CA2401070A1

CA2401070A1 - Compositions and methods for diagnosis and therapy of malignant mesothelioma

Info

Publication number: CA2401070A1
Application number: CA002401070A
Authority: CA
Inventors: Martin A. Cheever; Alexander Gaiger
Original assignee: Corixa Corp
Current assignee: Individual
Priority date: 2000-02-22
Filing date: 2001-02-22
Publication date: 2001-08-30
Also published as: JP2003524021A; NZ521430A; WO2001062920A3; AU2001247220A1; EP1261711A2; WO2001062920A2

Abstract

Disclosed are compositions and methods for the diagnosis and therapy of Wilm s' tumor antigen-associated cancers, and in particular, mesotheliomas. In particular embodiments, the invention provides methods, compositions and kit s for eliciting immune and T cell response to Wilms' tumor antigen polypeptide - derived antigenic fragments, and methods for the use of such compositions fo r diagnosis, detection, treatment, monitoring, and/or prevention of human malignant pleural mesothelioma.

Description

DESCRIPTION
COMPOSITIONS AND METHODS FOR DIAGNOSIS AND THERAPY
OF MALIGNANT MESOTHELIOMA
1. BACKGROUND OF THE INVENTION
The present application claims priority to United States Provisional Patent Applica-tion Serial No 60/184,070, filed February 22, 2000; the entire specification, claims and figures of which are incorporated herein by reference without disclaimer.
Portions of this research were conducted in part through funding from the United States Department of Health and Human Services under grant number SBIR R43 CA81752.
1.1 FIELD OF THE INVENTION
The present invention relates generally to the fields of cancer diagnosis and therapy.
More particularly, it concerns the surprising discovery of compositions and methods for the detection and immunotherapy of mesotheliomas, and particularly, malignant pleural mesothelioma. The invention provides new, effective method, compositions and kits for eliciting immune and T-cell response to Wilms' tumor antigen polypeptide-derived antigenic fragments, and methods for the use of such compositions for diagnosis, detection, treatment, monitoring, and/or prevention of human malignant pleural mesothelioma.
1.2 DESCRIPTION OF RELATED ART
1.2.1 WILMS' TUMOR ANTIGEN
The Wilms' tumor gene encodes a nuclear-expressed polypeptide designated WT1, which is possesses the structural features of a DNA binding transcription factor. WT1 has alternatively spliced variants, including a 429-amino acid polypeptide comprising four contiguous zinc finger domains at its carboxy terminus, and a glutamine/proline-rich region at its amino terminus, that mediates transcriptional suppression or activation in transient transfection assays.

A variety of diagnostic reagents for the detection of WT1 peptides exist, including rabbit polyclonal sera that specifically recognize large internal amino acid fragments of the wild type WTl polypeptide. Commercially available WTl polyclonal antibodies exist, but they have particular disadvantages including cross-reactivity with closely related proteins, and inconsistent results in antigen specificity and binding affinity studies, because of their nature as polyclonal sera. Such sera are therefore not particularly desirable for diagnostic uses, and are not useful for developing therapeutic reagents for in vivo inhibition of WTl polypeptide.
Commercially-available mouse monoclonal antibody have also been reported, however most are unsuitable for most therapeutic and diagnostic applications because they either (a) recognize only particular unique splice variant sequences (which are expressed in only a subpopulation of the alternatively-spliced WT1 mRNA); or (b) broadly cross-react with homologous, but functionally unrelated peptides, polypeptides, or proteins.
1 S 1.2.2 DETECTION OF WTl POLYPEPTIDES IN MALIGNANT MESOTHELIOMA
Malignant pleural mesothelioma is an increasingly common cancer, caused primarily by exposure to asbestos. The millions of workers who were exposed to asbestos dust prior to the implementation of asbestos regulation and improved control measures axe at risk for the disease. In addition, workers continue to be exposed to significant amounts of asbestos, when asbestos materials are disturbed during renovation, repair or demolition.
Asbestos-containing materials continue to be found in industrial, commercial and residential settings throughout the U. S., resulting in a sizeable population that remains at risk for malignant mesothelioma.
The prognosis for malignant mesothelioma is influenced by the stage of the disease.
