 166) 
               
               
                   
               
               
                 14 
                 101 
                 TGTAGACRY 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 224) 
               
               
                   
               
               
                 15 
                 375 
                 RRHTGVKPF 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 188) 
               
               
                   
               
               
                 16 
                 263 
                 GQSNHSTGY 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 100) 
               
               
                   
               
               
                 17 
                 163 
                 TPSHHAAQF 
                 3.000 
               
               
                   
                   
                 (SEQ ID NO: 228) 
               
               
                   
               
               
                 18 
                 33 
                 QWAPVLDFA 
                 2.688 
               
               
                   
                   
                 (SEQ ID NO: 174) 
               
               
                   
               
               
                 19 
                 130 
                 NAPYLPSCL 
                 2.640 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 20 
                 84 
                 HEEQCLSAF 
                 2.400 
               
               
                   
                   
                 (SEQ ID NO: 107) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXXIV 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Mouse MHC Class I Db 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 235 
                 CMTWNQMNL 
                 5255.712 
               
               
                   
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                 2 
                 126 
                 RMFPNAPYL 
                 1990.800 
               
               
                   
                   
                 (SEQ ID NO: 185) 
               
               
                   
               
               
                 3 
                 221 
                 YSSDNLYQM 
                 930.000 
               
               
                   
                   
                 (SEQ ID NO: 253) 
               
               
                   
               
               
                 4 
                 228 
                 QMTSQLECM 
                 33.701 
               
               
                   
                   
                 (SEQ ID NO: 169) 
               
               
                   
               
               
                 5 
                 239 
                 NQMNLGATL 
                 21.470 
               
               
                   
                   
                 (SEQ ID NO: 151) 
               
               
                   
               
               
                 6 
                 441 
                 NMTKLQLAL 
                 19.908 
               
               
                   
                   
                 (SEQ ID NO: 149) 
               
               
                   
               
               
                 7 
                 437 
                 MHQRNMTKL 
                 19.837 
               
               
                   
                   
                 (SEQ ID NO: 143) 
               
               
                   
               
               
                 8 
                 136 
                 SCLESQPAI 
                 11.177 
               
               
                   
                   
                 (SEQ ID NO: 198) 
               
               
                   
               
               
                 9 
                 174 
                 HSFKHEDPM 
                 10.800 
               
               
                   
                   
                 (SEQ ID NO: 110) 
               
               
                   
               
               
                 10 
                 302 
                 RVPGVAPTL 
                 10.088 
               
               
                   
                   
                 (SEQ ID NO: 195) 
               
               
                   
               
               
                 11 
                 130 
                 NAPYLPSCL 
                 8.400 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 12 
                 10 
                 ALLPAVPSL 
                 5.988 
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
               
                 13 
                 208 
                 SCTGSQALL 
                 4.435 
               
               
                   
                   
                 (SEQ ID NO: 202) 
               
               
                   
               
               
                 14 
                 209 
                 CTGSQALLL 
                 3.548 
               
               
                   
                   
                 (SEQ ID NO: 52) 
               
               
                   
               
               
                 15 
                 238 
                 WNQMNLGAT 
                 3.300 
               
               
                   
                   
                 (SEQ ID NO: 245) 
               
               
                   
               
               
                 16 
                 218 
                 RTPYSSDNL 
                 3.185 
               
               
                   
                   
                 (SEQ ID NO: 194) 
               
               
                   
               
               
                 17 
                 24 
                 CALPVSGAA 
                 2.851 
               
               
                   
                   
                 (SEQ ID NO: 43) 
               
               
                   
               
               
                 18 
                 18 
                 LGGGGGCAL 
                 2.177 
               
               
                   
                   
                 (SEQ ID NO: 134) 
               
               
                   
               
               
                 19 
                 142 
                 PAIRNQGYS 
                 2.160 
               
               
                   
                   
                 (SEQ ID NO: 152) 
               
               
                   
               
               
                 20 
                 30 
                 GAAQWAPVL 
                 1.680 
               
               
                   
                   
                 (SEQ ID NO: 86) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXXV 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Mouse MHC Class I Dd 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 112 
                 FGPPPPSQA 
                 48.000 
               
               
                   
                   
                 (SEQ ID NO: 76) 
               
               
                   
               
               
                 2 
                 122 
                 SGQARMFPN 
                 36.000 
               
               
                   
                   
                 (SEQ ID NO: 212) 
               
               
                   
               
               
                 3 
                 104 
                 AGACRYGPF 
                 30.000 
               
               
                   
                   
                 (SEQ ID NO: 31) 
               
               
                   
               
               
                 4 
                 218 
                 RTPYSSDNL 
                 28.800 
               
               
                   
                   
                 (SEQ ID NO: 194) 
               
               
                   
               
               
                 5 
                 130 
                 NAPYLPSCL 
                 20.000 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 6 
                 302 
                 RVPGVAPTL 
                 20.000 
               
               
                   
                   
                 (SEQ ID NO: 195) 
               
               
                   
               
               
                 7 
                 18 
                 LGGGGGCAL 
                 20.000 
               
               
                   
                   
                 (SEQ ID NO: 134) 
               
               
                   
               
               
                 8 
                 81 
                 AEPHEEQCL 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 30) 
               
               
                   
               
               
                 9 
                 29 
                 SGAAQWAPV 
                 7.200 
               
               
                   
                   
                 (SEQ ID NO: 211) 
               
               
                   
               
               
                 10 
                 423 
                 KKFARSDEL 
                 7.200 
               
               
                   
                   
                 (SEQ ID NO: 122) 
               
               
                   
               
               
                 11 
                 295 
                 RGIQDVRRV 
                 7.200 
               
               
                   
                   
                 (SEQ ID NO: 179) 
               
               
                   
               
               
                 12 
                 390 
                 RKFSRSDHL 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 183) 
               
               
                   
               
               
                 13 
                 332 
                 KRYFKLSHL 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 127) 
               
               
                   
               
               
                 14 
                 362 
                 RRFSRSDQL 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 187) 
               
               
                   
               
               
                 15 
                 417 
                 RWPSCQKKF 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 196) 
               
               
                   
               
               
                 16 
                 160 
                 YGHTPSHHA 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 249) 
               
               
                   
               
               
                 17 
                 20 
                 GGGGCALPV 
                 6.000 
               
               
                   
                   
                 (SEQ ID NO: 92) 
               
               
                   
               
               
                 18 
                 329 
                 GCNKRYFKL 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 90) 
               
               
                   
               
               
                 19 
                 372 
                 RHQRRHTGV 
                 4.500 
               
               
                   
                   
                 (SEQ ID NO: 181) 
               
               
                   
               
               
                 20 
                 52 
                 GGPAPPPAP 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 93) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXXVI 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Mouse MHC Class I Kb 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 329 
                 GCNKRYFKL 
                 24.000 
               
               
                   
                   
                 (SEQ ID NO: 90) 
               
               
                   
               
               
                 2 
                 225 
                 NLYQMTSQL 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 147) 
               
               
                   
               
               
                 3 
                 420 
                 SCQKKFARS 
                 3.960 
               
               
                   
                   
                 (SEQ ID NO: 200) 
               
               
                   
               
               
                 4 
                 218 
                 RTPYSSDNL 
                 3.630 
               
               
                   
                   
                 (SEQ ID NO: 194) 
               
               
                   
               
               
                 5 
                 437 
                 MHQRNMTKL 
                 3.600 
               
               
                   
                   
                 (SEQ ID NO: 143) 
               
               
                   
               
               
                 6 
                 387 
                 TCQRKFSRS 
                 3.600 
               
               
                   
                   
                 (SEQ ID NO: 219) 
               
               
                   
               
               
                 7 
                 302 
                 RVPGVAPTL 
                 3.300 
               
               
                   
                   
                 (SEQ ID NO: 195) 
               
               
                   
               
               
                 8 
                 130 
                 NAPYLPSCL 
                 3.000 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 9 
                 289 
                 HTHGVFRGI 
                 3.000 
               
               
                   
                   
                 (SEQ ID NO: 113) 
               
               
                   
               
               
                 10 
                 43 
                 PGASAYGSL 
                 2.400 
               
               
                   
                   
                 (SEQ ID NO: 153) 
               
               
                   
               
               
                 11 
                 155 
                 DGTPSYGHT 
                 2.400 
               
               
                   
                   
                 (SEQ ID NO: 56) 
               
               
                   
               
               
                 12 
                 273 
                 SDNHTTPIL 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 204) 
               
               
                   
               
               
                 13 
                 126 
                 RMFPNAPYL 
                 2.200 
               
               
                   
                   
                 (SEQ ID NO: 185) 
               
               
                   
               
               
                 14 
                 128 
                 FPNAPYLPS 
                 2.000 
               
               
                   
                   
                 (SEQ ID NO: 79) 
               
               
                   
               
               
                 15 
                 3 
                 SDVRDLNAL 
                 1.584 
               
               
                   
                   
                 (SEQ ID NO: 206) 
               
               
                   
               
               
                 16 
                 207 
                 DSCTGSQAL 
                 1.584 
               
               
                   
                   
                 (SEQ ID NO: 61) 
               
               
                   
               
               
                 17 
                 332 
                 KRYFKLSHL 
                 1.500 
               
               
                   
                   
                 (SEQ ID NO: 127) 
               
               
                   
               
               
                 18 
                 18 
                 LGGGGGCAL 
                 1.320 
               
               
                   
                   
                 (SEQ ID NO: 134) 
               
               
                   
               
               
                 19 
                 233 
                 LECMTWNQM 
                 1.320 
               
               
                   
                   
                 (SEQ ID NO: 131) 
               
               
                   
               
               
                 20 
                 441 
                 NMTKLQLAL 
                 1.200 
               
               
                   
                   
                 (SEQ ID NO: 149) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXXVII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Mouse MHC Class I Kd 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 285 
                 QYRIHTHGV 
                 600.000 
               
               
                   
                   
                 (SEQ ID NO: 175) 
               
               
                   
               
               
                 2 
                 424 
                 KFARSDELV 
                 288.000 
               
               
                   
                   
                 (SEQ ID NO: 119) 
               
               
                   
               
               
                 3 
                 334 
                 YFKLSHLQM 
                 120.000 
               
               
                   
                   
                 (SEQ ID NO: 248) 
               
               
                   
               
               
                 4 
                 136 
                 SCLESQPTI 
                 115.200 
               
               
                   
                   
                 (SEQ ID NO: 199) 
               
               
                   
               
               
                 5 
                 239 
                 NQMNLGATL 
                 115.200 
               
               
                   
                   
                 (SEQ ID NO: 151) 
               
               
                   
               
               
                 6 
                 10 
                 ALLPAVSSL 
                 115.200 
               
               
                   
                   
                 (SEQ ID NO: 35) 
               
               
                   
               
               
                 7 
                 47 
                 AYGSLGGPA 
                 86.400 
               
               
                   
                   
                 (SEQ ID NO: 41) 
               
               
                   
               
               
                 8 
                 180 
                 DPMGQQGSL 
                 80.000 
               
               
                   
                   
                 (SEQ ID NO: 59) 
               
               
                   
               
               
                 9 
                 270 
                 GYESDNHTA 
                 72.000 
               
               
                   
                   
                 (SEQ ID NO: 105) 
               
               
                   
               
               
                 10 
                 326 
                 AYPGCNKRY 
                 60.000 
               
               
                   
                   
                 (SEQ ID NO: 42) 
               
               
                   
               
               
                 11 
                 192 
                 QYSVPPPVY 
                 60.000 
               
               
                   
                   
                 (SEQ ID NO: 176) 
               
               
                   
               
               
                 12 
                 272 
                 ESDNHTAPI 
                 57.600 
               
               
                   
                   
                 (SEQ ID NO: 70) 
               
               
                   
               
               
                 13 
                 289 
                 HTHGVFRGI 
                 57.600 
               
               
                   
                   
                 (SEQ ID NO: 113) 
               
               
                   
               
               
                 14 
                 126 
                 DVRDLNALL 
                 57.600 
               
               
                   
                   
                 (SEQ ID NO: 62) 
               
               
                   
               
               
                 15 
                 4 
                 CTGSQALLL 
                 57.600 
               
               
                   
                   
                 (SEQ ID NO: 52) 
               
               
                   
               
               
                 16 
                 208 
                 SCTGSQALL 
                 48.000 
               
               
                   
                   
                 (SEQ ID NO: 202) 
               
               
                   
               
               
                 17 
                 441 
                 NMTKLQLAL 
                 48.000 
               
               
                   
                   
                 (SEQ ID NO: 149) 
               
               
                   
               
               
                 18 
                 207 
                 DSCTGSQAL 
                 48.000 
               
               
                   
                   
                 (SEQ ID NO: 61) 
               
               
                   
               
               
                 19 
                 130 
                 NAPYLPSCL 
                 48.000 
               
               
                   
                   
                 (SEQ ID NO: 144) 
               
               
                   
               
               
                 20 
                 235 
                 CMTWNQMNL 
                 48.000 
               
               
                   
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXXVIII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides to 
               
               
                 Mouse MHC Class I Kk 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score (Estimate 
               
               
                   
                   
                   
                 of Half Time of 
               
               
                   
                   
                   
                 Disassociation of 
               
               
                   
                 Start 
                 Subsequence 
                 a Molecule Containing 
               
               
                 Rank 
                 Position 
                 Residue Listing 
                 This Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 81 
                 AEPHEEQCL 
                 40.000 
               
               
                   
                   
                 (SEQ ID NO: 30) 
               
               
                   
               
               
                 2 
                 85 
                 EEQCLSAFT 
                 40.000 
               
               
                   
                   
                 (SEQ ID NO: 65) 
               
               
                   
               
               
                 3 
                 429 
                 DELVRHHNM 
                 20.000 
               
               
                   
                   
                 (SEQ ID NO: 53) 
               
               
                   
               
               
                 4 
                 315 
                 SETSEKRPF 
                 20.000 
               
               
                   
                   
                 (SEQ ID NO: 209) 
               
               
                   
               
               
                 5 
                 261 
                 TEGQSNHST 
                 20.000 
               
               
                   
                   
                 (SEQ ID NO: 221) 
               
               
                   
               
               
                 6 
                 410 
                 SEKPFSCRW 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 207) 
               
               
                   
               
               
                 7 
                 272 
                 ESDNHTTPI 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 71) 
               
               
                   
               
               
                 8 
                 318 
                 SEKRPFMCA 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 208) 
               
               
                   
               
               
                 9 
                 138 
                 LESQPAIRN 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 132) 
               
               
                   
               
               
                 10 
                 233 
                 LECMTWNQM 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 131) 
               
               
                   
               
               
                 11 
                 298 
                 QDVRRVPGV 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 164) 
               
               
                   
               
               
                 12 
                 84 
                 HEEQCLSAF 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 107) 
               
               
                   
               
               
                 13 
                 349 
                 GEKPYQCDF 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 91) 
               
               
                   
               
               
                 14 
                 289 
                 HTHGVFRGI 
                 10.000 
               
               
                   
                   
                 (SEQ ID NO: 113) 
               
               
                   
               
               
                 15 
                 179 
                 EDPMGQQGS 
                 8.000 
               
               
                   
                   
                 (SEQ ID NO: 64) 
               
               
                   
               
               
                 16 
                 136 
                 SCLESQPAI 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 198) 
               
               
                   
               
               
                 17 
                 280 
                 ILCGAQYRI 
                 5.000 
               
               
                   
                   
                 (SEQ ID NO: 116) 
               
               
                   
               
               
                 18 
                 273 
                 SDNHTTPIL 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 204) 
               
               
                   
               
               
                 19 
                 428 
                 SDELVRHHN 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 203) 
               
               
                   
               
               
                 20 
                 3 
                 SDVRDLNAL 
                 4.000 
               
               
                   
                   
                 (SEQ ID NO: 206) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XXXIX 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Mouse MHC Class I Ld 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of a 
               
               
                   
                   
                   
                 Molecule 
               
               
                   
                 Start 
                   
                 Containing 
               
               
                   
                 Posi- 
                 Subsequence 
                 This 
               
               
                 Rank 
                 tion 
                 Residue Listing 
                 Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 163 
                 TPSHHAAQF (SEQ ID NO: 228) 
                 360.000 
               
               
                   
               
               
                 2 
                 327 
                 YPGCNKRYF (SEQ ID NO: 250) 
                 300.000 
               
               
                   
               
               
                 3 
                 180 
                 DPMGQQGSL (SEQ ID NO: 59) 
                 150.000 
               
               
                   
               
               
                 4 
                 26 
                 LPVSGAAQW (SEQ ID NO: 138) 
                 93.600 
               
               
                   
               
               
                 5 
                 278 
                 TPILCGAQY (SEQ ID NO: 227) 
                 72.000 
               
               
                   
               
               
                 6 
                 141 
                 QPAIRNQGY (SEQ ID NO: 170) 
                 60.000 
               
               
                   
               
               
                 7 
                 219 
                 TPYSSDNLY (SEQ ID NO: 231) 
                 60.000 
               
               
                   
               
               
                 8 
                 303 
                 VPGVAPTLV (SEQ ID NO: 242) 
                 60.000 
               
               
                   
               
               
                 9 
                 120 
                 ASSGQARMF (SEQ ID NO: 40) 
                 50.000 
               
               
                   
               
               
                 10 
                 63 
                 PPPPPPHSF (SEQ ID NO: 158) 
                 45.000 
               
               
                   
               
               
                 11 
                 113 
                 GPPPPSQAS (SEQ ID NO: 97) 
                 45.000 
               
               
                   
               
               
                 12 
                 157 
                 TPSYGHTPS (SEQ ID NO: 229) 
                 39.000 
               
               
                   
               
               
                 13 
                 207 
                 DSCTGSQAL (SEQ ID NO: 61) 
                 32.500 
               
               
                   
               
               
                 14 
                 110 
                 GPFGPPPPS (SEQ ID NO: 96) 
                 30.000 
               
               
                   
               
               
                 15 
                 82 
                 EPHEEQCLS (SEQ ID NO: 68) 
                 30.000 
               
               
                   
               
               
                 16 
                 412 
                 KPFSCRWPS (SEQ ID NO: 123) 
                 30.000 
               
               
                   
               
               
                 17 
                 418 
                 WPSCQKKFA (SEQ ID NO: 246) 
                 30.000 
               
               
                   
               
               
                 18 
                 221 
                 YSSDNLYQM (SEQ ID NO: 253) 
                 30.000 
               
               
                   
               
               
                 19 
                 204 
                 TPTDSCTGS (SEQ ID NO: 230) 
                 30.000 
               
               
                   
               
               
                 20 
                 128 
                 FPNAPYLPS (SEQ ID NO: 79) 
                 30.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XL 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Human WT1 Peptides 
               
               
                 to Cattle HLA A20 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of a 
               
               
                   
                   
                   
                 Molecule 
               
               
                   
                 Start 
                   
                 Containing 
               
               
                   
                 Posi- 
                 Subsequence 
                 This 
               
               
                 Rank 
                 tion 
                 Residue Listing 
                 Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 350 
                 EKPYQCDFK (SEQ ID NO: 66) 
                 1000.00 
               
               
                   
               
               
                 2 
                 319 
                 EKRPFMCAY (SEQ ID NO: 67) 
                 500.000 
               
               
                   
               
               
                 3 
                 423 
                 KKFARSDEL (SEQ ID NO: 122) 
                 500.000 
               
               
                   
               
               
                 4 
                 345 
                 RKHTGEKPY (SEQ ID NO: 184) 
                 500.000 
               
               
                   
               
               
                 5 
                 390 
                 RKFSRSDHL (SEQ ID NO: 183) 
                 500.000 
               
               
                   
               
               
                 6 
                 137 
                 CLESQPAIR (SEQ ID NO: 47) 
                 120.000 
               
               
                   
               
               
                 7 
                 380 
                 VKPFQCKTC (SEQ ID NO: 239) 
                 100.000 
               
               
                   
               
               
                 8 
                 407 
                 GKTSEKPFS (SEQ ID NO: 95) 
                 100.000 
               
               
                   
               
               
                 9 
                 335 
                 FKLSHLQMH (SEQ ID NO: 78) 
                 100.000 
               
               
                   
               
               
                 10 
                 247 
                 LKGVAAGSS (SEQ ID NO: 135) 
                 100.000 
               
               
                   
               
               
                 11 
                 370 
                 LKRHQRRHT (SEQ ID NO: 136) 
                 100.000 
               
               
                   
               
               
                 12 
                 258 
                 VKWTEGQSN (SEQ ID NO: 240) 
                 100.000 
               
               
                   
               
               
                 13 
                 398 
                 LKTHTRTHT (SEQ ID NO: 137) 
                 100.000 
               
               
                   
               
               
                 14 
                 331 
                 NKRYFKLSH (SEQ ID NO: 145) 
                 100.000 
               
               
                   
               
               
                 15 
                 357 
                 FKDCERRFS (SEQ ID NO: 77) 
                 100.000 
               
               
                   
               
               
                 16 
                 385 
                 CKTCQRKFS (SEQ ID NO: 46) 
                 100.000 
               
               
                   
               
               
                 17 
                 294 
                 FRGIQDVRR (SEQ ID NO: 81) 
                 80.000 
               
               
                   
               
               
                 18 
                 368 
                 DQLKRHQRR (SEQ ID NO: 60) 
                 80.000 
               
               
                   
               
               
                 19 
                 432 
                 VRHHNMHQR (SEQ ID NO: 243) 
                 80.000 
               
               
                   
               
               
                 20 
                 118 
                 SQASSGQAR (SEQ ID NO: 216) 
                 80.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XLI 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Mouse WT1 Peptides 
               
               
                 to Human HLA A 0201 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of a 
               
               
                   
                   
                   
                 Molecule 
               
               
                   