Surgery, as well as adjuvant immunological treatments (e.g., interferon or interleukin) can be effective treatment, but only in the rare event of an early stage diagnosis.
1.3 DEFICIENCIES IN THE PRIOR ART
A major obstacle to contemporary cancer treatment is the problem of selectivity; that is, the ability to inhibit the multiplication of cancerous cells, while leaving unaffected the function of normal cells. Unfortunately, most mesothelioma patients are diagnosed only in -, advanced stages, where neither radiation, nor chemotherapy, nor multimodality treatments can significantly alter the poor prognosis. Moreover, the absence of a standard effective therapy for these patients makes long-term survival unlikely (Von Bultzingslowen, 1999;
Gemaro et al., 2000).
The poor survival rate for patients afflicted with malignant mesothelioma, however, could be greatly improved by diagnostic methods that provide more accurate and earlier detection, as well as improved therapies that selectively inhibit the hyperproliferating meothelioma cells. The need also exists for effective treatment regimens for mesotheliomas, and in particular, human malignant pleural mesothelioma, that circumvent the toxic side effects of existing therapies and provide more specific gene expression of the therapeutic constructs directly in the cancerous cells. Development of suitable treatment regimens for human malignant pleural mesothelioma would represent a significant advance for those of skill in the oncologic arts, and would facilitate improved diagnostic and therapeutic modalities for this aggressive cancer.
2. SUMMARY OF THE INVENTION
The present invention addresses the foregoing long-felt need and other deficiencies in the art by identifying new and effective strategies for the diagnosis, detection, prophylaxis, therapy, and immunomodulation of WT1-associated cancers, and in particular, malignant pleural mesothelioma. The present invention is based, in part, upon the surprising and unexpected discovery that immune and T cell responses to particular antigenic peptide fragments of the Wilms' tumor (WT) gene product (e.8., WT1) can provide particularly advantageous compositions and methods for the diagnosis, prophylaxis and/or therapy for an animal having, suspected of having, or at risk for developing one or more malignant diseases characterized by increased WT1 gene expression, and in particular, malignant pleural mesothelioma in a human. The WT1 gene was originally identified and isolated on the basis of a cytogenetic deletion at chromosome l 1p13 in patients with Wilms' tumor (U. S. Patent No. 5,350,840). The gene consists of 10 exons and encodes a zinc forger transcription factor, and sequences of mouse and human WTl polypeptides are provided in FIG. 1 (SEQ
ID
NO:319 and SEQ ID N0:320, respectively).

In a first embodiment, the invention provides a method of generating an immune or a T-cell response in an animal, and in particular in a mammal such as a human.
The method concerns in a general sense the administration of at least a first composition to the animal that comprises at least a first isolated peptide of from 9 to about 60 amino acids in length, or at least a first nucleic acid segment that encodes such a peptide, wherein the peptide comprises a first contiguous amino acid sequence according to any one of SEQ ID NO:I to SEQ ID
N0:4, SEQ ID NO:I3 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID N0:3I l, SEQ ID
N0:313, SEQ ID N0:314, SEQ ID N0:3I6 to SEQ ID N0:318, and SEQ ID N0:321 to SEQ ID N0:326, and more particularly, a contiguous amino acid sequence according to any one of SEQ ID N0:28 through SEQ ID N0:318, with peptides comprising one or more of the primary amino acid sequences disclosed in SEQ ID N0:2, SEQ ID N0:34, SEQ ID
N0:35, SEQ ID N0:49, SEQ ID N0:88, SEQ ID N0:144, SEQ ID N0:147, SEQ ID N0:185, SEQ
ID N0:198, SEQ ID NO:I99, SEQ ID N0:255, SEQ ID N0:282, SEQ ID N0:283, and SEQ
ID N0:293 being particularly preferred.
The invention encompasses peptides that may be of any intermediate length in the preferred ranges, such as for example, those peptides of about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, or even about 15 amino acids or so in length, as well as those peptides having intermediate lengths including all integers within these ranges (e.g., the peptides may be about 54, about 53, about 52, about 51, about 49, about 48, about 47, about 46, about 44, about 43, about 42, about 4I, about 39, about 38, about 27, or even about 36 or so amino acids in length, etc.). In particular embodiments, when smaller peptides are preferred, the length of the peptide may be 9, or about 10, or about 1 I, or about 12, or about 13, or about 14 or even about 15 or so amino acids in Length, so long as the peptide comprises at least a first contiguous amino acid sequence according to any one of SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:13, SEQ ID
N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, and SEQ ID N0:20, as well as any one of SEQ ID N0:28 to SEQ ID N0:311, SEQ ID
N0:3I3, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:318, SEQ ID N0:321, SEQ ID
N0:322, SEQ ID N0:323, SEQ ID N0:324, SEQ ID N0:325, and SEQ ID N0:326.
Likewise, when slightly longer peptides are preferred, the length of the peptide may be about 16, or about 17, or about 18, or about 19, or about 20, or about 21, or about 22, or about 23, or about 24, or even about 25 or so amino acids in length, so long as the peptide comprises at least a first contiguous amino acid sequence according to any one of SEQ ID
NO:l to SEQ
ID N0:4, SEQ ID NO:13 to SEQ ID N0:20, SEQ ID NO:28 to SEQ ID NO:31 l, SEQ ID
5 N0:313, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:318 and SEQ ID N0:321 to SEQ
ID N0:326. When intermediate-length antigenic peptides or antigen binding fragments are desired, the peptides may be on the order of about 26, or about 27, or about 28, or about 29, or about 30, or about 31, or about 32, or about 33, or about 34, or even about 35 Or SO am2210 acids in length, so long as they each comprise at least a first contiguous amino acid sequence IO according to any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID
N0:20, SEQ ID N0:28 to SEQ ID N0:311, SEQ ID NO:313, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:318, and SEQ ID N0:321 to SEQ ID N0:326.
These peptides comprise at least a first contiguous amino acid sequence according to any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID N0:20, SEQ ID
NO:28 to SEQ ID N0:318, and SEQ ID N0:321 to SEQ ID N0:326, but may also, optionally comprise at least a second, at least a third, or even at least a fourth or greater contiguous amino acid sequence according to any one of SEQ ID NO:1 to SEQ ID
N0:4, SEQ ID N0:13 to SEQ ID NO:20, SEQ ID NO:28 to SEQ ID NO:311, SEQ ID N0:313, SEQ ID NO:314, SEQ ID N0:316 to SEQ ID NO:318, and SEQ ID N0:321 to SEQ ID
NO:326. A single peptide may contain only one of the contiguous amino acid sequences disclosed herein, or alternatively, a single peptide may comprise a plurality of contiguous amino acid sequences according to any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ
ID
NO:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID NO:31I, SEQ ID N0:313, SEQ ID
N0:314, SEQ ID NO:316 to SEQ ID N0:318, and SEQ ID NO:321 to SEQ ID N0:326. In fact, the peptide may comprise a plurality of the same contiguous amino acid sequences, or they may comprise one or more different contiguous amino acid sequences disclosed in SEQ
ID NO:I to SEQ ID NO:4, SEQ ID N0:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID
N0:311, SEQ ID N0:313, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:318, and SEQ
ID NO:321 to SEQ ID N0:326. For example, a single peptide of from 9 to about 50 amino acids in length could comprise a single epitopic peptide disclosed herein, or could comprise 2, 3, 4, or even 5 distinct epitopic sequences as disclosed in any of SEQ ID
N0:1 to SEQ ID
N0:4, SEQ ID N0:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID N0:311, SEQ ID
N0:313, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:318, and SEQ ID N0:321 to SEQ ID N0:326. Alternatively, a single peptide of from 9 to about 50 amino acids in length could comprise 2, 3, 4, or even 5 identical epitopic sequences as disclosed in any one of SEQ
ID N0:1 to SEQ ID N0:4, SEQ ID NO:I3 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID
N0:311, SEQ ID N0:313, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:318, and SEQ
ID N0:321 to SEQ ID N0:326.