                 Start 
                   
                 Containing 
               
               
                   
                 Posi- 
                 Subsequence 
                 This 
               
               
                 Rank 
                 tion 
                 Residue Listing 
                 Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 126 
                 RMFPNAPYL (SEQ ID NO: 293) 
                 313.968 
               
               
                   
               
               
                 2 
                 187 
                 SLGEQQYSV (SEQ ID NO: 299) 
                 285.163 
               
               
                   
               
               
                 3 
                 10 
                 ALLPAVSSL (SEQ ID NO: 255) 
                 181.794 
               
               
                   
               
               
                 4 
                 225 
                 NLYQMTSQL (SEQ ID NO: 284) 
                 68.360 
               
               
                   
               
               
                 5 
                 292 
                 GVFRGIQDV (SEQ ID NO: 270) 
                 51.790 
               
               
                   
               
               
                 6 
                 93 
                 TLHFSGQFT (SEQ ID NO: 302) 
                 40.986 
               
               
                   
               
               
                 7 
                 191 
                 QQYSVPPPV (SEQ ID NO: 290) 
                 22.566 
               
               
                   
               
               
                 8 
                 280 
                 ILCGAQYRI (SEQ ID NO: 274) 
                 17.736 
               
               
                   
               
               
                 9 
                 441 
                 NMTKLHVAL (SEQ ID NO: 285) 
                 15.428 
               
               
                   
               
               
                 10 
                 235 
                 CMTWNQMNL (SEQ ID NO: 258) 
                 15.428 
               
               
                   
               
               
                 11 
                 7 
                 DLNALLPAV (SEQ ID NO: 261) 
                 11.998 
               
               
                   
               
               
                 12 
                 242 
                 NLGATLKGM (SEQ ID NO: 283) 
                 11.426 
               
               
                   
               
               
                 13 
                 227 
                 YQMTSQLEC (SEQ ID NO: 307) 
                 8.573 
               
               
                   
               
               
                 14 
                 239 
                 NQMNLGATL (SEQ ID NO: 286) 
                 8.014 
               
               
                   
               
               
                 15 
                 309 
                 TLVRSASET (SEQ ID NO: 303) 
                 7.452 
               
               
                   
               
               
                 16 
                 408 
                 KTSEKPFSC (SEQ ID NO: 277) 
                 5.743 
               
               
                   
               
               
                 17 
                 340 
                 LQMHSRKHT (SEQ ID NO: 280) 
                 4.752 
               
               
                   
               
               
                 18 
                 228 
                 QMTSQLECM (SEQ ID NO: 289) 
                 4.044 
               
               
                   
               
               
                 19 
                 37 
                 VLDFAPPGA (SEQ ID NO: 304) 
                 3.378 
               
               
                   
               
               
                 20 
                 302 
                 RVSGVAPTL (SEQ ID NO: 295) 
                 1.869 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XLII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding Prediction 
               
               
                 Analysis for Binding of Mouse WT1 Peptides 
               
               
                 to Mouse MHC Class I Db 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of a 
               
               
                   
                   
                   
                 Molecule 
               
               
                   
                 Start 
                   
                 Containing 
               
               
                   
                 Posi- 
                 Subsequence 
                 This 
               
               
                 Rank 
                 tion 
                 Residue Listing 
                 Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 221 
                 YSSDNLYQM (SEQ ID NO: 308) 
                 312.000 
               
               
                   
               
               
                 2 
                 126 
                 RMFPNAPYL (SEQ ID NO: 293) 
                 260.000 
               
               
                   
               
               
                 3 
                 235 
                 CMTWNQMNL (SEQ ID NO: 258) 
                 260.000 
               
               
                   
               
               
                 4 
                 437 
                 MHQRNMTKL (SEQ ID NO: 281) 
                 200.000 
               
               
                   
               
               
                 5 
                 238 
                 WNQMNLGAT (SEQ ID NO: 305) 
                 12.000 
               
               
                   
               
               
                 6 
                 130 
                 NAPYLPSCL (SEQ ID NO: 282) 
                 8.580 
               
               
                   
               
               
                 7 
                 3 
                 SDVRDLNAL (SEQ ID NO: 298) 
                 7.920 
               
               
                   
               
               
                 8 
                 136 
                 SCLESQPTI (SEQ ID NO: 296) 
                 7.920 
               
               
                   
               
               
                 9 
                 81 
                 AEPHEEQCL (SEQ ID NO: 254) 
                 6.600 
               
               
                   
               
               
                 10 
                 10 
                 ALLPAVSSL (SEQ ID NO: 255) 
                 6.600 
               
               
                   
               
               
                 11 
                 218 
                 RTPYSSDNL (SEQ ID NO: 294) 
                 6.000 
               
               
                   
               
               
                 12 
                 441 
                 NMTKLHVAL (SEQ ID NO: 285) 
                 3.432 
               
               
                   
               
               
                 13 
                 228 
                 QMTSQLECM (SEQ ID NO: 289) 
                 3.120 
               
               
                   
               
               
                 14 
                 174 
                 HSFKHEDPM (SEQ ID NO: 272) 
                 3.120 
               
               
                   
               
               
                 15 
                 242 
                 NLGATLKGM (SEQ ID NO: 283) 
                 2.640 
               
               
                   
               
               
                 16 
                 261 
                 TEGQSNHGI (SEQ ID NO: 301) 
                 2.640 
               
               
                   
               
               
                 17 
                 225 
                 NLYQMTSQL (SEQ ID NO: 284) 
                 2.640 
               
               
                   
               
               
                 18 
                 207 
                 DSCTGSQAL (SEQ ID NO: 263) 
                 2.600 
               
               
                   
               
               
                 19 
                 119 
                 QASSGQARM (SEQ ID NO: 288) 
                 2.600 
               
               
                   
               
               
                 20 
                 18 
                 LGGGGGCGL (SEQ ID NO: 279) 
                 2.600 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XLIII 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding 
               
               
                 Prediction Analysis for Binding of Mouse 
               
               
                 WT1 Peptides to Mouse MHC Class I Kb 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of a 
               
               
                   
                   
                   
                 Molecule 
               
               
                   
                 Start 
                   
                 Containing 
               
               
                   
                 Posi- 
                 Subsequence 
                 This 
               
               
                 Rank 
                 tion 
                 Residue Listing 
                 Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 329 
                 GCNKRYFKL (SEQ ID NO: 268) 
                 24.000 
               
               
                   
               
               
                 2 
                 225 
                 NLYQMTSQL (SEQ ID NO: 284) 
                 10.000 
               
               
                   
               
               
                 3 
                 420 
                 SCQKKFARS (SEQ ID NO: 297) 
                 3.960 
               
               
                   
               
               
                 4 
                 218 
                 RTPYSSDNL (SEQ ID NO: 294) 
                 3.630 
               
               
                   
               
               
                 5 
                 437 
                 MHQRNMTKL (SEQ ID NO: 281) 
                 3.600 
               
               
                   
               
               
                 6 
                 387 
                 TCQRKFSRS (SEQ ID NO: 300) 
                 3.600 
               
               
                   
               
               
                 7 
                 289 
                 HTHGVFRGI (SEQ ID NO: 273) 
                 3.000 
               
               
                   
               
               
                 8 
                 130 
                 NAPYLPSCL (SEQ ID NO: 282) 
                 3.000 
               
               
                   
               
               
                 9 
                 43 
                 PGASAYGSL (SEQ ID NO: 287) 
                 2.400 
               
               
                   
               
               
                 10 
                 155 
                 DGAPSYGHT (SEQ ID NO: 260) 
                 2.400 
               
               
                   
               
               
                 11 
                 126 
                 RMFPNAPYL (SEQ ID NO: 293) 
                 2.200 
               
               
                   
               
               
                 12 
                 128 
                 FPNAPYLPS (SEQ ID NO: 267) 
                 2.000 
               
               
                   
               
               
                 13 
                 207 
                 DSCTGSQAL (SEQ ID NO: 263) 
                 1.584 
               
               
                   
               
               
                 14 
                 3 
                 SDVRDLNAL (SEQ ID NO: 298) 
                 1.584 
               
               
                   
               
               
                 15 
                 332 
                 KRYFKLSHL (SEQ ID NO: 276) 
                 1.500 
               
               
                   
               
               
                 16 
                 233 
                 LECMTWNQM (SEQ ID NO: 278) 
                 1.320 
               
               
                   
               
               
                 17 
                 18 
                 LGGGGGCGL (SEQ ID NO: 279) 
                 1.320 
               
               
                   
               
               
                 18 
                 242 
                 NLGATLKGM (SEQ ID NO: 283) 
                 1.200 
               
               
                   
               
               
                 19 
                 123 
                 GQARMFPN (SEQ ID NO: 269)A 
                 1.200 
               
               
                   
               
               
                 20 
                 441 
                 NMTKLHVAL (SEQ ID NO: 285) 
                 1.200 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XLIV 
               
             
            
               
                   
               
               
                 Results of BIMAS HLA Peptide Binding 
               
               
                 Prediction Analysis for Binding of Mouse 
               
               
                 WT1 Peptides to Mouse MHC Class I Kd 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Score 
               
               
                   
                   
                   
                 (Estimate 
               
               
                   
                   
                   
                 of Half 
               
               
                   
                   
                   
                 Time of Dis- 
               
               
                   
                   
                   
                 association 
               
               
                   
                   
                   
                 of a 
               
               
                   
                   
                   
                 Molecule 
               
               
                   
                 Start 
                   
                 Containing 
               
               
                   
                 Posi- 
                 Subsequence 
                 This 
               
               
                 Rank 
                 tion 
                 Residue Listing 
                 Subsequence) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 285 
                 QYRIHTHGV (SEQ ID NO: 291) 
                 600.000 
               
               
                   
               
               
                 2 
                 424 
                 KFARSDELV (SEQ ID NO: 275) 
                 288.000 
               
               
                   
               
               
                 3 
                 334 
                 YFKLSHLQM (SEQ ID NO: 306) 
                 120.000 
               
               
                   
               
               
                 4 
                 136 
                 SCLESQPTI (SEQ ID NO: 296) 
                 115.200 
               
               
                   
               
               
                 5 
                 239 
                 NQMNLGATL (SEQ ID NO: 286) 
                 115.200 
               
               
                   
               
               
                 6 
                 10 
                 ALLPAVSSL (SEQ ID NO: 255) 
                 115.200 
               
               
                   
               
               
                 7 
                 47 
                 AYGSLGGPA (SEQ ID NO: 256) 
                 86.400 
               
               
                   
               
               
                 8 
                 180 
                 DPMGQQGSL (SEQ ID NO: 262) 
                 80.000 
               
               
                   
               
               
                 9 
                 270 
                 GYESDNHTA (SEQ ID NO: 271) 
                 72.000 
               
               
                   
               
               
                 10 
                 192 
                 QYSVPPPVY (SEQ ID NO: 292) 
                 60.000 
               
               
                   
               
               
                 11 
                 326 
                 AYPGCNKRY (SEQ ID NO: 257) 
                 60.000 
               
               
                   
               
               
                 12 
                 289 
                 HTHGVFRGI (SEQ ID NO: 273) 
                 57.600 
               
               
                   
               
               
                 13 
                 4 
                 DVRDLNALL (SEQ ID NO: 264) 
                 57.600 
               
               
                   
               
               
                 14 
                 126 
                 RMFPNAPYL (SEQ ID NO: 293) 
                 57.600 
               
               
                   
               
               
                 15 
                 209 
                 CTGSQALLL (SEQ ID NO: 259) 
                 48.000 
               
               
                   
               
               
                 16 
                 86 
                 EQCLSAFTL (SEQ ID NO: 265) 
                 48.000 
               
               
                   
               
               
                 17 
                 302 
                 RVSGVAPTL (SEQ ID NO: 295) 
                 48.000 
               
               
                   
               
               
                 18 
                 218 
                 RTPYSSDNL (SEQ ID NO: 294) 
                 48.000 
               
               
                   
               
               
                 19 
                 272 
                 ESDNHTAPI (SEQ ID NO: 266) 
                 48.000 
               
               
                   
               
               
                 20 
                 225 
                 NLYQMTSQL (SEQ ID NO: 284) 
                 48.000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE XLV 
               
             
            
               
                   
               
               
                 Results of TSites Peptide Binding Prediction 
               
               
                 Analysis for Human WT1 Peptides Capable 
               
               
                 of Eliciting a Helper T cell Response 
               
            
           
           
               
               
            
               
                 Peptide 
                 Sequence 
               
               
                   
               
               
                 p6-23 
                 RDLNALLPAVPSLGGGG (SEQ ID NO: 1) 
               
               
                   
               
               
                 p30-35 
                 GAAQWA (SEQ ID NO: 309) 
               
               
                   
               
               
                 p45-56 
                 ASAYGSLGGPAP (SEQ ID NO: 310) 
               
               
                   
               
               
                 p91-105 
                 AFTVHFSGQFTGTAG (SEQ ID NO: 311) 
               
               
                   
               
               
                 p117-139 
                 PSQASSGQARMFPNAPYLPSCLE (SEQ ID NO: 2) 
               
               
                   
               
               
                 p167-171 
                 HAAQF (SEQ ID NO: 312) 
               
               
                   
               
               
                 p202-233 
                 CHTPTDSCTGSQALLLRTPYSSDNLYQMTSQL 
               
               
                   
                 (SEQ ID NO: 313) 
               
               
                   
               
               
                 p244-262 
                 GATLKGVAAGSSSSVKWTE (SEQ ID NO: 4) 
               
               
                   
               
               
                 p287-318 
                 RIHTHGVFRGIQDVRRVPGVAPTLVRSASETS 
               
               
                   
                 (SEQ ID NO: 314) 
               
               
                   
               
               
                 p333-336 
                 RYFK (SEQ ID NO: 315) 
               
               
                   
               
               
                 p361-374 
                 ERRFSRSDQLKRHQ (SEQ ID NO: 316) 
               
               
                   
               
               
                 p389-410 
                 QRKFSRSDHLKTHTRTHTGKTS (SEQ ID NO: 317) 
               
               
                   
               
               
                 p421-441 
                 CQKKFARSDELVRHHNMHQRN (SEQ ID NO: 318) 
               
               
                   
               
            
           
         
       
     
     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 
               
             
            
               
                   
               
               
                 WT1 Peptide Sequences and HLA 
               
               
                 Peptide Binding Predictions 
               
            
           
           
               
               
               
            
               
                 Peptide 
                 Sequence 
                 Comments 
               
               
                   
               
               
                 p329-337 
                 GCNKRYFKL 
                 Score 24,000 
               
               
                   
                 (SEQ ID NOs: 90 and 
               
               
                   
                 268) 
               
               
                   
               
               
                 p225-233 
                 NLYQMTSQL 
                 binds also to class II 
               
               
                   
                 (SEQ ID NOs: 147 
                 and HLA A2, Kd, 
               
               
                   
                 and 284) 
                 score 10,000 
               
               
                   
               
               
                 p235-243 
                 CMTWNQMNL 
                 binds also to HLA A2, 
               
               
                   
                 (SEQ ID NOs: 49 and 
                 score 5,255,712 
               
               
                   
                 258) 
               
               
                   
               
               
                 p126-134 
                 RMFPNAPYL 
                 binds also to Kd, 
               
               
                   
                 (SEQ ID NOs: 185 
                 class II and HLA A2, 
               
               
                   
                 and 293) 
                 score 1,990,800 
               
               
                   
               
               
                 p221-229 
                 YSSDNLYQM 
                 binds also to Ld, 
               
               
                   
                 (SEQ ID NOs: 253 
                 score 312,000 
               
               
                   
                 and 308) 
               
               
                   
               
               
                 p228-236 
                 QMTSQLECM 
                 score 3,120 
               
               
                   
                 (SEQ ID NOs: 169 
               
               
                   
                 and 289) 
               
               
                   
               
               
                 p239-247 
                 NQMNLGATL 
                 binds also to HLA 
               
               
                   
                 (SEQ ID NOs: 151 
                 A 0201, Kd, score 
               
               
                   
                 and 286) 
                 8,015 
               
               
                   
               
               
                 mouse 
                 SCLESQPTI 
                 binds also to Kd, 
               
               
                 p136-144 
                 (SEQ ID NO: 296) 
                 1 mismatch 
               
               
                   
                   
                 to human 
               
               
                   
               
               
                 human 
                 SCLESQPAI 
                 score 7,920 
               
               
                 p136-144 
                 (SEQ ID NO: 198) 
               
               
                   
               
               
                 mouse 
                 ALLPAVSSL 
                 binds also to Kd, 
               
               
                 p10-18 
                 (SEQ ID NO: 255) 
                 HLA A2, 1 mismatch 
               
               
                   
                   
                 to human 
               
               
                   
               
               
                 human 
                 ALLPAVPSL 
                 score 6,600 
               
               
                 p10-18 
                 (SEQ ID NO: 34) 
               
               
                   
               
            
           
         
       
     
     Peptide binding to C57BI/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° C. in complete medium supplemented with 1% FCS. A total of 10 6  RMA-S cells were added into each well of a 24-well plate and incubated either alone or with the designated peptide (25 ug/ml) for 16 hours at 26° C. and additional 3 hours at 37° C. in complete medium. Cells were then washed three times and stained with fluorescein isothiocyanate-conjugated anti D b  or anti-K b  antibody (PharMingen, San Diego, Calif.). Labeled cells were washed twice, resuspended and fixed in 500 ul of PBS with 1% paraformaldehyde and analyzed for fluorescence intensity in a flow cytometer (Becton-Dickinson FACSCALIBUR™). The percentage of increase of D b  or K b  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 WT1 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 150 μCi of sodium  51 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, 10 4 51 Cr-labeled target cells were incubated with different concentrations of effector cells in a final volume of 200 μl in U-bottomed 96-well plates. Supernatants were removed after 4 to 7 hours at 37° C., and the percentage specific lysis was determined by the formula:
 
% specific lysis=100×(experimental release−spontaneous release)/(maximum release−spontaneous release).
 
     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 ( FIGS. 9A and 9B ), as determined using chromium release assays. Following immunization to CTL peptides p10-18 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 WT1 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 5 
     Use of A WT1 Polypeptide to Elicit WT1 Specific CTL in Mice 
     This Example illustrates the ability of a representative WT1 polypeptide to elicit CTL immunity capable of killing WT1 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 WT1 peptides, as well as WT1 positive and negative tumor cells. CTL were evaluated with a standard chromium release assay. The results, presented in  FIGS. 10A-10D , show that P117 can elicit WT1 specific CTL capable of killing WT1 positive tumor cells, whereas no killing of WT1 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 WT1. 
     Similar immunizations were performed using the 9-mer class I MHC binding peptides p136-144, p225-233, p235-243 as well as the 23-mer peptide p117-139. Following immunization, spleen cells were stimulated in vitro with each of the 4 peptides and tested for ability to lyse targets incubated with WT1 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  FIGS. 11A and 11B . Data for peptides p136-144 and p225-233 are not depicted. 
     CTL lysis demands that the target WT1 peptides are endogenously processed and presented in association with tumor cell class I MHC molecules. The above WT1 peptide specific CTL were tested for ability to lyse WT1 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 WT1 proteins ( FIG. 11A ). By marked contrast, CTL specific for p117-139 lysed targets incubated with p117-139 peptides and also lysed malignant cells expressing WT1 ( FIG. 11B ). As a negative control, CTL specific for p117-139 did not lyse WT1 negative EL-4 (also referred to herein as E10). 
     Specificity of WT1 specific lysis was confirmed by cold target inhibition ( FIGS. 12A-12B ). Effector cells were plated for various effector: target ratios in 96-well U-bottom plates. A ten-fold excess (compared to hot target) of the indicated peptide-coated target without  51 Cr labeling was added. Finally, 10 4 51 Cr-labeled target cells per well were added and the plates incubated at 37° C. for 4 hours. The total volume per well was 200 μl. 
     Lysis of TRAMP-C by p117-139 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 ( FIG. 12A ). Similarly, lysis of BLK-SV40 was blocked from 18% to 0% by EL-4 incubated with the relevant peptide p117-139 ( FIG. 12B ). Results validate that WT1 peptide specific CTL specifically kill malignant cells by recognition of processed WT1. 
     Several segments with putative CTL motifs are contained within p117-139. 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-2 b  class I molecules (Table XLVIII). CTL generated by immunization with p117-139 lysed targets incubated with p126-134 and p130-138, but not the other 9-mer peptides within p117-139 ( FIG. 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 showed lysis of a WT1 positive tumor cell line ( FIGS. 13B and 13C ). Thus, p130-138 appears to be the naturally processed epitope. 
     