In one exemplary embodiment, the peptide composition comprises at least a first isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes such a peptide; wherein the peptide comprises at least a first contiguous amino acid sequence selected from the group consisting of SEQ ID N0:34, SEQ ID
N0:35, SEQ ID N0:49, SEQ ID N0:88, SEQ ID N0:144, SEQ ID N0:147, SEQ ID N0:185, SEQ
ID N0:198, SEQ ID N0:199, and SEQ ID N0:282.
Preferred peptides of the present invention likewise encompass those from 10 to about 60 amino acids in length, those from 11 to about 60 amino acids in length, those from 12 to about 60 amino acids in length, those from 13 to about 60 amino acids in length, as well as those from 14 to about 60 amino acids in length, and those from 15 to about 60 amino acids in length. Likewise, preferred peptides of the present invention encompass those from 16 to about 60 amino acids in length, and any and all lengths, and sub-ranges of lengths within the overall preferred range of peptides of from 9 to about 60 amino acids or so in length. In similar fashion, the invention also encompasses those peptides having a length of from 10 or 11 to about 55 or 60 amino acids in length, and those having a length of from 12 or 13 to about 45 or 50 amino acids in length, as well as those peptides having a length of from 14 or 15 to about 35 or 40 amino acids in length, those peptides having a length of from 16 or 17 to about 25 or 30 amino acids in length, and those peptides having a length of from 18 or 19 to about 20 or so amino acids in length, and so on, to include all sub-ranges within the overall range of from 9 to about 60 amino acids in length.
Throughout this disclosure, a phrase such as "a sequence as disclosed in SEQ
ID
NO:1 to SEQ ID N0:4" is intended to encompass any and all contiguous amino acid sequences disclosed by any of these sequence identifiers, and particularly, the peptide sequences disclosed in Table 2 through Table 49 of the present specification.
That is to say, "a sequence as disclosed in any of SEQ ID NO:l through SEQ ID N0:4" means a sequence that is disclosed in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, or SEQ ID N0:4.
S Likewise, "SEQ ID NOs:2S to 37" means any and alI such sequences as disclosed in SEQ ID
N0:2S, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID N0:3S, SEQ ID
N0:36, and SEQ ID N0:37, and so forth. In fact, the invention encompasses peptides and polynucleotides encoding them that comprise at least a first contiguous amino acid sequence as disclosed in any one of the sequences identified as SEQ ID NO:1 to SEQ ID
N0:4, SEQ
ID NO:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID NO:311, SEQ ID N0:313, SEQ ID
NO:314, SEQ ID NO:316 to SEQ ID NO:318, and SEQ ID N0:321 to SEQ ID N0:326.
The invention also encompasses polynucleotides that comprise at least a first sequence region that encodes one or more of the peptides or peptide variants as disclosed 1 S herein. Such polynucleotides may comprise a sequence region of 27 to about nucleotides in length, or a sequence region of 27 to about 2000 nucleotides in length, or a sequence region of 27 to about 1000 nucleotides in length, or a sequence region of 27 to about 900, or about 800, or about 700, or about 600, or about 500, or about 400, or about 300, or about 200, or even about 100 or so nucleotides in length.
As in the case of the peptides, the length of the sequence region that encodes the peptide may be of any intermediate length in these ranges, such as those polynucleotides that comprise at Ieast a first sequence region of from about 30 to about 7S0 nucleotides in length, those that comprise at least a first sequence region of from about 3S to about 6S0 nucleotides in length, and those that compxise at least a first sequence region of from about 40 to about 2S SSO, about 450, about 350, about 250, about 150, or even about S0 or so nucleotides in length. Such sequence regions may be on the order of about 27, or about 28, or about 29, or about 30, or about 31, or about 32, or about 33, or about 34, or even about 3S
or so nucleotides in length, so long as the sequence region encodes at least a first peptide that comprises at least a first contiguous amino acid sequence according to any one of SEQ ID
NO:1 to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID

N0:31 l, SEQ ID N0:313, SEQ ID N0:314, SEQ ID NO:316 to SEQ ID N0:318, and SEQ
ID N0:321 to SEQ ID N0:326. When intermediate-length antigenic peptides or antigen binding fragments are desired, the nucleic acids that encode them may be on the order of about 40, about 45, or about 50, or about 55, or about 60, or about 65, or about 70, or about 75, or about 80, or even about 85 or 90 or so nucleotides in length, so long as they each encode at least a first peptide that comprises at least a first contiguous amino acid sequence according to any one of SEQ ID NO:l to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID
NO:20, SEQ ID N0:28 to SEQ ID N0:311, SEQ ID NO:313, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:318, and SEQ ID NO:321 to SEQ ID N0:326. When polynucleotides are contemplated that comprise sequence regions encoding larger antigenic peptides or antigen-binding fragments, the nucleic acid sequence region encoding them will necessarily be longer in length. For example, a nucleic acid sequence region encoding a peptide or antigen binding fragment on the order of about 40 to 50 amino acids in length, will necessarily be at least from about 120 to about 150 or so nucleotides in length, given the fact that a triplet codon is required to encode a single amino acid.