       
         
           
               
             
               
                 TABLE XLVIII 
               
             
            
               
                   
               
               
                 Binding of WT1 CTL 9mer Peptides within 
               
               
                 p117-139 to mouse B6 class I antigens 
               
            
           
           
               
               
            
               
                   
                 Binding 
               
               
                   
                 Affinity 
               
               
                   
                 to 
               
               
                   
                 Mouse 
               
               
                   
                 MHC 
               
               
                 Peptide 
                 Class I 
               
               
                   
               
            
           
           
               
               
               
            
               
                 P117-125 
                 PSQASSGQA (SEQ ID NO: 221) 
                 2% 
               
               
                   
               
               
                 P118-126 
                 SQASSGQAR (SEQ ID NO: 216) 
                 2% 
               
               
                   
               
               
                 P119-127 
                 QASSGQARM (SEQ ID Nos: 161 and 
                 2% 
               
               
                   
                 288) 
               
               
                   
               
               
                 P120-128 
                 ASSGQARMF (SEQ ID NO: 40 
                 1% 
               
               
                   
               
               
                 P121-129 
                 SSGQARMFP (SEQ ID NO: 222) 
                 1% 
               
               
                   
               
               
                 P122-130 
                 SGQARMFPN (SEQ ID NO: 212) 
                 1% 
               
               
                   
               
               
                 P123-131 
                 GQARMFPNA (SEQ ID Nos: 98 and 
                 1% 
               
               
                   
                 269) 
               
               
                   
               
               
                 P124-132 
                 QARMFPNAP (SEQ ID NO: 223) 
                 1% 
               
               
                   
               
               
                 P125-133 
                 ARMFPNAPY (SEQ ID NO: 38) 
                 1% 
               
               
                   
               
               
                 P126-134 
                 RMFPNAPYL (SEQ ID NOs: 185 and 
                 79%  
               
               
                   
                 293) 
               
               
                   
               
               
                 P127-135 
                 MFPNAPYLP (SEQ ID NO: 224) 
                 2% 
               
               
                   
               
               
                 P128-136 
                 FPNAPYLPS (SEQ ID NOs: 79 and 267) 
                 1% 
               
               
                   
               
               
                 P129-137 
                 PNAPYLPSC (SEQ ID NO: 225) 
                 1% 
               
               
                   
               
               
                 P130-138 
                 NAPYLPSCL (SEQ ID NOs: 144 and 
                 79%  
               
               
                   
                 282) 
               
               
                   
               
               
                 P131-139 
                 APYLPSCLE (SEQ ID NO: 226) 
                 1% 
               
               
                   
               
            
           
         
       
     
     Example 6 
     Identification of WT1 Specific mRNA in Mouse Tumor Cell Lines 
     This Example illustrates the use of RT-PCR to detect WT1 specific mRNA in cells and cell lines. 
     Mononuclear cells were isolated by density gradient centrifugation, and were immediately frozen and stored at −80° C. until analyzed by RT-PCR for the presence of WT1 specific mRNA. RT-PCR was generally performed as described by Fraizer et al.,  Blood  86:4704-4706, 1995. Total RNA was extracted from 10 7  cells according to standard procedures. RNA pellets were resuspended in 25 μL diethylpyrocarbonate treated water and used directly for reverse transcription. The zinc-finger 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, Conn.), 2.5 mM MgCl 2  and 20 μmol of each primer in a total reaction volume of 50 μl were used. Twenty μL 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 β-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 CM 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 MT GTC ACG CAC GAT TTC 3′ (antisense primer; SEQ ID NO:24) 
     Primers for use in amplifying human WT1 include: P117: 954-974: 5′ GGC ATC TGA GAC CAG TGA GAA 3′ (SEQ ID NO:25); and P118: 1434-1414: 5′ 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 MG CGC CAG CTG GAG TTT 3′ (SEQ ID NO:27). 
     Table XLVIII shows the results of WT1 PCR analysis of mouse tumor cell lines. Within Table IV, (+++) indicates a strong WT1 PCR amplification product in the first step RT PCR, (++) indicates a WT1 amplification product that is detectable by first step WT1 RT PCR, (+) indicates a product that is detectable only in the second step of WT1 RT PCR, and (−) indicates WT1 PCR negative. 
     
       
         
           
               
             
               
                 TABLE XLIX 
               
             
            
               
                   
               
               
                 Detection of WT1 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 
               
               
                 BLK-SV40 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, B/C); 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, B/C); Glynn et al., Cancer Res. 
                 − 
               
               
                 28: 434-439, 1968 
               
               
                 E10/EL-4 (lymphoma, B6); Glynn et al., Cancer Res. 
                 − 
               
               
                 28: 434-439, 1968 
               
               
                   
               
            
           
         
       
     
     Example 7 
     Expression in  E. Coli  of WT1 TRX Fusion Construct 
     The truncated open reading frame of WT1 (WT1B) was PCR amplified with the following primers: 
     
       
         
           
               
            
               
                 Forward Primer starting at amino acid 2 
               
               
                 P-37 
               
            
           
           
               
            
               
                 (SEQ ID NO. 347) 
               
            
           
           
               
            
               
                 5′ ggctccgacgtgcgggacctg 3′ Tm 64° C. 
               
               
                   
               
               
                 Reverse Primer creating EcoRI site after stop 
               
               
                 codon 
               
               
                 P-23 
               
            
           
           
               
            
               
                 (SEQ ID NO. 348) 
               
            
           
           
               
            
               
                 5′ gaattctcaaagcgccagctggagtttggt 3′ Tm 63° C. 
               
            
           
         
       
     
     The PCR was performed under the following conditions: 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 10 
                 μl 
                 10X Pfu buffer 
               
               
                 1 
                 μl 
                 10 mM dNTPs 
               
               
                 2 
                 μl 
                 10 μM each oligo 
               
               
                 83 
                 μL 
                 sterile water 
               
               
                 1.5 
                 μl 
                 Pfu DNA polymerase (Stratagene, La Jolla, CA) 
               
               
                 50 
                 ng 
                 DNA (pPDM FL WT1) 
               
               
                 96° 
                 C. 
                 2 minutes 
               
            
           
           
               
               
               
               
               
            
               
                 96° 
                 C. 
                 20 seconds 
                 63° C. 15 seconds 
                 72° C. 3 minutes × 40 cycles 
               
               
                 72° 
                 C. 
                 4 minutes 
               
               
                   
               
            
           
         
       
     
     The PCR product was digested with EcoRI restriction enzyme, gel purified and then cloned into pTrx 2H vector (a modified pET28 vector with a Trx fusion on the N-terminal and two His tags surrounding the Trx fusion. After the Trx fusion there exists protease cleavage sites for thrombin and enterokinase). The pTrx2H construct was digested with StuI and EcoRI restriction enzymes. The correct constructs were confirmed by DNA sequence analysis and then transformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells. 
     Example 8 
     Expression in  E. Coli  of WT1 A His Tag Fusion Constructs 
     The N-terminal open reading frame of WT1 (WT1A) was PCR amplified with the following primers: 
     
       
         
           
               
            
               
                 Forward Primer starting at amino acid 2 
               
               
                 P-37 
               
            
           
           
               
            
               
                 (SEQ ID NO. 347) 
               
            
           
           
               
            
               
                 5′ggctccgacgtgcgggacctg 3′ Tm 64° C. 
               
               
                   
               
               
                 Reverse Primer creating EcoRI site after an 
               
               
                 artificial stop codon put after amino acid 249. 
               
               
                 PDM-335 
               
            
           
           
               
            
               
                 (SEQ ID NO. 348) 
               
            
           
           
               
            
               
                 5′gaattctcaaagcgccagctggagtttggt 3′ Tm 64° C. 
               
            
           
         
       
     
     The PCR was performed under the following conditions: 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 10 
                 μl 
                 10X Pfu buffer 
               
               
                 1 
                 μl 
                 10 mM dNTPs 
               
               
                 2 
                 μl 
                 10 μM each oligo 
               
               
                 83 
                 μL 
                 sterile water 
               
               
                 1.5 
                 μl 
                 Pfu DNA polymerase (Stratagene, La Jolla, CA) 
               
               
                 50 
                 ng 
                 DNA (pPDM FL WT1) 
               
               
                 96° 
                 C. 
                 2 minutes 
               
            
           
           
               
               
               
               
               
            
               
                 96° 
                 C. 
                 20 seconds 
                 63° C. 15 seconds 
                 72° C. 1 minute 20 seconds × 
               
               
                   
                   
                   
                   
                 40 cycles 
               
               
                 72° 
                 C. 
                 4 minutes 
               
               
                   
               
            
           
         
       
     
     The PCR product was digested with EcoRI restriction enzyme, gel purified and then cloned into pPDM, a modified pET28 vector with a His tag in frame, which had been digested with Eco72I and EcoRI restriction enzymes. The PCR product was also transformed into pTrx 2H vector. The pTrx2H construct was digested with StuI and EcoRI restriction enzymes. The correct constructs were confirmed by DNA sequence analysis and then transformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells. 
     Example 9 
     Expression in  E. Coli  of WT1 B His Tag Fusion Constructs 
     The truncated open reading frame of WT1 (WT1A) was PCR amplified with the following primers: 
     
       
         
           
               
               
            
               
                   
                 Forward Primer starting at amino acid 250 
               
               
                   
                 PDM-346 
               
            
           
           
               
            
               
                 (SEQ ID NO. 351) 
               
            
           
           
               
               
            
               
                   
                 5′ cacagcacagggtacgagagc 3′ Tm 58° C. 
               
               
                   
                   
               
               
                   
                 Reverse Primer creating EcoRI site after stop 
               
               
                   
                 codon 
               
               
                   
                 P-23 
               
            
           
           
               
            
               
                 (SEQ ID NO. 348) 
               
            
           
           
               
               
            
               
                   
                 5′gaattctcaaagcgccagctggagtttggt 3′ Tm 63° C. 
               
            
           
         
       
     
     The PCR was performed under the following conditions: 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 10 
                 μl 
                 10X Pfu buffer 
               
               
                 1 
                 μl 
                 10 mM dNTPs 
               
               
                 2 
                 μl 
                 10 μM each oligo 
               
               
                 83 
                 μL 
                 sterile water 
               
               
                 1.5 
                 μl 
                 Pfu DNA polymerase (Stratagene, La Jolla, CA) 
               
               
                 50 
                 ng 
                 DNA (pPDM FL WT1) 
               
               
                 96° 
                 C. 
                 2 minutes 
               
            
           
           
               
               
               
               
               
            
               
                 96° 
                 C. 
                 20 seconds 
                 63° C. 15 seconds 
                 72° C. 1 minute 30 seconds × 
               
               
                   
                   
                   
                   
                 40 cycles 
               
               
                 72° 
                 C. 
                 4 minutes 
               
               
                   
               
            
           
         
       
     
     The PCR product was digested with EcoRI restriction enzyme, gel purified and then cloned into pPDM, a modified pET28 vector with a His tag in frame, which had been digested with Eco72I and EcoRI restriction enzymes. The PCR product was also transformed into pTrx 2H vector. The pTrx 2H construct was digested with StuI and EcoRI restriction enzymes. The correct constructs were confirmed by DNA sequence analysis and then transformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells. 
     For Examples 7-9, the following SEQ ID NOs. are disclosed:
     SEQ ID NO. 327 is the determined cDNA sequence for Trx_WT1_B   SEQ ID NO. 328 is the determined cDNA sequence for Trx_WT1_A   SEQ ID NO. 329 is the determined cDNA sequence for Trx_WT1   SEQ ID NO. 330 is the determined cDNA sequence for WT1_A   SEQ ID NO. 331 is the determined cDNA sequence for WT1_B   SEQ ID NO. 332 is the predicted amino acid sequence encoded by SEQ ID No.   SEQ ID NO. 333 is the predicted amino acid sequence encoded by SEQ ID No. 328   SEQ ID NO. 334 is the predicted amino acid sequence encoded by SEQ ID No. 329   SEQ ID NO. 335 is the predicted amino acid sequence encoded by SEQ ID No. 330   SEQ ID NO. 336 is the predicted amino acid sequence encoded by SEQ ID No. 331   

     Example 10 
     Truncated Forms of WT1 Expressed in  E. Coli    
     Three reading frames of WT1 were amplified by PCR using the following primers: 
     
       
         
           
               
            
               
                 For WT1 Tr2: 
               
               
                 PDM-441 
               
            
           
           
               
            
               
                 (SEQ ID NO. 353) 
               
            
           
           
               
            
               
                 5′ cacgaagaacagtgcctgagcgcattcac 3′ Tm 63° C. 
               
               
                   
               
               
                 PDM-442 
               
            
           
           
               
            
               
                 (SEQ ID NO. 354) 
               
            
           
           
               
            
               
                 5′ ccggcgaattcatcagtataaattgtcactgc 3′ TM 62° C. 
               
               
                   
               
               
                 For WT1 Tr3: 
               
               
                 PDM-443 
               
            
           
           
               
            
               
                 (SEQ ID NO. 355) 
               
            
           
           
               
            
               
                 5′ caggctttgctgctgaggacgccc 3′ Tm 64° C. 
               
               
                   
               
               
                 PDM-444 
               
            
           
           
               
            
               
                 (SEQ ID NO. 356) 
               
            
           
           
               
            
               
                 5′ cacggagaattcatcactggtatggtttctcacc Tm 64° C. 
               
               
                   
               
               
                 For WT1 Tr4: 
               
               
                 PDM-445 
               
            
           
           
               
            
               
                 (SEQ ID NO. 357) 
               
            
           
           
               
            
               
                 5′ cacagcaggaagcacactggtgagaaac 3′ Tm 63° C. 
               
               
                   
               
               
                 PDM-446 
               
            
           
           
               
            
               
                 (SEQ ID NO. 358) 
               
            
           
           
               
            
               
                 5′ ggatatctgcagaattctcaaagcgccagc 3′ TM 63° C. 
               
            
           
         
       
     
     The PCR was performed under the following conditions: 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 10 
                 μl 
                 10X Pfu buffer 
               
               
                 1 
                 μl 
                 10 mM dNTPs 
               
               
                 2 
                 μl 
                 10 μM each oligo 
               
               
                 83 
                 μL 
                 sterile water 
               
               
                 1.5 
                 μl 
                 Pfu DNA polymerase (Stratagene, La Jolla, CA) 
               
               
                 50 
                 ng 
                 DNA (pPDM FL WT1) 
               
               
                 96° 
                 C. 
                 2 minutes 
               
            
           
           
               
               
               
               
               
            
               
                 96° 
                 C. 
                 20 seconds 
                 63° C. 15 seconds 
                 72° C. 30 seconds × 40 cycles 
               
               
                 72° 
                 C. 
                 4 minutes 
               
               
                   
               
            
           
         
       
     
     The PCR products were digested with EcoRI and cloned into pPDM His (a modified pET28 vector with a His tag in frame on the 5′ end) which has been digested with Eco72I and EcoRI. The constructs were confirmed to be correct through sequence analysis and transformed into BL21 pLys S and BL21-CODONPLUS® cells or BLR pLys S and BL21-CODONPLUS® cells. 
     Example 11 
     WT1 C (Amino Acids 76-437) and WT1 D (Amino Acids 91-437) Expression in  E. Coli    
     The WT1 C reading frame was amplified by PCR using the following primers: 
     
       
         
           
               
            
               
                 PDM-504 
               
            
           
           
               
            
               
                 (SEQ ID NO. 359) 
               
            
           
           
               
            
               
                 5′ cactccttcatcaaacaggaac 3′ Tm 61° C. 
               
               
                   
               
               
                 PDM-446 
               
            
           
           
               
            
               
                 (SEQ ID NO. 358) 
               
            
           
           
               
            
               
                 5′ ggatatctgcagaattctcaaagcgccagc 3′ Tm 63° C. 
               
            
           
         
       
     
     The PCR was performed under the following conditions: 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 10 
                 μl 
                 10X Pfu buffer 
               
               
                 1 
                 μl 
                 10 mM dNTPs 
               
               
                 2 
                 μl 
                 10 μM each oligo 
               
               
                 83 
                 μL 
                 sterile water 
               
               
                 1.5 
                 μl 
                 Pfu DNA polymerase (Stratagene, La Jolla, CA) 
               
               
                 50 
                 ng 
                 DNA (pPDM FL WT1) 
               
               
                 96° 
                 C. 
                 2 minutes 
               
            
           
           
               
               
               
               
               
            
               
                 96° 
                 C. 
                 20 seconds 
                 63° C. 15 seconds 
                 72° C. 2 minutes × 40 cycles 
               
               
                 72° 
                 C. 
                 4 minutes 
               
               
                   
               
            
           
         
       
     
     The PCR product was digested with EcoRI and cloned into pPDM His which had been digested with Eco72I and EcoRI. The sequence was confirmed through sequence analysis and then transformed into BLR pLys S and BLR which is co-transformed with CODONPLUS® RP. 
     Example 12 
     Synthetic Production of WT1 TR-1 by Annealing Overlapping Oligos 
     This example was performed to determine the effect of changing proline codon usage on expression. 
     The following pairs of oligos were annealed: 
     
       
         
           
               
            
               
                 1. PDM-505 
               
            
           
           
               
            
               
                 (SEQ ID NO. 361) 
               
            
           
           
               
            
               
                 5′ ggttccgacgtgcgggacctgaacgcactgctg 3′ 
               
               
                   
               
               
                 PDM-506 
               
            
           
           
               
            
               
                 (SEQ ID NO. 362) 
               
            
           
           
               
            
               
                 5′ ctgccggcagcagtgcgttcaggtcccgcacgtcggaacc 3′ 
               
               
                   
               
               
                 2. PDM-507 
               
            
           
           
               
            
               
                 (SEQ ID NO. 363) 
               
            
           
           
               
            
               
                 5′ ccggcagttccatccctgggtggcggtggaggctg 3′ 
               
               
                   
               
               
                 PDM-508 
               
            
           
           
               
            
               
                 (SEQ ID NO. 364) 
               
            
           
           
               
            
               
                 5′ cggcagtgcgcagcctccaccgccacccagggatggaa 3′ 
               
               
                   
               
               
                 3. PDM-509 
               
            
           
           
               
            
               
                 (SEQ ID NO. 365) 
               
            
           
           
               
            
               
                 5′ cgcactgccggttagcggtgcagcacagtgggctc 3′ 
               
               
                   
               
               
                 PDM-510 
               
            
           
           
               
            
               
                 (SEQ ID NO. 366) 
               
            
           
           
               
            
               
                 5′ cagaactggagcccactgtgctgcaccgctaac 3′ 
               
               
                   
               
               
                 4. PDM-511 
               
            
           
           
               
            
               
                 (SEQ ID NO. 367) 
               
            
           
           
               
            
               
                 5′ cagttctggacttcgcaccgcctggtgcatccgcatac 3′ 
               
               
                   
               
               
                 PDM-512 
               
            
           
           
               
            
               
                 (SEQ ID NO. 368) 
               
            
           
           
               
            
               
                 5′ cagggaaccgtatgcggatgcaccaggcggtgcgaagtc 3′ 
               
               
                   
               
               
                 5. PDM-513 
               
            
           
           
               
            
               
                 (SEQ ID NO. 369) 
               
            
           
           
               
            
               
                 5′ ggttccctgggtggtccagcacctccgcccgcaacgcc 3′ 
               
               
                   
               
               
                 PDM-514 
               
            
           
           
               
            
               
                 (SEQ ID NO. 370) 
               
            
           
           
               
            
               
                 5′ ggcggtgggggcgttgcgggcggaggtgctggaccacc 3′ 
               
               
                   
               
               
                 6. PDM-515 
               
            
           
           
               
            
               
                 (SEQ ID NO. 371) 
               
            
           
           
               
            
               
                 5′ cccaccgcctccaccgcccccgcactccttcatcaaacag 3′ 
               
               
                   
               
               
                 PDM-516 
               
            
           
           
               
            
               
                 (SEQ ID NO. 372) 
               
            
           
           
               
            
               
                 5′ ctaggttcctgtttgatgaaggagtgcgggggcggtgga 3′ 
               
               
                   
               
               
                 7. PDM-517 
               
            
           
           
               
            
               
                 (SEQ ID NO. 373) 
               
            
           
           
               
            
               
                 5′ gaacctagctggggtggtgcagaaccgcacgaagaaca 3′ 
               
               
                   
               
               
                 PDM-518 
               
            
           
           
               
            
               
                 (SEQ ID NO. 374) 
               
            
           
           
               
            
               
                 5′ ctcaggcactgttcttcgtgcggttctgcaccaccccag 3′ 
               
               
                   
               
               
                 8. PDM-519 
               
            
           
           
               
            
               
                 (SEQ ID NO. 375) 
               
            
           
           
               
            
               
                 5′ gtgcctgagcgcattctgagaattctgcagat 3′ 
               
               
                   
               
               
                 PDM-520 
               
            
           
           
               
            
               
                 (SEQ ID NO. 376) 
               
            
           
           
               
            
               
                 5′ gtgtgatggatatctgcagaattctcagaatgcg 3′ 
               
            
           
         
       
     
     Each oligo pair was separately combined then annealed. The pairs were then ligated together and one μl of ligation mix was used for PCR conditions below: 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 10 
                 μl 
                 10X Pfu buffer 
               
               
                 1 
                 μl 
                 10 mM dNTPs 
               
               
                 2 
                 μl 
                 10 μM each oligo 
               
               
                 83 
                 μL 
                 sterile water 
               
               
                 1.5 
                 μl 
                 Pfu DNA polymerase (Stratagene, La Jolla, CA) 
               
               
                 96° 
                 C. 
                 2 minutes 
               
            
           
           
               
               
               
               
               
            
               
                 96° 
                 C. 
                 20 seconds 
                 63° C. 15 seconds 
                 72° C. 30 seconds × 40 cycles 
               
               
                 72° 
                 C. 
                 4 minutes 
               
               
                   
               
            
           
         
       
     
     The PCR product was digested with EcoRI and cloned into pPDM His which had been digested with Eco72I and EcoRI. The sequence was confirmed and then transformed into BLR pLys S and BLR which is co-transformed with CODONPLUS® RP. 
     For examples 10-12, the following SEQ ID NOs. are disclosed:
     SEQ ID NO:337 is the determined cDNA sequence for WT1_Tr1   SEQ ID NO:338 is the determined cDNA sequence for WT1_Tr2   SEQ ID NO:339 is the determined cDNA sequence for WT1_Tr3   SEQ ID NO:340 is the determined cDNA sequence for WT1_Tr4   SEQ ID NO:341 is the determined cDNA sequence for WT1_C   SEQ ID NO:342 is the predicted amino acid sequence encoded by SEQ ID NO:337   SEQ ID NO:343 is the predicted amino acid sequence encoded by SEQ ID NO:338   SEQ ID NO:344 is the predicted amino acid sequence encoded by SEQ ID NO:339   SEQ ID NO:345 is the predicted amino acid sequence encoded by SEQ ID NO:340   SEQ ID NO:346 is the predicted amino acid sequence encoded by SEQ ID NO:341   

     The WT1 C sequence represents a polynucleotide having the coding regions of TR2, TR3 and TR4. 
     The WT1 TR-1 synthetic sequence represents a polynucleotide in which alternative codons for proline were substituted for the native codons, producing a polynucleotide capable of expressing WT1 TR-1 in  E. coli.    
     Example 13 
     Evaluation of the Systemic Histopathological and Toxicological Effects of WT1 Immunization in Mice 
     The purpose of this example is to analyze the immunogenicity and potential systemic histopathological and toxicological effects of WT1 protein immunization in a multiple dose titration in mice. 
     The experimental design for immunization of mice with WT1 protein is outlined in Table L. 
     
       
         
           
               
             
               
                 TABLE L 
               
             
            
               
                   
               
               
                 Experimental Design of WT1 Immunization in Mice 
               
            
           
           
               
               
               
               
               
            
               
                 His- 
                   
                   
                   
                   
               
               
                 tology 
                 Corixa 
                   
                 Dose 
                 Total No. 
               
               
                 Group 
                 Group 
                 Treatment Description 
                 Level 
                 (Females) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 0 
                 No treatment 
                 0 
                   
                 4 
               
               
                 2 
                 1.1 
                 MPL ®-SE (adjuvants alone), 
                 10 
                 ug 
                 4 
               
               
                   
                   
                 6x, 1 week apart 
               
               
                 3 
                 1.2 
                 MPL ®-SE, 3x, 2 weeks apart 
                 10 
                 ug 
                 4 
               
               
                 4 
                 2.1 
                 Ra12-WT1 + MPL ®-SE, 6x 
                 25 
                 ug 
                 4 
               
               
                 5 
                 2.2 
                 Ra12-WT1 + MPL ®-SE, 3x 
                 25 
                 ug 
                 4 
               
               
                 6 
                 3.1 
                 Ra12-WT1 + MPL ®-SE, 6x 
                 100 
                 ug 
                 4 
               
               
                 7 
                 3.2 
                 Ra12-WT1 + MPL ®-SE, 3x 
                 100 
                 ug 
                 4 
               
               
                 8 
                 4.1 
                 Ra12-WT1 + MPL ®-SE, 6x 
                 1000 
                 ug 
                 4 
               
               
                 9 
                 4.2 
                 Ra12-WT1 + MPL ®-SE, 3x 
                 1000 
                 ug 
                 4 
               
               
                   
               
               
                 Vaccination to WT1 protein using MPL ®-SE as adjuvant, in a multiple dose titration study (doses ranging from 25 μg, 100 μg to 1000 μg WT1 protein) in female C57/B6 mice elicited a strong WT1-specific antibody response (FIG. 19) and cellular T-cell responses (FIG. 20). 
               
            
           
         
       
     
     No systemic histopathological or toxicological effects of immunization with WT1 protein was observed. No histological evidence for toxicity was seen in the following tissues: adrenal gland, brain, cecum, colon, duodenum, eye, femur and marrow, gall bladder, heart, ileum, jejunum, kidney, larynx, lacrimal gland, liver, lung, lymph node, muscle, esophagus, ovary, pancreas, parathyroid, salivary gland, sternum and marrow, spleen, stomach, thymus, trachea, thyroid, urinary bladder and uterus. 
     Special emphasis was put on evaluation of potential hematopoietic toxicity. The myeloid/erythroid ratio in sternum and femur marrow was normal. All evaluable blood cell counts and blood chemistry (BUN, creatinine, bilirubin, albumin, globulin) were within the normal range (Table LI). 
     Given that existent immunity to WT1 is present in some patients with leukemia and that vaccination to WT1 protein can elicit WT1 specific Ab and cellular T-cell responses in mice without toxicity to normal tissues, these experiments validate WT1 as a tumor/leukemia vaccine. 
     
       
         
           
               
             
               
                 TABLE LI 
               
               
                   
               
               
                 Clinical Chemistry and Hematology Analysis 
               
               
                 Table LI: WT1 Dose Titration Study 
               
               
                 Clinical Chemistry and Hematology Analysis 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 K/uL 
                 M/uL 
                 g/dl 
                 % 
                 fL 
                 pg 
                 % 
               
               
                   
                 WBC 
                 RBC 
                 Hg. 
                 HCT 
                 MCV 
                 MCH 
                 MCHC 
               
            
           
           
               
               
            
               
                   
                 Normal 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Animal # 
                 5.4-16.0 
                 6.7-12.5 
                 10.2-16.6 
                 32-54 
                 31-62 
                 9.2-20.8 
                 22.0-35.5 
               
               
                   
               
               
                 Group 1 
               
               
                  1 (0) 
                 5.6 
                 8.41 
                 12.8 
                 43.5 
                 53 
                 15.2 
                 29.4 
               
               
                  2 (0) 
                 5.5 
                 9.12 
                 13.4 
                 47.5 
                 53 
                 14.7 
                 28.2 
               
               
                  3 (0) 
                 7.5 
                 9.22 
                 13.5 
                 48 
                 54 
                 14.7 
                 28.1 
               
               
                  4 (0) 
                 3.9 
                 9.27 
                 13.6 
                 46 
                 52 
                 14.7 
                 29.6 
               
               
                 Mean 
                 5.6 
                 9.0 
                 13.3 
                 46.3 
                 53.0 
                 14.8 
                 28.8 
               
               
                 STD 
                 1.5 
                 0.4 
                 0.4 
                 2.0 
                 0.8 
                 0.3 
                 0.8 
               
               
                 Group 2 
               
               
                  5 (1.5) 
                 6.6 
                 9 
                 13.1 
                 46 
                 54 
                 14.5 
                 28.5 
               
               
                  6 (1.6) 
                 5.2 
                 8.58 
                 12.6 
                 44 
                 53 
                 14.7 
                 28.6 
               
               
                  7 (1.7) 
                 7.8 
                 9.21 
                 13.6 
                 46 
                 53 
                 14.7 
                 29.6 
               
               
                  8 (1.8) 
                 6.3 
                 NA 
                 NA 
                 41 
                 NA 
                 NA 
                 NA 
               
               
                 Mean 
                 6.5 
                 8.9 
                 13.1 
                 44.3 
                 53.3 
                 14.6 
                 28.9 
               
               
                 STD 
                 1.1 
                 0.3 
                 0.5 
                 2.4 
                 0.6 
                 0.1 
                 0.6 
               
               
                 Group 3 
               
               
                  9 (2.5) 
                 8.3 
                 9.16 
                 13.6 
                 50.3 
                 55 
                 14.9 
                 27.1 
               
               
                 10 (2.6) 
                 5 
                 8.78 
                 13 
                 44.2 
                 50 
                 14.8 
                 29.3 
               
               
                 11 (2.7) 
                 4 
                 8.94 
                 13.2 
                 48.3 
                 54 
                 14.7 
                 27.3 
               
               
                 12 (2.8) 
                 8.2 
                 NA 
                 NA 
                 41 
                 NA 
                 NA 
                 NA 
               
               
                 Mean 
                 6.4 
                 9.0 
                 13.3 
                 46.0 
                 53.0 
                 14.8 
                 27.9 
               
               
                 STD 
                 2.2 
                 0.2 
                 0.3 
                 4.2 
                 2.6 
                 0.1 
                 1.2 
               
               
                 Group 4 
               
               
                 13 (3.5) 
                 6.1 
                 8.82 
                 13.1 
                 46 
                 54 
                 14.9 
                 28.5 
               
               
                 14 (3.6) 
                 6.1 
                 8.64 
                 12.9 
                 46 
                 54 
                 15 
                 28 
               
               
                 15 (3.7) 
                 9.3 
                 8.93 
                 13.2 
                 48 
                 55 
                 14.8 
                 27.5 
               
               
                 16 (3.8) 
                 4.8 
                 8.19 
                 12.6 
                 44 
                 55 
                 15.3 
                 28.6 
               
               
                 Mean 
                 6.6 
                 8.6 
                 13.0 
                 46.0 
                 54.5 
                 15.0 
                 28.2 
               
               
                 STD 
                 1.9 
                 0.3 
                 0.3 
                 1.6 
                 0.6 
                 0.2 
                 0.5 
               
               
                 Group 5 
               
               
                 17 (4.5) 
                 3.1 
                 8.48 
                 12.6 
                 46 
                 54 
                 14.9 
                 27.5 
               
               
                 18 (4.6) 
                 5.7 
                 9.12 
                 13.7 
                 48 
                 54 
                 15 
                 28.5 
               
               
                 19 (4.7) 
                 5.3 
                 8.58 
                 13 
                 44.5 
                 55 
                 15.2 
                 29.2 
               
               
                 20 (4.8) 
                 5.3 
                 NA 
                 NA 
                 40 
                 NA 
                 NA 
                 NA 
               
               
                 Mean 
                 4.9 
                 8.7 
                 13.1 
                 44.6 
                 54.3 
                 15.0 
                 28.4 
               
               
                 STD 
                 1.2 
                 0.3 
                 0.6 
                 3.4 
                 0.6 
                 0.2 
                 0.9 
               
               
                 Group 6 
               
               
                 21 (1.1) 
                 3.5 
                 9.36 
                 13.5 
                 37.6 
                 40 
                 14.4 
                 35.9 
               
               
                 22 (1.2) 
                 6.9 
                 8.93 
                 13.6 
                 37.3 
                 42 
                 15.3 
                 36.6 
               
               
                 23 (1.3) 
                 3.6 
                 8.3 
                 12.5 
                 35.3 
                 43 
                 15.1 
                 35.5 
               
               
                 24 (1.4) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 Mean 
                 4.7 
                 8.9 
                 13.2 
                 36.7 
                 41.7 
                 14.9 
                 36.0 
               
               
                 STD 
                 1.9 
                 0.5 
                 0.6 
                 1.3 
                 1.5 
                 0.5 
                 0.6 
               
               
                 Group 7 
               
               
                 25 (2.1) 
                 4 
                 NA 
                 NA 
                 40 
                 NA 
                 NA 
                 NA 
               
               
                 26 (2.2) 
                 7.4 
                 9.12 
                 13.2 
                 38.5 
                 42 
                 14.5 
                 34.3 
               
               
                 27 (2.3) 
                 4.5 
                 8.19 
                 12.1 
                 34.5 
                 42 
                 14.8 
                 35.1 
               
               
                 28 (2.4) 
                 5.8 
                 8.25 
                 12.3 
                 34.1 
                 41 
                 14.9 
                 36.1 
               
               
                 Mean 
                 5.4 
                 8.5 
                 12.5 
                 36.8 
                 41.7 
                 14.7 
                 35.2 
               
               
                 STD 
                 1.5 
                 0.5 
                 0.6 
                 2.9 
                 0.6 
                 0.2 
                 0.9 
               
               
                 Group 8 
               
               
                 29 (3.1) 
                 5.1 
                 8.53 
                 12.6 
                 34.9 
                 41 
                 14.7 
                 36 
               
               
                 30 (3.2) 
                 7.6 
                 8.42 
                 13 
                 36.1 
                 43 
                 15.4 
                 35.9 
               
               
                 31 (3.3) 
                 3.4 
                 8.45 
                 12.6 
                 34.9 
                 41 
                 14.9 
                 36.1 
               
               
                 32 (3.4) 
                 6.1 
                 8.11 
                 12.3 
                 34.8 
                 43 
                 15.2 
                 35.5 
               
               
                 Mean 
                 5.6 
                 8.4 
                 12.6 
                 35.2 
                 42.0 
                 15.1 
                 35.9 
               
               
                 STD 
                 1.8 
                 0.2 
                 0.3 
                 0.6 
                 1.2 
                 0.3 
                 0.3 
               
               
                 Group 9 
               
               
                 33 (4.1) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 34 (4.2) 
                 4.5 
                 8.63 
                 12.8 
                 36.2 
                 42 
                 14.8 
                 35.2 
               
               
                 35 (4.3) 
                 3.9 
                 8.85 
                 13 
                 36.6 
                 41 
                 14.7 
                 35.6 
               
               
                 36 (4.4) 
                 4.7 
                 8.14 
                 12.3 
                 33.8 
                 42 
                 15.1 
                 36.3 
               
               
                 Mean 
                 4.4 
                 8.5 
                 12.7 
                 35.5 
                 41.7 
                 14.9 
                 35.7 
               
               
                 STD 
                 0.4 
                 0.4 
                 0.4 
                 1.5 
                 0.6 
                 0.2 
                 0.6 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 yes/no 
                 K/uL 
                 Abs. 
                 Abs. 
                 Abs. 
                 Abs. 
                 Abs. 
                 Abs. 
               
               
                   
                 Plt. clump 
                 Platelets 
                 Baso 
                 Eos 
                 Bands 
                 Polys 
                 Lymph 
                 Mono 
               
            
           
           
               
               
            
               
                   
                 Normal 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 0.0-0.15 
                 0.0-0.51 
                 0.0-0.32 
                 8.0-42.9 
                 8.0-18.0 
                 0.0-1.5 
               
               
                 Animal # 
                 no 
                 150-1500 
                 K/uL 
                 K/uL 
                 K/uL 
                 K/uL 
                 K/uL 
                 K/uL 
               
               
                   
               
               
                 Group 1 
               
               
                  1 (0) 
                 yes 
                 726 
                 0 
                 56 
                 0 
                 336 
                 5208 
                 0 
               
               
                  2 (0) 
                 no 
                 860 
                 0 
                 0 
                 0 
                 55 
                 5445 
                 0 
               
               
                  3 (0) 
                 no 
                 875 
                 0 
                 375 
                 0 
                 525 
                 6525 
                 75 
               
               
                  4 (0) 
                 yes 
                 902 
                 0 
                 0 
                 0 
                 156 
                 3744 
                 0 
               
               
                 Mean 
                   
                 840.8 
                 0.0 
                 107.8 
                 0.0 
                 268.0 
                 5230.5 
                 18.8 
               
               
                 STD 
                   
                 78.4 
                 0.0 
                 180.1 
                 0.0 
                 207.0 
                 1144.8 
                 37.5 
               
               
                 Group 2 
               
               
                  5 (1.5) 
                 no 
                 1193 
                 0 
                 132 
                 0 
                 792 
                 5214 
                 462 
               
               
                  6 (1.6) 
                 no 
                 1166 
                 0 
                 52 
                 0 
                 624 
                 4472 
                 52 
               
               
                  7 (1.7) 
                 no 
                 1087 
                 0 
                 234 
                 0 
                 1170 
                 6396 
                 0 
               
               
                  8 (1.8) 
                 yes 
                 NA 
                 0 
                 126 
                 0 
                 126 
                 5922 
                 126 
               
               
                 Mean 
                   
                 1148.7 
                 0.0 
                 136.0 
                 0.0 
                 678.0 
                 5501.0 
                 160.0 
               
               
                 STD 
                   
                 55.1 
                 0.0 
                 74.8 
                 0.0 
                 433.1 
                 840.5 
                 207.9 
               
               
                 Group 3 
               
               
                  9 (2.5) 
                 no 
                 705 
                 0 
                 166 
                 0 
                 664 
                 7387 
                 83 
               
               
                 10 (2.6) 
                 no 
                 1140 
                 0 
                 150 
                 0 
                 500 
                 4350 
                 0 
               
               
                 11 (2.7) 
                 no 
                 952 
                 0 
                 120 
                 0 
                 680 
                 3200 
                 0 
               
               
                 12 (2.8) 
                 yes 
                 NA 
                 0 
                 164 
                 0 
                 656 
                 7216 
                 164 
               
               
                 Mean 
                   
                 932.3 
                 0.0 
                 150.0 
                 0.0 
                 625.0 
                 5538.3 
                 61.8 
               
               
                 STD 
                   
                 218.2 
                 0.0 
                 21.2 
                 0.0 
                 83.9 
                 2090.6 
                 78.6 
               
               
                 Group 4 
               
               
                 13 (3.5) 
                 no 
                 785 
                 0 
                 488 
                 0 
                 732 
                 4636 
                 244 
               
               
                 14 (3.6) 
                 yes 
                 973 
                 0 
                 0 
                 0 
                 488 
                 5307 
                 305 
               
               
                 15 (3.7) 
                 yes 
                 939 
                 0 
                 465 
                 0 
                 558 
                 7812 
                 465 
               
               
                 16 (3.8) 
                 yes 
                 1622 
                 0 
                 192 
                 0 
                 480 
                 4080 
                 48 
               
               
                 Mean 
                   
                 1079.8 
                 0.0 
                 286.3 
                 0.0 
                 564.5 
                 5458.8 
                 265.5 
               
               
                 STD 
                   
                 370.6 
                 0.0 
                 233.4 
                 0.0 
                 117.0 
                 1647.1 
                 172.4 
               
               
                 Group 5 
               
               
                 17 (4.5) 
                 no 
                 892 
                 0 
                 31 
                 0 
                 620 
                 2449 
                 0 
               
               
                 18 (4.6) 
                 yes 
                 966 
                 57 
                 114 
                 0 
                 855 
                 4674 
                 0 
               
               
                 19 (4.7) 
                 yes 
                 883 
                 0 
                 53 
                 0 
                 742 
                 4452 
                 53 
               
               
                 20 (4.8) 
                 yes 
                 NA 
                 0 
                 106 
                 0 
                 53 
                 5141 
                 0 
               
               
                 Mean 
                   
                 913.7 
                 14.3 
                 76.0 
                 0.0 
                 567.5 
                 4179.0 
                 13.3 
               
               
                 STD 
                   
                 45.5 
                 28.5 
                 40.4 
                 0.0 
                 356.2 
                 1188.5 
                 26.5 
               
               
                 Group 6 
               
               
                 21 (1.1) 
                 yes 
                 784 
                 0 
                 35 
                 0 
                 385 
                 2870 
                 210 
               
               
                 22 (1.2) 
                 yes 
                 806 
                 0 
                 69 
                 0 
                 207 
                 6486 
                 138 
               
               
                 23 (1.3) 
                 yes 
                 790 
                 0 
                 180 
                 0 
                 396 
                 2988 
                 36 
               
               
                 24 (1.4) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 Mean 
                   
                 793.3 
                 0.0 
                 94.7 
                 0.0 
                 329.3 
                 4114.7 
                 128.0 
               
               
                 STD 
                   
                 11.4 
                 0.0 
                 75.8 
                 0.0 
                 106.1 
                 2054.5 
                 87.4 
               
               
                 Group 7 
               
               
                 25 (2.1) 
                 yes 
                 NA 
                 0 
                 80 
                 0 
                 200 
                 3720 
                 0 
               
               
                 26 (2.2) 
                 yes 
                 753 
                 0 
                 0 
                 0 
                 518 
                 6734 
                 148 
               
               
                 27 (2.3) 
                 yes 
                 725 
                 0 
                 90 
                 0 
                 225 
                 4140 
                 45 
               
               
                 28 (2.4) 
                 yes 
                 792 
                 0 
                 232 
                 0 
                 754 
                 4814 
                 0 
               
               
                 Mean 
                   
                 756.7 
                 0.0 
                 100.5 
                 0.0 
                 424.3 
                 4852.0 
                 48.3 
               
               
                 STD 
                   
                 33.7 
                 0.0 
                 96.5 
                 0.0 
                 263.0 
                 1333.1 
                 69.8 
               
               
                 Group 8 
               
               
                 29 (3.1) 
                 yes 
                 784 
                 0 
                 153 
                 0 
                 561 
                 4233 
                 153 
               
               
                 30 (3.2) 
                 yes 
                 512 
                 0 
                 152 
                 0 
                 304 
                 6992 
                 152 
               
               
                 31 (3.3) 
                 yes 
                 701 
                 0 
                 0 
                 0 
                 238 
                 3094 
                 68 
               
               
                 32 (3.4) 
                 yes 
                 631 
                 0 
                 305 
                 0 
                 305 
                 5368 
                 122 
               
               
                 Mean 
                   
                 657.0 
                 0.0 
                 152.5 
                 0.0 
                 352.0 
                 4921.8 
                 123.8 
               
               
                 STD 
                   
                 115.1 
                 0.0 
                 124.5 
                 0.0 
                 142.8 
                 1663.3 
                 39.9 
               
               
                 Group 9 
               
               
                 33 (4.1) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 34 (4.2) 
                 yes 
                 724 
                 0 
                 125 
                 0 
                 540 
                 3780 
                 45 
               
               
                 35 (4.3) 
                 yes 
                 758 
                 0 
                 117 
                 0 
                 429 
                 3315 
                 39 
               
               
                 36 (4.4) 
                 yes 
                 808 
                 0 
                 47 
                 0 
                 329 
                 4089 
                 235 
               
               
                 Mean 
                   
                 763.3 
                 0.0 
                 96.3 
                 0.0 
                 432.7 
                 3728.0 
                 106.3 
               
               
                 STD 
                   
                 42.3 
                 0.0 
                 42.9 
                 0.0 
                 105.5 
                 389.6 
                 111.5 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 mg/dl 
                 mg/dl 
                 g/dl 
                 g/dl 
                 g/dl 
                 mg/dl 
               
               
                   
                 BUN 
                 Creatinine 
                 T. protein 
                 Albumin 
                 Globulin 
                 T. Bilirubin 
               
            
           
           
               
               
            
               
                   
                 Normal 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Animal # 
                 13.9-28.3 
                 0.3-1.0 
                 4.0-8.6 
                 2.5-4.8 
                 1.5-3.8 
                 0.10-0.90 
               
               
                   
               
               
                 Group 1 
               
               
                  1 (0) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                  2 (0) 
                 28 
                 0.5 
                 4.9 
                 3.7 
                 1.2 
                 0.3 
               
               
                  3 (0) 
                 25 
                 0.5 
                 4.9 
                 3.8 
                 1.1 
                 0.2 
               