Likewise, the polynucleotides comprising such sequence regions can be substantially larger than the coding region itself, particularly when the sequence region is operably linked to one or more promoters, or to one or more sequence regions that encode one or more signal sequences, and/or peptide fusion products. In those embodiments, the polynucleotide may be on the order of about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or even about 1500, 1600, 1700, 1800, 1900, or even 2000 or so nucleotides in length, even up to and including those sequences that are on the order of about 10,000 or so nucleotides in length. Such polynucleotides are particularly useful in the preparation of expression vectors, delivery vehicles, viral vectors, and transformed host cells that express the particular encoded peptides) and/or antigen-binding fragments) encoded by the sequence region comprised within the polynucleotide and/or genetic construct or expression element.
In another exemplary embodiment, the peptide comprises at least a first isolated peptide of from 9 to about 11 amino acids in length, or at least a first nucleic acid segment that encodes the peptide; wherein the peptide consists essentially of the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID N0:20, SEQ ID
N0:28 to SEQ ID N0:311, SEQ ID N0:313, SEQ ID NO:314, SEQ ID N0:316 to SEQ ID
N0:318, and SEQ ID NO:321 to SEQ ID N0:326.
Similarly, in another related embodiment, the peptide comprises at least a first isolated peptide of from 9 to about 10 or 11 or so amino acids in length, or at least a first nucleic acid segment that encodes the peptide; wherein the peptide consists of the amino acid sequence of any one of SEQ ID N0:13 to SEQ ID N0:20, SEQ ID NO:28 to SEQ ID
N0:311, SEQ ID N0:313, SEQ ID N0:314, and SEQ ID N0:316 to SEQ ID NO:318, and particularly wherein the peptide consists of the amino acid sequence of any one of SEQ ID
N0:34, SEQ ID NO:35, SEQ ID N0:49, SEQ ID N0:88, SEQ ID N0:144, SEQ ID NO:147, SEQ ID N0:185, SEQ ID N0:198, SEQ ID NO:199, and SEQ ID N0:282.
In addition to peptides and compositions that comprise a single peptide species, the invention also concerns compositions that comprise 2, 3, 4, or more peptide species and/or the polynucleotides that encode such peptides. Such pluralities of peptide and/or polynucleotide species are particularly desirable in the formulation of therapeutic agents that comprise pluralities of peptides having two or more different contiguous amino acid sequence as disclosed in the amino acid sequences of SEQ ID NO:l to SEQ ID
N0:4, SEQ
ID N0:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID N0:311, SEQ ID NO:313, SEQ ID
N0:314, SEQ ID NO:316 to SEQ ID NO:318, and SEQ ID N0:321 to SEQ ID N0:326, and/or a plurality of polynucleotides that encode such peptides. Irrespective of the source of the particular antigenic WT1-derived peptide and polynucleotide compounds, the invention particularly contemplates the use of one, two, three or four distinct peptides, polynucleotides or derivatives thereof, up to and including a plurality of such compounds.
This exemplifies the use of singular terminology throughout the entire application" wherein the terms "a" and "an" axe used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated or would be understood by one of ordinary skill in the art. The operable limits and parameters of combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present disclosure.

The additional peptides in such compositions may all be of approximately the same size and/or approximately the same primary amino acid sequence, or alternatively, the peptides may differ considerably in length and/or primary amino acid sequence.