               
                  4 (0) 
                 27 
                 0.5 
                 4.7 
                 3.7 
                 1   
                 0.2 
               
               
                 Mean 
                 26.7 
                 0.5 
                 4.8 
                 3.7 
                 1.1 
                 0.2 
               
               
                 STD 
                 1.5 
                 0.0 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
               
               
                 Group 2 
               
               
                  5 (1.5) 
                 34 
                 0.5 
                 4.6 
                 3.6 
                 1   
                 0.2 
               
               
                  6 (1.6) 
                 31 
                 0.4 
                 4.6 
                 3.3 
                 1.3 
                 0.2 
               
               
                  7 (1.7) 
                 34 
                 0.6 
                 4.9 
                 4   
                 0.9 
                 0.3 
               
               
                  8 (1.8) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 Mean 
                 33.0 
                 0.5 
                 4.7 
                 3.6 
                 1.1 
                 0.2 
               
               
                 STD 
                 1.7 
                 0.1 
                 0.2 
                 0.4 
                 0.2 
                 0.1 
               
               
                 Group 3 
               
               
                  9 (2.5) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 10 (2.6) 
                 33 
                 0.5 
                 4.6 
                 3.6 
                 1   
                 0.3 
               
               
                 11 (2.7) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 12 (2.8) 
                 31 
                 0.5 
                 4.8 
                 3.7 
                 1.1 
                 0.2 
               
               
                 Mean 
                 32.0 
                 0.5 
                 4.7 
                 3.7 
                 1.1 
                 0.3 
               
               
                 STD 
                 1.4 
                 0.0 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
               
               
                 Group 4 
               
               
                 13 (3.5) 
                 32 
                 0.7 
                 4.6 
                 3.4 
                 1.2 
                 0.2 
               
               
                 14 (3.6) 
                 34 
                 0.4 
                 4.8 
                 3.8 
                 1   
                 0.2 
               
               
                 15 (3.7) 
                 30 
                 0.4 
                 4.7 
                 3.4 
                 1.3 
                 0.2 
               
               
                 16 (3.8) 
                 24 
                 0.3 
                 5.1 
                 3.8 
                 1.3 
                 0.2 
               
               
                 Mean 
                 30.0 
                 0.5 
                 4.8 
                 3.6 
                 1.2 
                 0.2 
               
               
                 STD 
                 4.3 
                 0.2 
                 0.2 
                 0.2 
                 0.1 
                 0.0 
               
               
                 Group 5 
               
               
                 17 (4.5) 
                 22 
                 0.4 
                 4.6 
                 3.3 
                 1.3 
                 0.2 
               
               
                 18 (4.6) 
                 31 
                 0.5 
                 4.9 
                 3.7 
                 1.2 
                 0.2 
               
               
                 19 (4.7) 
                 23 
                 0.6 
                 4.8 
                 3.6 
                 1.2 
                 0.2 
               
               
                 20 (4.8) 
                 28 
                 0.5 
                 4.5 
                 3.4 
                 1.1 
                 0.2 
               
               
                 Mean 
                 26.0 
                 0.5 
                 4.7 
                 3.5 
                 1.2 
                 0.2 
               
               
                 STD 
                 4.2 
                 0.1 
                 0.2 
                 0.2 
                 0.1 
                 0.0 
               
               
                 Group 6 
               
               
                 21 (1.1) 
                 28 
                 0.3 
                 5.1 
                 3.4 
                 1.7 
                 0.2 
               
               
                 22 (1.2) 
                 36 
                 0.3 
                 5.1 
                 3.8 
                 1.3 
                 0.2 
               
               
                 23 (1.3) 
                 32 
                 0.4 
                 4.9 
                 3.5 
                 1.4 
                 0.1 
               
               
                 24 (1.4) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 Mean 
                 32.0 
                 0.3 
                 5.0 
                 3.6 
                 1.5 
                 0.2 
               
               
                 STD 
                 4.0 
                 0.1 
                 0.1 
                 0.2 
                 0.2 
                 0.1 
               
               
                 Group 7 
               
               
                 25 (2.1) 
                 32 
                 0.2 
                 5   
                 3.4 
                 1.6 
                 0.2 
               
               
                 26 (2.2) 
                 24 
                 0.3 
                 4.2 
                 2.8 
                 1.4 
                 0.1 
               
               
                 27 (2.3) 
                 28 
                 0.3 
                 4.8 
                 3.2 
                 1.6 
                 0.2 
               
               
                 28 (2.4) 
                 27 
                 0.3 
                 5   
                 3.4 
                 1.6 
                 0.1 
               
               
                 Mean 
                 27.8 
                 0.3 
                 4.8 
                 3.2 
                 1.6 
                 0.2 
               
               
                 STD 
                 3.3 
                 0.0 
                 0.4 
                 0.3 
                 0.1 
                 0.1 
               
               
                 Group 8 
               
               
                 29 (3.1) 
                 32 
                 0.3 
                 4.9 
                 3.3 
                 1.6 
                 0.2 
               
               
                 30 (3.2) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 31 (3.3) 
                 18 
                 0.3 
                 4.8 
                 3.1 
                 1.7 
                 0.2 
               
               
                 32 (3.4) 
                 26 
                 0.2 
                 4.2 
                 2.9 
                 1.3 
                 0   
               
               
                 Mean 
                 25.3 
                 0.3 
                 4.6 
                 3.1 
                 1.5 
                 0.1 
               
               
                 STD 
                 7.0 
                 0.1 
                 0.4 
                 0.2 
                 0.2 
                 0.1 
               
               
                 Group 9 
               
               
                 33 (4.1) 
                 25 
                 0.2 
                 4.1 
                 2.7 
                 1.4 
                 0.3 
               
               
                 34 (4.2) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 35 (4.3) 
                 23 
                 0.2 
                 4.7 
                 3.1 
                 1.6 
                 0.2 
               
               
                 36 (4.4) 
                 29 
                 0.3 
                 4.7 
                 3.2 
                 1.5 
                 0.3 
               
               
                 Mean 
                 25.7 
                 0.2 
                 4.5 
                 3.0 
                 1.5 
                 0.3 
               
               
                 STD 
                 3.1 
                 0.1 
                 0.3 
                 0.3 
                 0.1 
                 0.1 
               
               
                   
               
               
                 Abbreviations: 
               
               
                 WBC: white blood cells; 
               
               
                 RBC: red blood cells; 
               
               
                 Hg.: hemoglobin; 
               
               
                 HCT: hematocrit; 
               
               
                 MCV: Mean corpuscular volume; 
               
               
                 MCH: mean corpuscular hemoglobin; 
               
               
                 MCHC: mean corpuscular hemoglobin concentration; 
               
               
                 Plt.: platelets; 
               
               
                 Abs.: Absolute; 
               
               
                 Baso: basophils; 
               
               
                 Eos: eosinophils; 
               
               
                 Abs. Bands: immature neutrophils; 
               
               
                 Polys: polymorphonuclear cells; 
               
               
                 Lymph: lymphocytes; 
               
               
                 Mono: monocytes; 
               
               
                 BUN: blood urea nitrogen. 
               
            
           
         
       
     
     Example 14 
     Elicitation of Human WT1-Specific T-Cell Responses by Whole Gene In Vitro Priming 
     This example demonstrates that WT1 specific T-cell responses can be generated from the blood of normal individuals. 
     Dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC of normal donors by growth for 4-10 days in RPMI medium containing 10% human serum, 50 ng/ml GMCSF and 30 ng/ml IL-4. Following culture, DC were infected 16 hours with recombinant WT1-expressing vaccinia virus at an M.O.I. of 5, or for 3 days with recombinant WT1-expressing adenovirus at an M.O.I. of 10 ( FIGS. 21 and 22 ). Vaccinia virus was inactivated by U.V. irradiation. CD8+ T-cells were isolated by positive selection using magnetic beads, and priming cultures were initiated in 96-well plates. Cultures were restimulated every 7-10 days using autologous dendritic cells adeno or vaccinia infected to express WT1. Following 3-6 stimulation cycles, CD8+ lines could be identified that specifically produced interferon-gamma when stimulated with autologous-WT1-expressing dendritic cells or fibroblasts. The WT1-specific activity of these lines could be maintained following additional stimulation cycles. These lines were demonstrated to specifically recognize adeno or vaccinia WT1 infected autologous dendritic cells but not adeno or vaccinia EGFP-infected autologous dendritic cells by Elispot assays ( FIG. 23 ). 
     Example 15 
     Formulation of RA12-WT1 for Injection: Use of Excipients to Stabilize Lyophilized Product 
     This example describes the formulation that allows the complete solubilization of lyophilized Ra12-WT1. 
     The following formulation allowed for the recombinant protein Ra12-WT1 to be dissolved into an aqueous medium after being lyophylized to dryness: 
     Recombinant Ra12-WT1 concentration: 0.5-1.0 mg/ml; Buffer: 10-20 mM Ethanolamine, pH 10.0; 1.0-5.0 mM Cysteine; 0.05% TWEEN®-80 (Polysorbate-80); Sugar: 10% Trehalose (T5251, Sigma, Mo.) 10% Maltose (M9171, Sigma, Mo.) 10% Sucrose (S7903, Sigma, Mo.) 10% Fructose (F2543, Sigma, Mo.) 10% Glucose (G7528, Sigma, Mo.). 
     The lyophilized protein with the sugar excipient was found to dissolve significantly more than without the sugar excipient. Analysis by coomassie stained SDS-PAGE showed no signs of remaining solids in the dissolved material. 
     Example 16 
     Formulation of a WT1 Protein Vaccine 
     This example describes the induction of WT1-specific immune responses following immunization with WT1 protein and 2 different adjuvant formulations. 
     According to this example, WT1 protein in combination with MPL®-SE induces a strong Ab and Interferon-γ(IFN-γ) response to WT1. Described in detail below are the methods used to induce WT1 specific immune responses following WT1 protein immunization using MPL®-SE or ENHANZYN® as adjuvant in C57/B6 mice. C57BL/6 mice were immunized with 20 μg rRa2-WT1 combined with either MPL®-SE or ENHANZYN® adjuvants. One group of control mice was immunized with rRa12-WT1 without adjuvant and one group was immunized with saline alone. Three intramuscular (IM) immunizations were given, three weeks apart. Spleens and sera were harvested 2 weeks post-final immunization. Sera were analyzed for antibody responses by ELISA on plates coated with Ra12-WT1 fusion, Ra12 or WT1 TRX. Similar levels of IgG2a and IgG1 antibody titers were observed in mice immunized with Ra12-WT1+MPL®-SE and Ra12-WT1+ENHANZYN®. Mice immunized with rRa12-WT1 without adjuvant showed lower levels of IgG2a antibodies. 
     CD4 responses were assessed by measuring Interferon-γ production following stimulation of splenocytes in vitro with rRa12-WT1, rRa12 or with WT1 peptides p6, p117 and p287. Both adjuvants improved the CD4 responses over mice immunized with rRA12-WT1 alone. Additionally, the results indicate that rRA12-WT1+MPL®-SE induced a stronger CD4 response than did rRA12-WT1+ENHANZYN®. IFN-γOD readings ranged from 1.4-1.6 in the mice immunized with rRA12-WT1+MPL®-SE as compared to 1-1.2 in the mice immunized with rRA12-WT1+ENHANZYN®. Peptide responses were only observed against p117, and then only in mice immunized with rRa12-WT1+MPL®-SE. Strong IFN-γ responses to the positive control, ConA, were observed in all mice. Only responses to ConA were observed in the negative control mice immunized with saline indicating that the responses were specific to rRA12-WT1. 
     Example 17 
     Construction of a Randomly Mutated WT1 Library 
     The nucleic acid sequence of human WT1 was randomly mutated using a polymerase chain reaction method in the presence of 8-oxo dGTP and dPTP (journal of Molecular Biology 1996; 255:589-603). The complete unspliced human WT1 gene is disclosed in SEQ ID NO:380 and the corresponding protein sequence is set forth in SEQ ID NO:404. A splice variant of WT1 was used as a template for the PCR reactions and is disclosed in SEQ ID NOs:381 (DNA) and 408 (protein). Conditions were selected so that the frequency of nucleic acid alterations led to a targeted change in the amino acid sequence, usually 5-30% of the PCR product. The mutated PCR product was then amplified in the absence of the nucleotide analogues using the four normal dNTPs. This PCR product was subcloned into mammalian expression vectors and viral vectors for immunization. This library, therefore, contains a mixed population of randomly mutated WT1 clones. Several clones were selected and sequenced. The mutated WT1 variant DNA sequences are disclosed in SEQ ID NOs:377-379 and the predicted amino acid sequences of the variants are set forth in SEQ ID NOs:405-407. These altered sequences, and others from the library, can be used as immunogens to induce stronger T cell responses against WT1 protein in cancer cells. 
     Example 18 
     Construction of WT1-Lamp Fusions 
     A tripartite fusion was constructed using the polymerase chain reaction and synthetic oligonucleotides containing the desired junctions of human lysosomal associated membrane protein-1 (LAMP-1) and a splice variant of the human WT1 sequence. The splice variant of WT1 and the LAMP-1 sequence used for these fusions are disclosed in SEQ ID NOs:381 and 383. Specifically, the signal peptide of LAMP-1 (base pairs 1-87 of LAMP) was fused to the 5-prime end of the human WT1 open reading frame (1,290 base pairs in length), then the transmembrane and cytoplasmic domain of LAMP-1 (base pairs 1161 to 1281 of LAMP) was fused to the 3-prime end of the WT1 sequence. The sequence of the resulting WT1-LAMP construct is set forth in SEQ ID NO:382 (DNA) and SEQ ID NO:409 (protein). The construct was designed so that when it is expressed in eukaryotic cells, the signal peptide directs the protein to the endoplasmic reticulum (ER) where the localization signals in the transmembrane and cytoplasmic domain of LAMP-1 direct transport of the fusion protein to the lysosomal location where peptides are loaded on to Class II MHC molecules. 
     Example 19 
     Construction of WT1-Ubiquitin Fusions for Enhanced MHC Class I Presentation 
     The human ubiquitin open reading frame (SEQ ID NO:384) was mutated such that the nucleotides encoding the last amino acid encode an alanine instead of a glycine. This mutated open reading frame was cloned in frame just upstream of the first codon of a splice variant of human WT1 (SEQ ID NOs:381 and 408, DNA and protein, respectively). The G-&gt;A mutation prevents co-translational cleavage of the ancient protein by the proteases that normally process poly-ubiquitin during translation. The DNA and predicted amino acid sequence for the resulting construct are set forth in SEQ ID NOs:385 and 410, respectively. The resulting protein demonstrated decreased cellular cytotoxicity when it was expressed in human cells. Whereas it was not possible to generate stable lines expressing native WT1, cell lines expressing the fusion protein were readily obtained. The resulting protein is predicted to be targeted to the proteosome by virtue of the added ubiquitin molecule. This should result in more efficient recognition of the protein by WT1 specific CD8+ T cells. 
     Example 20 
     Construction of an Adenovirus Vector Expressing Human WT1 
     A splice variant of human WT1 (SEQ ID NO:381) was cloned into an E1 and E3 deleted adenovirus serotype 5 vector. The expression of the WT1 gene is controlled by the CMV promoter mediating high levels of WT1 protein expression. Infection of human cells with this reagent leads to a high level of expression of the WT1 protein. The antigenic nature of the adenoviral proteins introduced into the host cell during and produced at low levels subsequent to infection can act to increase immune surveillance and immune recognition of WT1 as an immunological target. This vector can be also used to generate immune responses against the WT1 protein when innoculated into human subjects. If these subjects are positive for WT1 expressing tumor cells the immune response could have a therapeutic or curative effect on the course of the disease. 
     Example 21 
     Construction of a Vaccinia Virus Vector Expressing Human WT1 
     A splice variant of the full length human WT1 gene (SEQ ID NO:381) was cloned into the thymidine kinase locus of the Western Reserve strain of the vaccinia virus using the pSC11 shuttle vector. The WT1 gene is under the control of a hybrid vaccinia virus promoter that mediates gene expression throughout the course of vaccinia virus infection. This reagent can be used to express the WT1 protein in human cells in vivo or in vitro. WT1 is a self protein that is overexpressed on some human tumor cells. Thus, immunological responses to WT1 delivered as a protein are unlikely to lead to Major Histocompatibility Class I (MHC class I)-mediated recognition of WT1. However, expression of the protein in the intracellular compartment by the vaccinia virus vector will allow high level MHC class I presentation and recognition of the WT1 protein by CD8+ T cells. Expression of the WT1 protein by the vaccinia virus vector will also lead to presentation of WT1 peptides in the context of MHC class II and thus to recognition by CD4+ T cells. 
     The uses of this invention include its use as a cancer vaccine. Immunization of human subjects bearing WT1 positive tumors could lead to a therapeutic or curative response. The expression of WT1 within the cell will lead to recognition of the protein by both CD4 and CD8 positive T cells. 
     Example 22 
     Generation of WT1-Specific CD8+ T-Cell Clones Using Whole Gene Priming 
     Dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC of normal donors by growth for 4-6 days in RPMI medium containing 10% human serum, 50 ng/ml GM-CSF and 30 ng/ml IL-4. Following culture, DC were infected 16 hours with recombinant WT1-expressing vaccinia virus (described in Example 21) at a multiplicity of infection (MOI) of 5 or for 3 days with recombinat WT1-expressing adenovirus at an MOI of 10. Vaccinia virus was inactivated by U.V. irradiation. CD8+ T-cells were isolated by negative depletion using magnetic beads, and priming cultures were initiated in 96-well plates. Cultures were restimulated every 7-10 days using autologous dendritic cells infected with adeno or vaccinia virus engineered to express WT1. Following 4-5 stimulation cycles, CD8+ T-cell lines could be identified that specifically produced interferon-gamma when stimulated with autologous-WT1 expressing dendritic cells or fibroblasts. These lines were cloned and demonstrated to specifically recognize WT1 transduced autologous fibroblasts but not EGFP transduced fibroblasts by Elispot assays. 
     The Wilms&#39; tumor (WT1) gene participates in leukemogenesis and is overexpressed in most human leukemias as well as in several solid tumors. Previous studies in humans have demonstrated the presence of WT1 specific antibody (Ab) responses in 16/63 (25%) of AML and in 15/81 (19%) of CML patients studied. Previous studies in mice have shown that WT1 peptide based vaccines elicit WT1 specific Ab, Th and CTL responses. The use of peptides as vaccines in humans is limited by their HLA restriction and the tendency to elicit peptide specific responses and only in a minority of patients tumor specific CTL. The advantages of whole gene immunization are that several helper and CTL epitopes can be included in a single vaccine, thus not restricting the vaccine to specific HLA types. The data disclosed herein demonstrate the induction of WT1 specific immune responses using whole gene in vitro priming and that WT1 specific CD8+ T-cell clones can be generated. Given that existent immunity to WT1 is present in some patients with leukemia and that murine and human WT1 are 96% identical at the amino acid level and vaccination to WT1 protein, DNA or peptides can elicit WT1 specific Ab, and cellular T-cell responses in mice without toxicity to normal tissues in mice, these human in vitro priming experiments provide further validation of WT1 as a tumor/leukemia vaccine. Furthermore, the ability to generate WT1 specific CD8+ T-cell clones may lead to the treatment of malignancies associated with WT1 overexpression using genetically engineered T-cells. 
     Example 23 
     Recombinant Constructs for Clinical Manufacturing of WT1 
     Five constructs were made as described in detail below, for the production of clinical grade WT1. 
     Design of Ra12/WT-E (SEQ ID NOs:388 (cDNA) and 391 (protein)) and WT-1 E (SEQ ID NOs:386 (cDNA) and 395 (protein)) with No His tag: 
     The WT-1 E reading frame was PCR amplified with the following primers for the non-His non fusion construct: 
     
       
         
           
               
               
            
               
                   
                 PDM-780 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 396) 
               
            
           
           
               
               
            
               
                   
                 5′ gacgaaagcatatgcactccttcatcaaac 3′ Tm {tilde over (6)}° C. 
               
               
                   
                   
               
               
                   
                 PDM-779 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 397) 
               
            
           
           
               
               
            
               
                   
                 5′ cgcgtgaattcatcactgaatgcctctgaag 3′ Tm 63° C. 
               
            
           
         
       
     
     The following PCR cycling conditions were used: 1 μl 10×Pfu buffer, 1 μl 10 mM dNTPs, 2 μl 1M each oligo, 83 μl sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.), 50 μg DNA (pPDMRa12WT-1 No His). The reaction was denatured initially at 96° C. for 2 minutes, followed by 40 cycles of 96° C. for 20 seconds, 62° C. for 15 seconds, and 72° C. for 1 minute and 40 seconds. This was followed by a final extension of 72° C. for 4 minutes. The PCR product was digested with NdeI and EcoRI and cloned into pPDM His (a modified pET28 vector) that had been digested with NdeI and EcoRI. The construct was confirmed through sequence analysis and then transformed into BLR (DE3) pLys S and HMS 174 (DE3) pLys S cells. This construct—pPDM WT-1 E was then digested with NcoI and XbaI and used as the vector backbone for the NcoI and XbaI insert from pPDM Ra12 WT-1 F (see below). The construct was confirmed through sequence analysis and then transformed into BLR (DE3) pLys S and HMS174 (DE3) pLys S cells. Protein expression was confirmed by Coomassie stained SDS-PAGE and N-terminal protein sequence analysis. 
     Design of Ra12-WT-1-F (a.a. 1-281) with No His tag (SEQ ID NOs:389 (cDNA) and 393 (protein)): 
     The Ra12 WT-1 reading frame was PCR amplified with the following primers: 
     
       
         
           
               
               
            
               
                   
                 PDM-777 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 398) 
               
            
           
           
               
               
            
               
                   
                 5′ cgataagcatatgacggccgcgtccgataac 3′ Tm 66° C. 
               