Such compositions may further comprise one or more additional components, such as for example, 5 a pharmaceutically acceptable excipient, buffer, or reagent as described in detail hereinbelow.
Such compositions may also optionally further comprise at least a first immunostimulant or at least a first adjuvant as described herein. Such immunostimulants and adjuvants preferentially enhance a T-cell response in a human, and may preferably be selected from the group consisting of Montanide ISA50, Seppic Montanide ISA720, a cytokine, a microsphere, 10 a dimethyl dioctadecyl ammonium bromide adjuvant, AS-1, AS-2, Ribi Adjuvant, QS21, saponin, microfluidized Syntex adjuvant, MV, ddMV, an immune stimulating complex and an inactivated toxin. As described in more detail hereinbelow, and particularly in Section 4, the compositions may be formulated for diagnostic or therapeutic uses, including their incorporation into one or more diagnostic or therapeutic kits for clinical packaging and/or commercial resale, with those formulations suitable for administration to a mammal, such as a human, with parenteral, intravenous, intraperitoneal, subcutaneous, intranasal, transdermal, .
and oral routes being particularly preferred.
The compositions may further optionally comprise one or more detection reagents, one or more additional diagnostic reagents, one or more control reagents, and/or one or more therapeutic reagents. In the case of diagnostic reagents, the compositions may further optionally comprise one or more detectable labels that may be used in both i~c vitro and/or in vivo diagnostic and therapeutic methodologies. In the case of therapeutic compositions and formulations, the compositions of the invention may also further optionally comprise one or more additional anti-cancer, anti-mesothelioma or otherwise therapeutically-beneficial components as may be required in particular circumstances, and such like.
In another aspect, the invention also provides methods for inhibiting the development of malignant mesothelioma in a human patient, comprising administering to a human patient a pharmaceutical composition comprising: (a) a WT1 peptide that comprises an immunogenic portion of a native WTl 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 antibodies and/or T cell lines or clones is not substantially diminished;
and (b) a physiologically acceptable carrier or excipient. Within certain embodiments, the patient is afflicted with malignant mesothelioma. In other embodiments, the composition is administered prophylactically to a patient considered at risk for the development of malignant mesothelioma. The WT 1 peptide may, but need not, be present within a vaccine, which further comprises an immunostimulant, such as an adjuvant.
Within further aspects, methods are provided for inhibiting the development of malignant mesothelioma in a human patient, comprising administering to a human patient a pharmaceutical composition, comprising: (a) a polynucleotide encoding a WTI
peptide, wherein the peptide comprises an immunogenic portion of a native WTI 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 antibodies and/or T cell lines or clones is not substantially diminished; and (b) a pharmaceutically acceptable carrier or excipient.
Within certain embodiments, the patient is afflicted with malignant mesothelioma. In other I S embodiments, the composition is administered prophylactically to a patient considered at risk for the development of malignant mesothelioma. The WT1 polynucleotide may, but need not, be present within a vaccine, which further comprises an immunostimuIant, such as an adjuvant.
Methods are further provided for inhibiting the development of malignant mesothelioma in a human patient, comprising administering to a human patient a pharma-ceutical composition, comprising: (a) an antibody or antigen-binding fragment thereof that specifically binds to WT1; and (b) a pharmaceutically acceptable carrier or excipient. Within certain embodiments, the patient is afflicted with malignant mesothelioma. In other embodiments, the composition is administered prophylactically to a patient considered at risk for the development of malignant mesothelioma.
Within further aspects, methods are provided for inhibiting the development of malignant mesothelioma in a human patient, comprising administering to a human patient a pharmaceutical composition, comprising: (a) a T cell that specifically reacts with WTl; and (b) a pharmaceutically acceptable carrier or excipient. Within certain embodiments, the patient is afflicted with malignant mesothelioma. In other embodiments, the composition is administered prophylactically to a patient considered at risk fox the development of malignant mesothelioma.
Further methods for inhibiting the development of malignant mesothelioma in a human patient comprise administering to a human patient a pharmaceutical composition, comprising: (a) an antigen-presenting cell that expresses (i) a WT1 peptide that comprises an immunogenic portion of a native WTl or a variant thereof that differs in one or more substitutions, deletions, additions andlor insertions such that the abiwity of the variant to react with antigen-specific antibodies and/or T cell lines or clones is not substantially diminished;
and (b) a pharmaceutically acceptable carrier or excipient. Within certain embodiments, the patient is afflicted with malignant mesothelioma. In other embodiments, the composition is administered prophylactically to a patient considered at risk for the development of malignant mesothelioma. The antigen presenting cell may, but need not, be present within a vaccine, which further comprises an immunostimulant, such as an adjuvant.
Within other aspects, the present invention provides methods for inhibiting the development of malignant mesothelioma in a human patient, comprising administering to a human patient a preparation of stimulated and/or expanded T cells, wherein the T cells are stimulated and/or expanded by contact with a WT 1 peptide, a polynucleotide encoding a WT1 peptide and/or an antigen-presenting cell that expresses a WTl peptide.
The T cells may be present, for example, within bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood (e.g., obtained from a patient afflicted with malignant mesothelioma). The T cells may, but need not, be cloned prior to expansion.
Methods are further provided for inhibiting the development of malignant meso-thelioma in a patient, comprising the steps of (a) incubating CD4+ andlor CD8+
T cells isolated from a patient with one or more of: (i) a WTl peptide; (ii) a polynucleotide encoding a WT1 peptide; or (iii) an antigen-presenting cell that expresses a WT1 peptide; such that the T cells proliferate; and (b) administering to the patient an effective amount of the proliferated T cells.
Further methods for inhibiting the development of malignant mesothelioma in a patient, comprising the steps of: (a) incubating CD4''~ and/or CD8+ T cells isolated from a patient with orie or more of (i) a WTl peptide; (ii) a polynucleotide encoding a WT1 peptide; or (iii) an antigen-presenting cell that expresses a WT1 peptide;
such that the T cells proliferate; (b) cloning one or more cells that proliferated in the presence of WTl peptide;
and (c) administering to the patient an effective amount of the cloned T
cells.
Within other aspects, the present invention provides method for determining the presence or absence of malignant mesothelioma 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 peptide; (ii) a polynucleotide encoding a WT1 peptide; or (iii) an antigen-presenting cell that expresses a WT1 peptide; and (b) detecting the presence or absence of specific activation of the T cells. The step of detecting may comprise, for example, detecting the presence or absence of proliferation of the T cells or the generation of cytolytic activity.
The present invention further provides methods for determining the presence or absence of malignant mesothelioma in a patient, comprising the steps of: (a) incubating a biological sample obtained from a patient with one or more of: (i) a WT1 peptide; (ii) a polynucleotide encoding a WTl peptide; or (iii) an antigen-presenting cell that expresses a WTl peptide; 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 peptide and antibodies in the biological sample that specifically bind to the WT1 peptide. The step of detecting may comprise, for example, (a) incubating the immunocomplexes with a detection reagent that is capable of binding to the immuno-complexes, wherein the detection reagent comprises a reporter group, (b) removing unbound detection reagent, and (c) detecting the presence or absence of the reporter group.
Methods are further provided, within other aspects, for monitoring the effectiveness of an immunization or therapy for malignant mesothelioma in a patient, comprising the steps of: (a) incubating a first biological sample with one or more of: (i) a WT1 peptide; (ii) a polynucleotide encoding a WT1 peptide; or (iii) an antigen-presenting cell that expresses a WTl peptide, 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 immunocornplexes to form; (b) detecting immunocomplexes formed between the WT 1 peptide and antibodies in the biological sample that specifically bind to the WTl peptide; (c) repeating steps (a) and (b) using a second biological sample obtained from the patient following therapy or immunization; and (d) comparing the number of immunocomplexes detected in the first and second biological samples. The step of detecting may comprise, for example, (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.
Within further aspects, methods are provided for monitoring the effectiveness of an immunization or therapy for malignant mesothelioma in a patient, comprising the steps of (a) incubating a first biological sample with one or more of: (i) a WT1 peptide; (ii) a WT1 polynucleotide encoding a WT1 peptide; or (iii) an antigen-presenting cell that expresses a WT1 peptide; 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 in the biological sample; (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 biologi-cal 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.