               
                   
                   
               
               
                   
                 PDM-779 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 399) 
               
            
           
           
               
               
            
               
                   
                 5′ cgcgtgaattcatcactgaatgcctctgaag 3′ Tm 63° C. 
               
            
           
         
       
     
     The following PCR cycling conditions were used: 1 μl 10×Pfu buffer, 1 μl 10 mM dNTPs, 2 μl 1 μM each oligo, 83 μl sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.), 50 ηg DNA (pPDMRa12 WT-1 No His). The reaction was denatured initially at 96° C. for 2 minutes, followed by 40 cycles of 96° C. for 20 seconds, 58° C. for 15 seconds, and 72° C. for 3 minutes. This was followed by a final extension of 72° C. for 4 minutes. The PCR product was digested with NdeI and cloned into pPDM His that had been digested with NdeI and Eco72I. The sequence was confirmed through sequence analysis and then transformed into BLR (DE3) pLys S and HMS174 (DE3) pLysS cells. Protein expression was confirmed by Coomassie stained SDS-PAGE and N-terminal protein sequence analysis. 
     Design of Ra12-WT-1 with No His tag (SEQ ID NOs:390 (cDNA) and 392 (protein)): 
     The Ra12WT-1 reading frame was PCR amplified with the following primers: 
     
       
         
           
               
               
            
               
                   
                 PDM-777 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 400) 
               
            
           
           
               
               
            
               
                   
                 5′ cgataagcatatgacggccgcgtccgataac 3′ Tm 66° C. 
               
               
                   
                   
               
               
                   
                 PDM-778 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 401) 
               
            
           
           
               
               
            
               
                   
                 5′ gtctgcagcggccgctcaaagcgccagc 3′ Tm {tilde over (7)}° C. 
               
            
           
         
       
     
     The following PCR cycling conditions were used: 1 μl 10×Pfu buffer, 1 μl 10 mM dNTPs, 2 μl 1 μM each oligo, 83 μl sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.), 50 ηg DNA (pPDMRa12 WT-1 No His). The reaction was denatured initially at 96° C. for 2 minutes, followed by 40 cycles of 96° C. for 20 seconds, 68° C. for 15 seconds, and 72° C. for 2 minutes and 30 seconds. This was followed by a final extension of 72° C. for 4 minutes. The PCR product was digested with NotI and NdeI and cloned into pPDM His that had been digested with NdeI and NotI. The sequence was confirmed through sequence analysis and then transformed into BLR (DE3) pLys S and HMS174 (DE3) pLysS cells. Protein expression was confirmed by Coomassie stained SDS-PAGE and N-terminal protein sequence analysis. 
     Design of WT-1 C (a.a. 69-430) in  E. coli  without His tag (SEQ ID NOs:387 (cDNA) and 394 (protein)): 
     The WT-1 C reading frame was PCR amplified with the following primers: 
     
       
         
           
               
               
            
               
                   
                 PDM-780 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 402) 
               
            
           
           
               
               
            
               
                   
                 5′ gacgaaagcatatgcactccttcatcaaac 3′ Tm {tilde over (6)}° C. 
               
               
                   
                   
               
               
                   
                 PDM-778 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 403) 
               
            
           
           
               
               
            
               
                   
                 5′ gtctgcagcggccgctcaaagcgccagc 3′ Tm {tilde over (7)}° C. 
               
            
           
         
       
     
     The following PCR cycling conditions were used: 1 μl 10×Pfu buffer, 1 μl 10 mM dNTPs, 2 μl 1 μM each oligo, 83 μl sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.), 50 ηg DNA (pPDMRa12WT-1 No His). The reaction was denatured initially at 96° C. for 2 minutes, followed by 40 cycles of 96° C. for 20 seconds, 62° C. for 15 seconds, and 72° C. for 2 minutes. This was followed by a final extension of 72° C. for 4 minutes. The PCR product was digested with NdeI and cloned into pPDM His that had been digested with NdeI and Eco72I. The sequence was confirmed through sequence analysis and then transformed into BLR (DE3) pLys S and HMS174 (DE3) pLys S cells. Protein expression was confirmed by Coomassie stained SDS-PAGE and N-terminal protein sequence analysis. 
     Example 24 
     Generation of WT1-Specific CD8 +  T Cell Clones Using Whole Gene Priming and Identification of an HLA-A2-Restricted WT1 Epitope 
     In this example, Adeno and Vaccinia virus delivery vehicles were used to generate WT1-specific T cell lines. A T cell clone from the line was shown to be specific for WT1 and further, the epitope recognized by this clone was identified. 
     Dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC of normal donors by growth for 4-6 days in RPMI medium containing 10% human serum, 50 ng/ml GM-CSF and 30 ng/ml IL-4. Following culture, DC were infected 16 hours with recombinant WT1-expressing vaccinia virus at a multiplicity of infection (MOI) of 5 or for 2-3 days with recombinant WT1-expressing adeno virus at an MOI of 3-10. Vaccinia virus was inactivated by U.V. irradiation. CD8+ T-cells were isolated by negative depletion using antibodies to CD4, CD14, CD16, CD19 and CD56+ cells, followed by magnetic beads specific for the Fc portion of these Abs. 
     Priming cultures were initiated in 96-well plates. Cultures were restimulated every 7-14 days using autologous dendritic cells infected with adeno or vaccinia virus engineered to express WT1. Following 4-5 stimulation cycles, CD8+ T cell lines could be identified that specifically produced interferon-γ (IFN-γ) when stimulated with autologous-WT1 expressing dendritic cells or fibroblasts. These lines were cloned and demonstrated to specifically recognize WT1 transduced autologous fibroblasts but not control transduced fibroblasts by Elispot assays. 
     To further analyze HLA restriction of these WT1 specific CD8+ T-cell clones, fibroblasts derived from an additional donor (D475), sharing only the HLA-A2 allele with the donor (D349) from which the T-cell clone was established, were transduced with WT1. ELISPOT analysis demonstrated recognition of these D475 target cells by the T-cell clone. To further demonstrate HLA A2 restriction and demonstrate that this epitope is expressed by tumor cells “naturally” overexpressing WT1 (as part of their malignant transformation), the leukemia cell line K562 was tested. K562 was transduced with the HLA A2 molecule, and HLA-A2 negative K562 cells were used as controls for nonspecific IFN-γ release. ELISPOT analysis demonstrated that the T cells recognized the A2 positive K562 cell line, but not the A2 negative K562 cells. Further proof of specificity and HLA-A2 restriction of the recognition was documented by HLA-A2 antibody blocking experiments. 
     To further define the WT1 epitope, 4 truncated WT1 retroviral constructs were generated. Donor 475 fibroblasts were then transduced with these constructs. ELISPOT assays demonstrated recognition of D475 fibroblasts transduced with the WT1 Tr1 construct (aa2-aa92), thus demonstrating that the WT1 epitope is localized within the first 91 N-terminal amino acids of the WT1 protein. To fine map the epitope, 15mer peptides of the WT1 protein, overlapping by 11 amino acids, were synthesized. The WT1 specific T-cell clone recognized two overlapping 15mer peptides, peptide 9 (QWAPVLDFAPPGASA) (SEQ ID NO: 412) and peptide 10 (VLDFAPPGASAYGSL) (SEQ ID NO: 413). To further characterize the minimal epitope recognized, shared 9mer and 10 mer peptides of the 15mers (5 total) were used to analyse the specificity of the clone. The clone specifically recognized the 9mer, VLDFAPPGA (SEQ ID NO:241), and the 10 mer, VLDFAPPGAS (SEQ ID NO:411). 
     Example 25 
     Cloning and Sequencing of TCR Alpha and Beta Chains Derived from a CD8 T Cell Specific for WT1 
     T cell receptor (TCR) alpha and beta chains from CD8+ T cell clones specific for WT1 are cloned. Sequence analysis is carried to demonstrate the family origin of the alpha and beta chains of the TCR. Additionally, unique diversity and joining segments (contributing to the specificity of the response) are identified. 
     Total mRNA from 2×10 6  cells from a WT1 specific CD8+ T cell clone is isolated using TRIZOL® reagent (a mono-phasic solution of phenol and guanidine isothiocyanate) and cDNA is synthesized using READY-TO-GO® kits (Pharmacia). To determine Vα and Vβ sequences in a clone, a panel of Vα and Vβ subtype specific primers are synthesized (based on primer sequences generated by Clontech, Palo Alto, Calif.) and used in RT-PCR reactions with cDNA generated from each clone. The RT-PCR reactions demonstrate which Vβ and Vα sequence is expressed by each clone. 
     To clone the full-length TCR alpha and beta chains from a clone, primers are designed that span the initiator and terminator-coding TCR nucleotides. Standard 35 cycle RT-PCR reactions are established using cDNA synthesized from the CTL clone and the above primers using the proofreading thermostable polymerase PWO (Roche, Basel, Switzerland). The resultant specific bands (˜850 bp for alpha and ˜950 for beta) are ligated into the PCR blunt vector (Invitrogen, Carlsbad, Calif.) and transformed into  E. coli. E. coli  transformed with plasmids containing full-length alpha and beta chains are identified, and large scale preparations of the corresponding plasmids are generated. Plasmids containing full-length TCR alpha and beta chains are then sequenced using standard methods. The diversity-joining (DJ) region that contributes to the specificity of the TCR is thus determined. 
     Example 26 
     WT1 Specific CD8+ T-Cell Clone Lyses WT1-Expressing Leukemic Blasts 
     The CD8+ T cell clone initially disclosed in Example 24 that recognizes peptide sequence VLDFAPPGA (human WT1 residues 37-45; SEQ ID NO:241) was further tested for the ability to kill (lyse) WT1 expressing leukemia target cells in an HLA A2 restricted fashion. K562 target cells transduced with the HLA A2 molecule, GFP, A2 Kb, or untransduced, were used in a standard 4.5 hour  51 Chromium release assay with effector to target cell (E:T) ratios of 25:1 and 5:1. At an E:T ratio of 25:1, the CD8+ T-cell clone lysed the K562/A2 and K562/A2 Kb cells (40% and 49% specific lysis, respectively) while the control GFP transduced and the K562 cells were not lysed. At an E:T of 5:1, specific lysis of the K562/A2 and K562/A2 Kb cells was 21% and 24%, respectively. Thus, this CD8+ T cell clone recognizes and lyses leukemic cells expressing WT1 in an HLA-A2-restricted fashion. The ability to generate WT1 specific CD8+ T-cell clones has utility in the treatment of malignancies associated with WT1 overexpression using genetically engineered T-cells. 
     Example 27 
     Construction of HLA-A2-Peptide-MHC Tetrameric Complexes 
     This example describes the cloning and expression of soluble HLA-A2 in insect cells, and the purification and assembly of HLA-A2 into fluorescent, multivalent peptide-MHC tetramer complexes for the detection and isolation of antigen-specific CD8 T cells. 
     This system is similar to that developed and described by Altman, et al. (Altman, J., et al., Science, 1996 274(5284):94-6) in that soluble HLA-A2 was singly biotinylated at a birA recognition sequence and was subsequently assembled into multimers on a phycoerythrin-conjugated streptavidin scaffolding. The materials described herein differ in that the HLA-A2 was expressed in a glycosylated, soluble form from insect cells and the heterodimer was purified using an anti-human class I MHC antibody affinity column. 
     The HLA-A2 heavy chain gene, appended with the birA biotinylation sequence, and the human beta-2-microglobulin gene were cloned into the baculovirus expression vector pFASTBAC-dual. Upon infection of insect cells the genes were concomitantly transcribed from divergent promoters and fully assembled, glycosylated soluble HLA-A2 heterodimer was secreted into the growth medium. The infected insect cells were cultured in cell factories for 4 days at 21° C. before the supernatants were harvested. HLA-A2 production was monitored by a capture ELISA employing the W6/32 and biotinylated B9.12.1 antibodies. HLA-A2 was purified from the culture supernatant to &gt;90% purity in one step by affinity chromatography using 2 anti-human class I MHC monoclonal antibodies linked to Sepharose beads. The antibodies used were PA2.1 and W6/32. Purified HLA-A2 was singly biotinylated on the birA recognition sequence on the C-terminus of the heavy chain using the commercially available birA enzyme. The efficiency of biotinylation was evaluated essentially as described (Crawford et al (1998) Immunity June; 8(6):675-82.), and the material was further purified by size exclusion chromatography (SEC). Phycoerythrin-conjugated streptavidin was saturated with bio-HLA-A2 and the multivalent staining reagent was purified from free HLA-A2 by SEC. HLA-A2 tetramer was incubated for 48 hours at room temperature with a 10-fold molar excess of Her-2/neu E75 peptide or Influenza matrix MI peptide before the specific T cell clones were stained at 4° C. for 30 minutes in the presence of peptide loaded tetramer and anti-CD8 antibody. Results indicated that the tetramers incubated in the presence of molar excess of the M1 58-66 M1 influenza peptide specifically stained an influenza-specific T cell clone and the tetramers incubated with an excess of the Her-2/neu E75 peptide specifically stained the Her-2/new specific T cell clone. 
     Example 28 
     Detection of WT1 Specific T-Cells Using WT1 MHC-Peptide Tetramers 
     HLA-A2 tetramers described in Example 27 were incubated with a molar excess of the WT1 p37-45 peptide (VLDFAPPGA) (human WT1 residues 37-45; SEQ ID NO:241) previously shown in Example 24 to be restricted by HLA-A2. This tetramer was used to stain the WT1-specific CD8+ T cell clone described in Example 24. This clone was shown to specifically recognize the p37-45 epitope. When the tetramers were incubated with an excess of p37-45 peptide, they specifically stained the CD8+ T cell clone while those tetramers incubated with an excess of irrelevant HLA-A2 peptides (Her2/neu, WT1 p38-46, WT1 p39-47), the tetramers did not stain the CD8+ T cell clone. Thus, the WT1 p37-45-specific CD8+ T cell clone is specifically recognized by the HLA-A2-p37-45 peptide MHC tetramer. 
     A WT1-specific T cell line generated as described in Example 24 was then stained with the HLA-A2-p37-45, irrelevant Her2/neu or WT1 p37-46 tetramers. The HLA-A2-p37-45 tetramers stained 1% of the total population of this WT1-specific T cell line and 7% of the gated CD8+ population while the control HLA-A2-p37-46 tetramer stained at the same background levels as the control HLA-A2-Her2/neu tetramers. 
     These results indicate that MHC-peptide tetramers are a highly sensitive and specific tool for detecting WT1 specific immune responses. The peptide-MHC tetramers can be used for early detection of WT1 associated malignancies, monitoring WT1-specific responses, and for monitoring minimal residual disease. Detection of WT1 specific T-cells by tetramer staining is also a useful tool to identify groups within a patient population suffering from a WT1 associated disease at a higher risk for relapse or disease progression. 
     Example 29 
     Generation of a WT1-Specific CD8 +  T Cell Line from an HLA-A24-Positive Donor Using Whole Gene Priming 
     In this example, Adeno and Vaccinia virus delivery vehicles were used to generate WT1-specific T cell lines from an HLA-A24 positive donor. This T cell line was shown to be MHC class I restricted. These experiments further confirm the immunogenicity of the WT1 protein and support its use as a target for vaccine and/or other immunotherapeutic approaches. 
     Dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC of a normal HLA-A24-positive donor by growth for 4-6 days in RPMI medium containing 10% human serum, 50 ng/ml GM-CSF and 30 ng/ml IL-4. Following culture, DC were infected 16 hours with recombinant WT1-expressing vaccinia virus at a multiplicity of infection (MOI) of 5 or for 2-3 days with recombinant WT1-expressing adeno virus at an MOI of 3-10. Vaccinia virus was inactivated by U.V. irradiation. CD8+ T-cells were isolated by negative depletion using antibodies to CD4, CD14, CD16, CD19 and CD56+ cells, followed by magnetic beads specific for the Fc portion of these Abs. 
     Priming cultures were initiated in 96-well plates. Cultures were restimulated every 7-14 days using autologous dendritic cells infected with adeno or vaccinia virus engineered to express WT1. Following 4-5 stimulation cycles, CD8+ T cell lines could be identified that specifically produced interferon-γ (IFN-γ) when stimulated with autologous-WT1 expressing dendritic cells or fibroblasts. These lines were cloned and shown by Elispot assays to specifically recognize WT1 transduced autologous fibroblasts but not control transduced fibroblasts in an MHC class I-restricted manner. 
     These experiments show that the WT1 protein can be used to generate a T cell response and thus, further confirm the immunogenicity of the WT1 antigen and support its use as a target for vaccine and other immunotherapeutic approaches. 
     Example 30 
     Identification of HLA-A2 High Affinity WT1 Epitopes 
     This experiment describes the in silico identification of WT1 epitopes predicted to bind to HLA-A2 with higher affinity than naturally processed epitopes. The epitopes identified herein have utility in vaccine and/or immunotherapeutic strategies for the treatment of cancers associated with WT1 expression. 
     Peptide analogs of the naturally processed HLA A2 restricted WT1 epitope p37-45 (VLDFAPPGA; human WT1 residues 37-45; SEQ ID NO:241; previously shown in Example 24 to be restricted by HLA-A2) with motifs for binding to HLA-A2.1 with higher affinity than the naturally processed peptide were constructed as described in further detail below. 
     A peptide motif searching program based on algorithms developed by Rammensee, et al (Hans-Georg Rammensee, Jutta Bachmann, Niels Nikolaus Emmerich, Oskar Alexander Bachor, Stefan Stevanovic: SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics (1999) 50: 213-219) and by Parker, et al (Parker, K. C., M. A. Bednarek, and J. E. Coligan. 1994. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 152:163.) was used to identify analogs of the WT1 p37-45 peptide epitope that are predicted to bind to HLA-A2 with higher affinity than the natural p37-45 peptide. The peptides shown in Table LII have predicted peptide binding scores equal to or greater than the naturally processed p37-45 peptide. The binding score is derived from a predicted half-time of dissociation to the HLA-A2 class I molecule. The analysis is based on coefficient tables deduced from the published literature by Dr. Kenneth Parker kparker@atlas.niaid.nih.gov, NIAID, NIH. 
     
       
         
           
               
             
               
                 TABLE LII 
               
             
            
               
                   
               
               
                 p37-45 Peptide Analogs 
               
            
           
           
               
               
               
               
            
               
                 Position 
                   
                 Theoretical Binding 
                 SEQ ID 
               
               
                 Modified 
                 Sequence 
                 Score 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 
                   Wild Type 
                 
                 
                   VLDFAPPGA 
                 
                 
                   3.378 
                 
                 241 
               
               
                   
               
               
                 P1 
                   I LDFAPPGA 
                 3.378 
                 414 
               
               
                   
               
               
                 P1 
                   L LDFAPPGA 
                 3.378 
                 415 
               
               
                   
               
               
                 P1 
                   F LDFAPPGA 
                 9.141 
                 416 
               
               
                   
               
               
                 P1 
                   K LDFAPPGA 
                 6.955 
                 417 
               
               
                   
               
               
                 P1 
                   M LDFAPPGA 
                 3.378 
                 418 
               
               
                   
               
               
                 P1 
                   Y LDFAPPGA 
                 9.141 
                 419 
               
               
                   
               
               
                 P2 
                 V M DFAPPGA 
                 2.44 
                 420 
               
               
                   
               
               
                 P4 
                 VLD E APPGA 
                 13.85 
                 421 
               
               
                   
               
               
                 P4 
                 VLD K APPGA 
                 3.378 
                 422 
               
               
                   
               
               
                 P6 
                 VLDFA V PGA 
                 7.77 
                 423 
               
               
                   
               
               
                 P8 
                 VLDFAPP K A 
                 3.378 
                 424 
               
               
                   
               
               
                 P9 
                 VLDFAPPG V   
                 47.3 
                 425 
               
               
                   
               
               
                 P9 
                 VLDFAPPG L   
                 14.53 
                 426 
               
               
                   
               
               
                 P1 and P4 
                   F LD E APPGA 
                 37.48 
                 427 
               
               
                   
               
               
                 P1 and P4 
                   K LD E APPGA 
                 28.52 
                 428 
               
               
                   
               
               
                 P1 and P4 
                   Y LD E APPGA 
                 37.48 
                 429 
               
               
                   
               
               
                 P1 and P4 
                   F LD K APPGA 
                 9.141 
                 430 
               
               
                   
               
               
                 P1 and P4 
                   K LD K APPGA 
                 6.955 
                 431 
               
               
                   
               
               
                 P1 and P4 
                   Y LD K APPGA 
                 9.141 
                 432 
               
               
                   
               
               
                 P1 and P9 
                   F LDFAPPG V   
                 128 
                 433 
               
               
                   
               
               
                 P1 and P9 
                   K LDFAPPG V   
                 97.37 
                 434 
               
               
                   
               
               
                 P1 and P9 
                   Y LDFAPPG V   
                 128 
                 435 
               
               
                   
               
               
                 P1 and P9 
                   F LDFAPPG L   
                 39.31 
                 436 
               
               
                   
               
               
                 P1 and P9 
                   K LDFAPPG L   
                 29.91 
                 437 
               
               
                   
               
               
                 P1 and P9 
                   Y LDFAPPG L   
                 39.31 
                 438 
               
               
                   
               
               
                 P1, P4 and P9 
                   F LD E APPG V   
                 524.7 
                 439 
               
               
                   
               
               
                 P1, P4 and P9 
                   K LD E APPG V   
                 399.2 
                 440 
               
               
                   
               
               
                 P1, P4 and P9 
                   Y LD E APPG V   
                 524.7 
                 441 
               
               
                   
               
               
                 P1, P4 and P9 
                   F LD E APPG L   
                 161.2 
                 442 
               
               
                   
               
               
                 P1, P4 and P9 
                   K LD E APPG L   
                 122.6 
                 443 
               
               
                   
               
               
                 P1, P4 and P9 
                   Y LD E APPG L   
                 161.2 
                 444 
               
               
                   
               
            
           
         
       
     
     In a separate analysis, computer modeling was used to identify peptide epitope analogs of the p37-45 WT1 epitope. The coordinates of the HLA-A2 native structure were downloaded from the Brookhaven protein database (pdb I.D.: 3HLA) (L. L. Walsh, “Annotated PDB File Listing”, Protein Science 1:5, Diskette Appendix (1992). This file was used as a template for manipulations with the SwissModel (Peitsch MC (1996) ProMod and Swiss-Model: Internet-based tools for automated comparative protein modeling.  Biochem. Soc. Trans.  24:274-279.) program available through the Expasy web site (Appel R. D., Bairoch A., Hochstrasser D. F. A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server. Trends Biochem. Sci. 19:258-260(1994). The peptide bound to the protein was mutated manually to yield the bound WT p37-45 peptide. The new structure was submitted for three rounds of energy minimization with the GROMOS96 implementation of the Swiss-PdbViewer; two energy minimizations were performed on the whole structure, followed by one round with unfavorable residues selected. A final evaluation showed an overall favorable energy state for the model. Ramachandran plotting indicated that only one non-glycinyl residue is far in disallowed regions. Peptides identified using the modeling method described herein are set forth in Table LIII below. 
     
       
         
           
               
             
               
                 TABLE LIII 
               
             
            
               
                   
               
               
                 p37-45 Peptide Analogs Identified by 
               
               
                 Computer Modeling 
               
            
           
           
               
               
               
               
            
               
                   
                 Position 
                   
                 SEQ ID 
               
               
                   
                 Modified 
                 Sequence 
                 NO: 
               
               
                   
                   
               
               
                   
                 
                   Wild Type 
                 
                 
                   VLDFAPPGA 
                 
                 241 
               
               
                   
                   
               
               
                   
                 P6 
                 VLDFA G PGA 
                 445 
               
               
                   
                   
               
               
                   
                 P6 
                 VLDFA T PGA 
                 446 
               
               
                   
                   
               
               
                   
                 P6 and P9 
                 VLDFA T PG V   
                 447 
               
               
                   
                   
               
               
                   
                 P6 and P9 
                 VLDFA T PG L   
                 448 
               
               
                   
                   
               
               
                   
                 P6 and P9 
                 VLDFA T PG S   
                 449 
               
               
                   
                   
               
               
                   
                 P6 and P9 
                 VLDFA T PG A   
                 450 
               
               
                   
                   
               
            
           
         
       
     
     Several peptides identified using the two methods described above were then tested for the ability to be recognized by the p37-45 specific CTL clone (see Example 24). ELISPOT analysis showed that peptides p37-1 (SEQ ID NO:426) and model-1 (SEQ ID NO:445) were recognized by the p37-45 CTL clone. These results suggest that these 2 peptide analogs are predicted to bind to HLA-A2 with higher affinity than the naturally processed epitope and still be recognized by a native T cell receptor. 
     Thus, this experiment describes the in silico identification of WT1 epitopes predicted to bind to HLA-A2 with higher affinity than naturally processed epitopes. Two of the epitopes identified were tested and shown to be recognized by a CTL clone generated with the native WT1 p37-45 epitope. The epitopes identified herein have utility in vaccine and/or immunotherapeutic strategies for the treatment of cancers associated with WT1 expression. 
     Example 31 
     The In Vivo Immunogenicity of the WT1 Antigen 
     This example describes three in vivo immunogenicity studies to evaluate vaccination strategies with WT1 in mice. The three strategies comprised: 1) a naked DNA vaccine prime and boost; 2) an attenuated adenovirus prime followed by an attenuated alphavirus boost; or 3) a naked DNA prime followed by an adenovirus boost. The full-length cDNA of the splice variant of WT1 used in these studies is set forth in SEQ ID NO:381. The results described herein provide support for the use of WT1 DNA/DNA, DNA/adenovirus or adenovirus/alphavirus prime/boost regimens as vaccine strategies for treating cancers associated with WT1 expression. 
     In the first study, C57/BI6 mice were immunized 3 times at 2 week intervals with 100 μg of naked DNA encoding for WT1. Mice were sacrificed 2-3 weeks after the final immunization and CTL were evaluated by standard Chromium release assay. This first study showed that WT1 DNA immunization elicits WT1-specific cytotoxic T cell responses in these mice with a 25:1 E:T ratio showing 40% lysis. 
     In the second study, HLA-A2/Kb transgenic mice were immunized once with 5×10 8  PFU of attenuated adenovirus encoding WT1 (as described in Example 20) followed 4 weeks later by one boost with 5×10 6  PFU of alphavirus (AlphaVax) encoding WT1. Mice were sacrificed 2-3 weeks after the final immunization and CTL were evaluated by standard Chromium release assay. The results showed that WT1-specific CTL in HLA-A2/Kb transgenic mice specifically lysed dendritic cells (DC) transduced with WT1-expressing viral construct as well as DC pulsed with WT1 peptides. Thus, this immunization strategy also effectively elicits WT1-specific CTL in vivo. 
     In the third study, C57/BI6 and HLA-A2/Kb transgenic mice were immunized twice with 100 μg of naked WT1 DNA 2 weeks apart followed 3 weeks later by a boost with 7×10 8  PFU adenovirus encoding WT1. Mice were sacrificed 2-3 weeks after the final immunization and CTL were evaluated by IFN-γ ELISPOT assay. The results showed that the WT1 DNA and adenovirus prime-boost generates a WT1-specific CD8 T cell response in HLA-A2/Kb transgenic mice. About 42% of CD8 positive cells stained positive for IFN-γ following a 7 day stimulation with DCs transduced with WT-1. The results from the C57/BL6 mice showed that this immunization strategy generates CD8 responses detectable in fresh splenocytes. Splenocytes were stimulated for 6 hours with pools of 10 15-mer peptides overlapping by 11 amino acids that span the entire WT1 protein. Only cells stimulated with the p121-171 showed IFN-γ staining. About 1.1% of those CD8 T cells stimulated with the p121-171 peptide pool stained positive for IFN-γ. This peptide contains the p117-139 peptide (SEQ ID NO:2) shown in Example 3 to elicit CTL, T helper cell and antibody responses in mice. 
     In summary, these results show that the three immunization strategies tested herein generate T cell responses in vivo. Thus, these studies further confirm the immunogenicity of the WT1 protein and provide support for the use of WT1 DNA/DNA, DNA/adenovirus or adenovirus/alphavirus prime/boost regimens as vaccine strategies for treating cancers associated with WT1 expression. 
     Example 32 
     Reduction in WT1+Tumor Growth in HLA-A2/kb Transgenic Mice Immunized with WT1 Protein 
     This example describes the reduction of WT1+ tumors in transgenic mice immunized with a WT1 vaccine. These results further validate WT1 as a vaccine target and provide support for the use of WT1 in vaccine strategies for treating cancers associated with WT1 expression. 
     The murine dendritic cell (DC) line DC2.4. was stably transduced with a WT1-LAMP construct (see Example 18, cDNA and protein sequences set forth in SEQ ID NO:382 and 409, respectively). Mice were then inoculated either subcutaneously (s.c.) or intraperitoneally (i.p.) with 2×10 6  cells. This resulted in tumor growth in 80-100% of the mice. The tumors established in mice in vivo retained their WT1 expression. Thus, this model provides a system in which to validate the efficacy of WT1 vaccine strategies. 
     Three groups of A2/Kb mice were then immunized 3 times, 2 weeks apart as follows: 
     Group 1: saline alone s.c. (control, n=10 mice) 
     Group 2: MPL®-SE 10 μg alone s.c. (control, n=10 mice) 
     Group 3: Raa12/WT1 protein 100 μg+10 μg MPL®-SE s.c. (n=9 mice) 
     Two to three weeks after the last WT1 immunization, mice were inoculated with 2×10 6  A2/Kb DC2.4 tumor cells overexpressing WT1. After tumor challenge mice were monitored and tumor size measured twice per week up to 4 weeks after tumor challenge. 
     The results showed that the percentage of mice with tumor growth in the group that received the WT1 protein vaccine was reduced from about 100% (saline control) or 90% (MPL®-SE adjuvant control) to 45% (WT1 protein immunized group). Further, the average tumor volume was reduced in this group from an average tumor size of 1233 cmm (saline control) or 753 cmm (MPL®-SE adjuvant control) observed in the control group to 226 cmm in the WT1 protein immunized group. Histopathological analyses showed that tumor margins in vaccinated animals were mixed with host immunological reactions including histiocytes, eosinophils, lymphocytes, mast cells and plasmacytes. Taken together, the results demonstrate that WT1 protein immunization protects against or delays the growth of WT1-positive tumors in the animals immunized with WT1. Thus, these results support the use of WT1 protein as a vaccine for malignancies associated with WT1 expression. 
     Example 33 
     Identification of a Naturally Processed WT1 Cytotoxic T Cell Epitope 
     This example describes the identification of a naturally processed epitope of the WT1 protein recognized by cytotoxic T cells. This experiment further confirms the immunogenicity of the WT1 protein and provides support for its use as a target for vaccine and/or other immunotherapeutic approaches. Additionally, this experiment identifies epitopes of the WT1 protein that may be used in these applications. 
     HLA-A2/Kb transgenic mice were immunized twice with 100 ug of naked WT1 DNA 2 weeks apart followed 3 weeks later by a boost with 10 7  PFU adenovirus encoding WT1. Mice were sacrificed 2-3 weeks after the final immunization and CTL were evaluated by standard chromium release assay. As observed in previous experiments, immunization with WT1 DNA followed by adenoviral boost elicited a WT1-specific CTL response in HLA-A2 transgenic mice. In order to identify which epitopes were recognized by the T cells, CTL lines were generated and cloned by limiting dilution using standard protocols. A positive clone was then tested using as target cells DC2.4 A2/Kb cells pulsed with peptides corresponding to the top 20 predicted HLA-A2 restricted CTL epitopes. The results showed that the WT1 p10-18 9mer peptide (amino acids: ALLPAVPSL, set forth in SEQ ID NO:451) was recognized by this CTL clone. This epitope was previously predicted to be an epitope, as described in Table XLVI, SEQ ID NO:34. In an additional experiment, CTL responses to the p10 peptide were observed in 4 of 5 WT1 immunized animals tested. Thus, this experiment demonstrates that the predicted p10-18 WT1 epitope is naturally processed and recognized by CTLs. Moreover, this experiment confirms the immunogenicity of the WT1 protein and further defines a naturally processed HLA-A2-restricted CTL epitope that can be used in vaccine and immunotherapeutic strategies for the treatment of malignancies associated with WT1 overexpression. 
     Example 34 
     WT1 Expression Constructs Using Twin Arginine Translocator (TAT) Signal Peptide 
     This example describes the construction of WT1-TAT vectors and expression of WT1-TAT from these vectors. These constructs have utility in the expression of WT1-TAT molecules for the use in vaccination strategies. 
     WT-1-F (a.a. 2-281 of the WT1 protein; cDNA and amino acid sequence of 2-281 of WT1 are set forth in SEQ ID NOs:460 and 461, respectively) and full-length WT-1 were constructed as pTAT fusions with no His tag as described below. The cDNA sequence of the resulting fusion contructs pTAT-WT1F and the pTAT-WT1 full-length are set forth in SEQ ID NOs 452 and 453 respectively. The amino acid sequences of the resulting fusions are set forth in SEQ ID NOs:455 and 454, respectively. 
     The WT-1-F open reading frame was PCR amplified with the following primers: 
     
       
         
           
               
               
            
               
                   
                 PDM-439 (SEQ ID NO: 456): 
               
               
                   
                 5′ GGCTCCGACGTGCGGGACCTGAAC 3′ Tm 66° C. 
               
               
                   
                   
               
               
                   
                 PDM-779 (SEQ ID NO: 457): 
               
               
                   
                 5′ CGCGTGAATTCATCACTGAATGCCTCTGAAG 3′ Tm 63° C. 
               
            
           
         
       
     
     The WT-1 full-length open reading frame was amplified with the following primers: 
     
       
         
           
               
               
            
               
                   
                 p37 (SEQ ID NO: 458): 
               
               
                   
                 5′ GGCTCCGACGTGCGGGACCTG 3′ 
               
               
                   
                   
               
               
                   
                 p23 (SEQ ID NO: 459): 
               
               
                   
                 5′ GAATTCTCAAAGCGCCAGCTGGAGTTTGGT 3′ 
               
            
           
         
       
     
     The PCR conditions were as follows: 1 μl 10×Pfu buffer, 1 μl 10 mM dNTPs 2 μl 1 μM each oligo 83 μl sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.) 50 ng DNA (pPDM FL WT-1). The reaction was denatured at 96° C. for 2 minutes followed by 40 cycles of 96° C. for 20 seconds, 64° C. for 15 seconds, and 72° C. for 2 minutes, 30 seconds and a single, final extension of 4 minutes at 72° C. 
     The PCR products were digested with EcoRI and cloned into pTAT (a modified pET28 vector with a Twin Arginine Translocation (TAT) signal peptide from the TorA signal peptide in  E. coli  on the N-terminus; see J. Mol. Microbiol. (2000) 2(2): 179-189; Journal of Bacteriology, January 2001 p604-610 Vol 183, No 2; Journal of Biochemistry Vol 276, Mar. 16, 2001 pp 8159-8164) at the Eco72I and EcoRI sites. The sequences were confirmed through sequence analysis and then transformed into BLR (DE3) pLys S and HMS174 (DE3) pLysS cells. Expression of the WT1-TAT proteins was confirmed by Western analysis. 
     Example 35 
     The N-Terminus of WT1 is the Dominant In Vivo Immunogenic Portion of the Protein 
     In this Example, mice were immunized with different protein constructs of WT-1, (F truncation (2-281) and full length (2-430) as described in Example 34)) formulated with MPL®-SE adjuvant. Improved CD4 responses were elicited by the truncated constructs relative to the full length protein. Thus, this example demonstrates that the N-terminal portion of the WT1 protein spanning from amino acid 2 to 281 is the dominant immunogenic portion of the WT1 protein in vivo. 
     Groups of four C57BL/6 mice were immunized subcutaneously with 20 μg WT-1 proteins: WT-1-F or WT-1 full length (FL), with Ra12, HIS or TAT fusions. Immunizations were performed at weeks 0, 3 and 9, and spleens were harvested at week 11. Splenocytes were then stimulated in vitro for 6 hours with medium alone, with a 15-mer peptide “p32” (ARMFPNAPYLPSCLE, amino acids 125-139 of WT-1; found within the p117-139 peptide set forth in SEQ ID NO:2), with the DC2.4-WT-1/LAMP cell line, or with rRa12. CD4 cells were then stained for intracellular interferon-gamma and quantified by FACS analysis. A portion of these splenocytes were then stimulated in vitro for 8 days, after which CD4+ IFN+ cells were enumerated. After the 6 hour stimulation with p32, 0.33% of CD4-positive cells were positive for intracellular IFN-gamma staining in mice immunized with the truncated N-terminal construct rWT1-F-TAT. By contrast, only 0.10% of CD4-positive cells stained positive for intracellular IFN-gamma in mice immunized with rWT1-FL-TAT. After the 8 day stimulation, mice immunized with the rWT1-F-TAT construct showed IFN-gamma staining in 10.72% of the CD4+ cells. By contrast, 0.24% of CD4-positive cells from mice immunized with the full-length WT1-TAT construct stained positive for intracellular IFN-gamma. These data indicate that improved CD4 responses were elicited by the truncated rWT1-TAT construct relative to the full-length rWT1-TAT construct. 
     In a second assay splenocytes were stimulated in vitro with the 23-mer peptide, p117-139 (SEQ ID NO:2; PSQASSGQARMFPNAPYLPSCLE, containing a known CD4 epitope and encompassing “p32”), for 3 days, after which supernatants were assayed for secreted IFN-gamma by ELISA. There was no detectable IFN-gamma secretion from splenocytes from mice immunized with the full-length WT1 constructs. By contrast, an average of 2477 pg/ml IFN-gamma was detected from splenocytes from mice immunized with rWT1-F without a HIS tag. An average of 4658 pg/ml IFN-gamma was detected from splenocytes from mice immunized with rWT1-F-TAT. These data further support the observation that improved CD4 responses were elicited by the truncated N-terminal WT1 constructs relative to the full length protein. 
     The WT1 protein is a transcription factor which is composed of two functional domains: a proline-glutamine rich domain at the N-terminus, and a zinc finger domain composed of four zinc fingers at the C-terminus with homology to the EGR1/Sp1 family of transcription factors. WT1 is a self-protein. The C-terminus is homologous to other self-proteins and is thus less immunogenic, i.e. the subject of a greater degree of immunological tolerance. Of note, the 4 zinc-finger domains within the C-terminus have homology to EGR family members. The results described in this example indicate that tolerance will vary between different portions of a protein, possibly depending on sequence homologies and functional domains. 
     In summary, the data described in this example support the notion that the most efficient WT1 vaccine will comprise the WT1 N-terminus, either as a recombinant protein or gene-based construct. 
     Example 36 
     Baculovirus Expression Constructs for Expression of the N-Terminal Fragment of WT1 (WT-1-F: Amino Acids 1-281) and Large Scale Production of Protein Using Insect Cells 
     The cDNA for the N-terminal fragment of WT-1, together with a Kozak consensus sequence, were obtained by PCR using the WT1-F plasmid as a template (WT-1-F: amino acids 2-281 of the WT1 protein cloned downstream of a start methionine; cDNA and amino acid sequence of 2-281 of WT1 are set forth in SEQ ID NOs:460 and 461, respectively. The pTAT fusion construct used as template in the experiments described herein is described in Example 34. The cDNA of this construct is set forth in SEQ ID NO:452). The following primers were used for amplification: 
     
       
         
           
               
               
            
               
                   
                 WT1F1 (SEQ ID NO: 466): 
               
               
                   
                 5′ CGGCTCTAGAGCCGCCACCATGGGCTCCGACGTGCG 
               
               
                   
                   
               
               
                   
                 WT1RV4 (SEQ ID NO: 467): 
               
               
                   
                 5′ CGGCTCTAGACTACTGAATGCCTCTGAAGACACCGTG 
               
            
           
         
       
     
     The cDNA for the same ORF plus a C-terminal 10 residue His Tag was obtained by PCR similarly as above except using WT1 RV3 (SEQ ID NO:468) as reverse primer (5′ CGGCTCTAGACTAATGGTGATGGTGATGATGATGGTGATGATGCTGAATGCC TCTGAAGACACCGTG). 
     The purified PCR products were cloned into the Xba I site of the donor plasmid, pFastBac1. The recombinant donor plasmid pFBWT1-F (cDNA and amino acid sequences set forth in SEQ ID NOs:463 and 465, respectively) and pFBWT1-FH (with the 10×His Tag; cDNA and amino acid sequence set forth in SEQ ID NOs:462 and 464, respectively) were transformed into  E. coli  strain DH10Bac (Invitrogen, Carlsbad, Calif.) to make recombinant bacmids in  E. coli  through site-specific transposition. The recombinant bacmids were confirmed by PCR analysis and then transfected into Sf-9 insect cells to make recombinant baculoviruses BVWT1-F and BVWT1-FH. The recombinant viruses were amplified to high titer viral stock in Sf-9 cells. 
     The HIGH FIVE insect cell line was used to optimize conditions for the protein expression and for the large-scale production of the recombinant proteins. To optimize the conditions for protein expression, High 5 insect cell monolayers were infected with the recombinant baculoviruses BVWT1-F and BVWT1-FH at different multiplicities of infection (MOI) and harvested the transduced cells at different periods of time. The identities of the proteins were confirmed by Western blot analysis with a rabbit anti-WT1 polyclonal antibody [#942-32 (799L)]. Both WT1-F and WT1-FH recombinant proteins were well expressed at either 48 hours or 54 hours post-infection when HIGH FIVE cells were infected by the recombinant viruses at MOI 1.0 or 2.0. 
     The amplification of the recombinant baculoviruses and the expression of the recombinant WT1-F and WT1-FH proteins was then further optimized. Both BVWT1-F and BVWT1-FH were amplified in the Sf9 insect cell line in ESF921 medium containing 2% fatal calf serum and 0.5×PSF. The amplification was carried out for 4 days at MOI 0.05 at 28° C. The HIGH FIVE insect cell line was used to optimize conditions for the protein expression. High 5 insect cells were infected by the recombinant baculoviruses at MOIs of 0.5, 1 or 2 for 30, 48, 54, or 72 hours before harvesting. The identity of the recombinant protein was confirmed by Western blot analysis with a specific rabbit polyclonal antibody against WT1 (Antibody #942-32), and by capillary LC-ESI-tandem mass spectrometry. These experiments indicate that optimal protein expression for large scale production of WT1-F and WT1-FH occurs at 43 hours post-infection when HIGH FIVE cells were infected by the recombinant viruses at an MOI 0.5-1.0. 
     In summary, the above WT1 baculovirus can be used for large-scale protein production of the N-terminal portion of WT1 for use in a variety of vaccine strategies for the treatment of malignancies associated with WT1 expression. 
     Example 37 
     Induction of In Vivo CD4 and CD8 T Cell Responses in Mice Using Recombinant Tricom Vaccinia and Fowl Pox Vectors 
     This example describes in vivo immunogenicity studies to evaluate vaccination strategies with WT1 in mice. The purpose of these experiments was to test the ability to rV-WT1/TRICOM® and rF-WT1/TRICOM® to induce immunity, in particular T cell immunity, to WT1. The results described herein provide support for the use of TRICOM® vaccinia and fowlpox vectors expressing WT1 and containing a triad of costimulatory molecules (B7-1, ICAM-1 and LFA-3) in vaccine strategies for treating cancers associated with WT1 expression. 
     In the first study, C57BI/6 mice (12 mice per group) were immunized two or three times with 14 days between the primary, secondary and tertiary immunizations as shown below in Table 1. Mice were harvested at 21 days following the secondary and tertiary immunizations. CD8 and CD4 T cell responses were assayed by IFN-γ intracellular cytokine staining of WT1-peptide activated spleen cells as described in further detail below. CD4 T cell responses were additionally assayed by IFN-γ release from rWT1 protein stimulated spleen cells. Serum IgG 1  and IgG 2b  antibody responses were assayed by ELISA. T cell responses were evaluated using pooled splenocyte cultures (4 mice/group/time point). Antibody titers were determined for individual mice (4 mice/group/time point). 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Vaccination Strategy 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 Groups: 
               
               
                   
                 A. Non-immune 
               
               
                   
                 B. rWT1 + SE-Vehicle 
               
               
                   
                 C. rWT1 + MPL ®-SE 
               
               
                   
                 D. 1°: WT1-DNA, 2°: WT1-DNA, 3°: WT1-Adeno 
               
               
                   
                 E. rF-WT1/TRICOM ® 
               
               
                   
                 F. 1°: rV-WT1/TRICOM ®, 2°: rF-WT1/TRICOM ®, 3°: rF- 
               
               
                   
                 WT1/TRICOM ® 
               
               
                   
                 Dose: 
               
               
                   
                 rWT1: 50 ug 
               
               
                   
                 MPL ®-SE: 10 ug 
               
               
                   
                 WT1-DNA: 100 ug 
               
               
                   
                 WT1-Adeno: 5 × 10 5  pfu 
               
               
                   
                 rF-WT1/TRICOM ®: 1 × 10 8  pfu 
               
               
                   
                 rV-WT1/TRICOM ®: 1 × 10 8  pfu 
               
               
                   
                 Route: 
               
               
                   
                 Subcutaneous (200 ul) immunization for 
               
               
                   
                 protein/adjuvant and vectors. 
               
               
                   
                 Intramuscular immunization (50 ul) for WT1-DNA. 
               
               
                   
                   
               
            
           
         
       
     
     The full-length cDNA of the splice variant of WT1 used in these studies is set forth in SEQ ID NO:381. The WT1-adenovirus used herein is as described in Example 20. The rF-WT1/TRICOM® recombinant fowlpox and the rV-WT1/TRICOM® recombinant vaccinia vectors both expressing WT1 and containing a triad of costimulatory molecules (B7-1, ICAM-1 and LFA-3) were generated by Therion Biologics (Cambridge, Mass., USA). 
     To evaluate T cell responses, splenocytes were stimulated in vitro with WT1 peptide “p32” (ARMFPNAPYLPSCLE [SEQ ID NO:509], amino acids 125-139 of WT-1; found within the p117-139 peptide set forth in SEQ ID NO:2) known to contain a CTL and a helper T cell epitope. Intracellular cytokine staining for IFN-γ of p125-139 activated splenocytes at 21 days following the secondary immunization showed a significant percentage WT1 responsive CD4 +  and CD8 +  T cells in mice immunized with rV-WT1/TRICOM® (prime) and rF-WT1/TRICOM® (boost) whereas no other groups showed significant WT1 specific T cell immune responses. Note that the DNA group was primed and boosted with DNA only. The same group subsequently had an rWT1-Adv tertiary immunization. 
     Following tertiary immunization, responses to pools of overlapping 15-mer peptides were evaluated rather than responses to the single peptide #32. WT1 peptide CD8 +  and CD4 +  T-cells specific for peptide pool #32-36 were found to respond at levels similar to the responses following secondary immunization in mice vaccinated with rV-WT1/TRICOM® (prime) and rF-WT1/TRICOM® (boost X2). In addition, CD4 +  and CD8 +  T-cells from these mice were found to respond, albeit at a much lower level than peptide #32, to a second WT1 epitope contained within two overlapping 15-mer peptides #57-58 (Peptide 57: DNLYQMTSQLECMTWN (amino acids 224-239; SEQ ID NO:510); Peptide 58: MTSQLECMTWNQMNL (amino acids 229-243; SEQ ID NO:511; overlap corresponds to amino acid residues 224-243 of WT1). 
     Antibody responses were evaluated using a standard ELISA. Low levels of serum IgG 2b  antibodies to WT1 were measurable in all 8 mice 21 days post secondary immunization (titer of 1:1350) and 21 days post tertiary immunization (titer of 1:3325) in mice immunized with rWT1+MPL®-SE. By contrast, in all other groups serum IgG 2b  antibodies titers were &lt;1:100 (Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Antibody Responses in WT1-Immunized Mice 
               
            
           
           
               
               
            
               
                   
                 Serum Titers* 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Secondary 
                   
                 Tertiary 
                   
               
               
                 Group 
                 IgG 1   
                 IgG 2b   
                 IgG 1   
                 IgG 2b   
               
               
                   
               
               
                 Non-immune 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 WT1 + Vehicle-SE 
                 &lt;75 
                 &lt;100 
                 475 
                  &lt;50 
               
               
                 WT1 + MPL ®-SE 
                 &lt;50 
                 1350 
                 338 
                 3325 
               
               
                 WT1-DNA, WT1-DNA, 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 WT1-Adeno 
               
               
                 rF-WT1/TRICOM ® 
                 ND 
                 ND 
                 &lt;50 
                  &lt;50 
               
               
                 rV-WT/TRICOM ®, 
                 ND 
                  &lt;50 
                 &lt;50 
                 ND 
               
               
                 rFWT1/TRICOM ® 
               
               
                   
               
               
                 *Serum titers are the average of 4 mice per group. Titers written as &lt;50, &lt;75, or &lt;100 were also averages, but in all instances less than 4 mice had detectable titers. 
               
               
                 ND indicates antibody was not detected in any of the mice. 
               
            
           
         
       
     
     In summary, immunization of C57BI/6 mice with rV-WT1/TRICOM® (prime) followed by rF-WT1/TRICOM® (boost) or by WT1-DNA (prime) followed by WT1-Adeno (boost) elicited both CD8 and CD4 T cell responses against WT1. The T cell responses to rV-WT1/TRICOM® followed by rF-WT1/TRICOM® versus WT1-DNA followed by WT1-Adeno were equivalent within the power of the experimental design. Without being bound by theory, a major advantage of rF-WT1/TRICOM® is that multiple boosts can continue to increase the level of immunity. Thus, these studies further confirm the immunogenicity of the WT1 protein and provide support for the use of WT1 DNA/adenovirus or rv-WT1/TRICOM®/rF-WT1/TRICOM® immunization regimens in vaccine strategies for treating cancers associated with WT1 expression. 
     Example 38 
     Construction of the Stumpy-WT1-F Vector for Expression of WT1-F in  E. Coli    
     This example describes the construction of an expression vector containing a truncated twin arginine translocator (TAT) signal peptide fused to the WT1-F reading frame (2-281 N-terminal portion of the WT1 protein). This vector can be used to produce a single species truncated TAT-WT1-F protein for use in immunization strategies for the treatment of malignancies associated with expression of WT1. 
     As described previously in Example 34, the TAT signal sequence was used to make various WT1 vectors. When these TAT vectors were used in expression, multiple forms of the protein were observed. N-terminal sequencing of these forms showed that each of the three separate proteins being expressed were truncations of the TAT peptide. These cleavages were occurring at each of the twin arginine sites. Thus, a truncated TAT vector was constructed to shorten the TAT signal peptide from 39 amino acids to 12 amino acids to avoid generation of these cleavage products during expression. The TAT “Stumpy” vector was generated by maintaining the first 12 residues of the TAT signal peptide up to the first twin arginine. This vector was constructed as follows: 
     The following pairs of oligonucleotides were combined and then annealed. 
     
       
         
           
               
               
            
               
                   
                 Pair 1: 
               
               
                   
                 Stumpy 1 (SEQ ID NO: 486): 
               
               
                   
                 5′ tatgaacaataacgatctgtttcaggc 3′ 
               
               
                   
                   
               
               
                   
                 Stumpy 3 (SEQ ID NO: 487): 
               
               
                   
                 5′ CTGAAACAGATCGTTATTGTTCA 3′ 
               
               
                   
                   
               
               
                   
                 Pair 2: 
               
               
                   
                 Stumpy 4 (SEQ ID NO: 488): 
               
               
                   
                 5′ aattcttggtcattcacgtggcggctcgc 3′ 
               
               
                   
                   
               
               
                   
                 Stumpy 2 (SEQ ID NO: 489): 
               
               
                   
                 5′ gagccgccacgtgaatgaccaag 3′ 
               
            
           
         
       
     
     The pairs were then ligated together with pPDM, a modified pET28 vector, that had been digested with NdeI and EcoRI. The resulting plasmid, pStumpy, is set forth in SEQ ID NO:471. The amino acid sequence of the truncated TAT protein is set forth in SEQ ID NO:506. The full-length TAT polynucleotide is set forth in SEQ ID NO:503 and encodes the amino acid sequence of the TAT polypeptide set forth in SEQ ID NO:504. 
     The pStumpy vector was then used to construct the Stumpy-WT1-F vector to express the N-terminal portion of WT1 without cleavage products. The Stumpy-WT1-F vector was constructed as follows: 
     The coding region of WT1-F was PCR amplified with the following primer set: 
     
       
         
           
               
               
            
               
                   
                 PDM-1005 (SEQ ID NO: 484): 
               
               
                   
                 5′ GGCTCCGACGTTCGGGACCTGAACGCACTG 3′ 
               
               
                   
                   
               
               
                   
                 PDM-1004 (SEQ ID NO: 485): 
               
               
                   
                 5′ CTGCAGAATTCATCACTGAATGCCTCTGAAG 3′ 
               
            
           
         
       
     
     The amplification reaction contained 10 ul 10×Pfu buffer, 1 ul 10 mM dNTPs, 2 ul each 10 uM primer, 83 ul sterile water, and 1.5 ul Pfu DNA polymerase (Stratagene, La Jolla, Calif.). The reaction was first denatured for 2 minutes at 96° C. followed by 40 cycles of 96° C. for 20 seconds, 63° C. for 15 seconds, and 72° C. for 30 seconds. The reaction was then extended for a final extension of 72° C. for 4 minutes. 
     The PCR product was then digested with EcoRI and cloned into the pStumpy vector that had been digested with Eco72I and EcoRI. The construct was confirmed through sequence analysis and then transformed into BLR (DE3) pLys S cells. The resulting truncated TAT WT1-F fusion protein expressed as a single species. The polynucleotide sequence of the coding region of pStumpy WT1-F is set forth in SEQ ID NO:469 which encodes the amino acid sequence set forth in SEQ ID NO:470. 
     Example 39 
     Construction of WT1-F GMP, WT1-G, and WT1-Delta G Vectors for Expression in  E. Coli    
     This example describes the construction of WT1-F, WT1-G, and WT1-Delta G vectors optimized for protein production in  E. coli.    
     The TAT WT-1 F DNA sequence was codon optimized and synthetically engineered by Blue Heron Biotechnology (Bothel, Wash.) to be optimized for expression in  E. coli . This codon optimized cDNA sequence of WT1-F is set forth in SEQ ID NOs:477 and encodes the amino acid sequence set forth in SEQ ID NO:479. The plasmid containing this sequence was then digested with NdeI and EcoRI and sub-cloned into a modified pET28 vector that had been digested with NdeI and EcoRI for expression in  E. coli . This construct was confirmed to be correct by DNA sequence analysis and then transformed into BLR (DE3) pLys S cells for expression. This construct is a purely synthetic traceable source of TAT WT1-F template DNA. 
     The codon optimized sequence was then used as template for PCR to delete a proline rich region of fourteen amino acids from the N-terminal region (deletion of amino acids 54-67 of WT-1 F sequence set forth in SEQ ID NO:461; corresponding to amino acids 55-68 of full-length WT1), creating the WT-1 G cDNA sequence set forth in SEQ ID NO:473 which encodes the amino acid sequence set forth in SEQ ID NO:478. This was done by first generating the pTAT-WT-1 G construct as follows: 
     The 5′ coding region of WT-1 G was PCR amplified using the following primer set: 
     
       
         
           
               
            
               
                 PDM-1007 
               
            
           
           
               
            
               
                 (SEQ ID NO: 490) 
               
            
           
           
               
            
               
                 5′ caagcgctcatgagcccgaagtggcgagccc 3′ Tm 76° C. 
               
               
                   
               
               
                 PDM-1006 
               
            
           
           
               
            
               
                 (SEQ ID NO: 491) 
               
            
           
           
               
            
               
                 5′ cataaatttgcccgggcccgcccagagagccg 3′ Tm 76° C. 
               
            
           
         
       
     
     The 3′ coding region of the WT-1 G region was PCR amplified using the following primer set: 
     
       
         
           
               
               
            
               
                   
                 PDM-1008 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 492) 
               
            
           
           
               
               
            
               
                   
                 5′ cattcattcatcaaacaggagcc 3′ Tm 61° C. 
               
               
                   
                   
               
               
                   
                 PDM-1009 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 493) 
               
            
           
           
               
               
            
               
                   
                 5′ ccattaagaattcatcactgaatacc 3′ Tm 60° C. 
               
            
           
         
       
     
     The PCR reaction for amplification of WT1-G contained 10 ul 10×Pfu buffer, 1 ul 10 mM dNTPs, 2 ul each 10 uM primer, 83 ul sterile water, 1.5 ul Pfu DNA polymerase (Stratagene, La Jolla, Calif.), and 50 ng DNA template (pTAT WT1-F codon optimized as set forth in SEQ ID NO: 477). The reaction was first denatured for 2 minutes at 96° C. followed by 40 cycles of 96° C. for 20 seconds, 63° C. for 15 seconds, and 72° C. for 30 seconds. The reaction was then extended for a final extension of 72° C. for 4 minutes. 
     The first PCR product was then digested with XbaI and cloned into pPDM (a modified pET28 vector) that had been digested with XbaI and Eco72I. The resulting construct (pTAT WT1-GA) was then digested with SrfI and EcoRI. The second PCR construct was digested with EcoRI and cloned into the pTAT WT1-GA plasmid construct. At the same time a three-way ligation was carried out by digesting the first PCR construct with XbaI and SrfI and digesting the second PCR construct with EcoRI and cloning into pPDM that had been digested with XbaI and EcoRI. The resulting pTAT-WT1-G construct was confirmed to be correct through sequence analysis and then transformed into an expression host. The cDNA of pTAT-WT1-G is set forth in SEQ ID NO:475 and encodes the amino acid sequence of SEQ ID NO:482. 
     Due to incomplete SrfI digestion during the second method described above, an unexpected form of WT-1 G (WT-1 Delta G was created that deleted 14 amino acids (amino acids 55-68 of WT-1 F) and added three additional amino acids (WT-1 Delta G polynucleotide sequence set forth in SEQ ID NO:505 which encodes the polypeptide sequence set forth in SEQ ID NO:502). This TAT WT-1 Delta G construct resulted in an unexpected 5-fold over expression as compared to the TAT WT-1 F constructs. The cDNA of the pTAT-WT1 Delta G construct is set forth in SEQ ID NO:476 and encodes the amino acid sequence set forth in SEQ ID NO:481. 
     Following generation of the pTAT-WT-1 G, pStumpy-WT1 G and WT1-G with and without a His tag were constructed using the pTAT-WT1 G as a template in the PCR reactions described below. 
     The coding region of the WT1-G region was PCR amplified using the following primer sets: 
     
       
         
           
               
            
               
                 PCR 1: 
               
               
                 PDM-1010 
               
            
           
           
               
            
               
                 (SEQ ID NO: 494) 
               
            
           
           
               
            
               
                 5′ ggttcggatgtacgcgatctgaacg 3′ Tm 68° C. 
               
               
                   
               
               
                 PDM-1011 
               
            
           
           
               
            
               
                 (SEQ ID NO: 495) 
               
            
           
           
               
            
               
                 5′ caaagaattcatcactgaataccgcg 3′ Tm 63° C. 
               
               
                   
               
               
                 PCR 2: 
               
               
                 PDM-1023 
               
            
           
           
               
            
               
                 (SEQ ID NO: 496) 
               
            
           
           
               
            
               
                 5′ gcttttggcatatgggttcggatgtacgcgatc 3′ Tm 71° C. 
               
               
                   
               
               
                 PDM-1011 
               
            
           
           
               
            
               
                 (SEQ ID NO: 497) 
               
            
           
           
               
            
               
                 5′ caaagaattcatcactgaataccgcg 3′ Tm 63° C. 
               
            
           
         
       
     
     The PCR reaction and amplification conditions were as described above using the pTAT-WT1 G set forth in SEQ ID NO:475 as template. The first PCR product was digested with EcoRI and cloned into pPDM (a modified pET28 vector with a his tag in-frame) and pSTUMPY (a modified pET28 vector with a 12 amino acid truncated TAT leader peptide in frame set forth in SEQ ID NO:471) that were digested with EcoRI and Eco72I. The second PCR product was digested with NdeI and EcoRI and cloned into a modified pET28 vector (pPDM) that had been digested with NdeI and EcoRI. The constructs were confirmed to be correct through sequence analysis and then transformed into an expression host. The resulting constructs are as follows: The polynucleotide of WT1-G with His tag is set forth in SEQ ID NO:472 and encodes the amino acid sequence set forth in SEQ ID NO:480. The polynucleotide of WT1-G without His tag is set forth in SEQ ID NO:473 which encodes the amino acid sequence set forth in SEQ ID NO:478. The pStumpy-WT1-G polynucleotide sequence is set forth in SEQ ID NO:474 and encodes the amino acid sequence set forth in SEQ ID NO:483. 
     In a related experiment, the pStumpy WT-1 F GMP (codon optimized) vector was constructed as follows: 
     The coding region of the WT-1 F region was amplified using the following primers 
     
       
         
           
               
               
            
               
                   
                 PDM-1010 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 500) 
               
            
           
           
               
               
            
               
                   
                 5′ ggttcggatgtacgcgatctgaacg 3′ Tm 68° C. 
               
               
                   
                   
               
               
                   
                 PDM-1011 
               
            
           
           
               
               
            
               
                   
                 (SEQ ID NO: 501) 
               
            
           
           
               
               
            
               
                   
                 5′ caaagaattcatcactgaataccgcg 3′ Tm 63° C. 
               
            
           
         
       
     
     The PCR reaction and amplification conditions were as described above using pTAT WT-1F GMP as template. The PCR product was digested with EcoRI and cloned into the pStumpy vector described in Example 38 that was digested with EcoRI and Eco72I. The construct was confirmed to be correct through sequence analysis and then transformed into an expression host. The polynucleotide sequence of pStumpy WT-1 F GMP is set forth in SEQ ID NO:498 which encodes the amino acid sequence of SEQ ID NO:499. 
     In summary, the expression vectors described herein can be used for optimal expression and production of WT1, truncated WT1, and WT1 fusion proteins for use in vaccine strategies for the treatment of malignancies associated with expression of WT1. 
     Example 40 
     Construction of an Adenovirus Vector Expressing Human WT1-F 
     WT1-F including a start codon (the DNA sequence of WT1-F including the start codon is set forth in SEQ ID NO:508) was cloned into an E1 and E3 deleted adenovirus serotype 5 vector. The resulting adenovirus vector is set forth in SEQ ID NO:507. The expression of WT1-F is controlled by the CMV promoter mediating high levels of WT1-F protein expression. Infection of human cells with this reagent leads to a high level of expression of the WT1-F protein. The antigenic nature of the adenoviral proteins introduced into the host cell during and produced at low levels subsequent to infection can act to increase immune surveillance and immune recognition of WT1 as an immunological target. This vector can be also used to generate immune responses against the WT1 protein when innoculated into mammalian subjects. If these subjects are positive for WT1 expressing tumor cells the immune response could have a therapeutic or curative effect on the course of the disease. 
     Example 41 
     CTL Responses in HLA-A2 Transgenic Mice WITH WT-1 Protein Plus Various Adjuvants 
     Groups of four HLA-A2/Kb transgenic mice were immunized intradermally three times at three week intervals with 20 μg of WT1-F truncation protein (see Example 35) admixed with one of the following adjuvants, 10 μg RC-529™-SE, 10 μg ENHANZYN®, or 10 μg RC-529™-AF+10 μg QuilA. A positive control group were immunized twice with WT1-F DNA followed by a boost with 10 7  PFU adenovirus encoding WT1-F (see example 40). Mice were sacrificed 2 weeks after the final immunization and CTL were measured. Spleens were harvested and stimulated in vitro with CD.24-A2 Kb-WT-1-LAMP cells (see Example 18) and assayed against Jurkat A2Kb cells expressing full length WT1-F or pulsed with WT-1 CD8 epitope peptides (p10-18 (ALLPAVPSL, SEQ ID NO:451), p32 (ARMFPNAPYLPSCLE [SEQ ID NO:509], aa 125-139 found within p117-139 SEQ ID NO:2, and WT1 p37-45 peptide (VLDFAPPGA, human WT1 residues 37-45; SEQ ID NO:241). A standard chromium release assay was used to assay for CTL activity. As observed in previous experiments, immunization with WT1 DNA followed by adenoviral boost elicited a WT1-specific CTL response in HLA-A2 transgenic mice. CTL responses were also detected from the spleens of mice immunized with WT1-F protein in combination with ENHANZYN®, RC-529™-SE and the combination of RC-529™-AF and QuilA. The magnitude of the responses induced was comparable to that seen in the DNA-Adenovirus immunization. 
     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.