Throughout the methods of the invention, an "effective inhibitory amount" is an amount of at least a first WT1 compound effective to inhibit, and preferably to significantly inhibit, mesothelioma in an animal afflicted with such a disorder. The effective inhibitory amounts are thus also amounts effective to inhibit, and preferably to significantly inhibit, a biological activity of native WT1 polypeptide. More preferably, the effective inhibitory amounts are amounts of WT1 compounds effective to inhibit, and preferably to significantly inhibit, the biological activity of native WT1 polypeptide in a human having or suspected of having malignant pleural mesothelioma. Any degree of inhibition is sufficient to satisfy the invention, although those of ordinary skill in the art will understand the inhibition levels that are sufficient to indicate preferred it2 vitro and in vivo inhibition.

"Inhibition" requires a "reproducible," i.e., consistently observed, inhibition in one or more of the foregoing parameters. A "significant inhibition" is a reproducible or consistently observed significant inhibition in one or more of the foregoing parameters, such as a reproducible inhibition of at least about SO%, about 55%, about 60%, about 65%, about 70%, 5 about 75%, about 80%, or about 85% in comparison to control levels, i.e., in the absence of the WT1 therapeutic composition. Although not required to practice the invention, inhibition levels of at Least about 90%, about 92%, about 94%, about 96%, or even about 98% or higher are by no means excluded.
Execution of one or more of the therapeutic methods disclosed herein gives rise to 10 effective therapies for preventing or treating malignant mesothelioma.
These methods, which typically comprise providing to an animal or patient having, suspected of having, or at risk for developing malignant mesothelioma, an amount of at least a first WT1 peptide, antibody, antigen presenting cell, T cell, antigen binding fragment, or polynucleotide effective to inhibit malignant mesothelioma within cells of the animal or patient, thereby 15 preventing or treating malignant mesothelioma.
The foregoing "prophylactically and therapeutically effective amounts" are thus encompassed within the terms "biologically effective amounts" and "effective inhibitory amounts" of WT 1 peptide, antibody, antigen presenting cell, T cell, antigen binding fragment, or polynucleotide compositions. All such "effective amounts" are amounts of the disclosed WT1 compounds effective to produce some, and preferably some significant, benefit upon administration to an animal or patient. The benefzts include reducing symptoms, severity andlor duration, as well as lessening the chance of transmission and other veterinary and clinical benefits.
The routes of administration that may be used in the present invention are virtually limitless, so long as an effective amount of at least a first WTl peptide, antibody, antigen resentin cell T cell anti en bindin fra 'ment or of nucleotide com osition can be p g > > g g g ~ p y p provided thereby. Exemplary means for therapeutic delivery of the disclosed compositions, including e.g., ingestion, inhalation, transdermal, parenteral administration, intranasal administration, subcutaneous injection, intravenous injection, continuous infusion, and the like are discussed in more detail hereinbelow.

All such compositions and methods of the invention may be combined for use with one or more other anti-cancer agents, such as at least a second, third, fourth or fifth, anti-mesothelioma agent or at Least a first, second, third or fourth anti-cancer therapeutic agent. A
plurality of distinct anti-cancer or anti-mesothelioma therapeutic agents may be administered to an animal or patient, up to and including the dose limiting toxicity of the combination.
The invention can thus be used to form synergistic combinations with other therapies and/or known agents, particularly those methods and agents that previously failed to achieve maximal effectiveness in vivo, perhaps due to dose-limiting toxicity and/or resistance.
In such combination therapies, the at Least a first WTl peptide, antibody, antigen presenting cell, T cell, antigen binding fragment, or polynucleotide, and at least a second anti-mesothelioma or anti-cancer therapeutic agent may be administered to the animal or patient substantially simultaneously, such as from a single pharmaceutical formulation or two distinct pharmaceutical formulations. Alternatively, the at least a first WT1 peptide, antibody, antigen presenting cell, T cell, antigen binding fragment, or polynucleotide, and at least a second anti-mesothelioma or anti-cancer therapeutic agent may be administered to the animal or patient sequentially, such as on alternate days.
In further embodiments, the invention provides a range of therapeutic kits.
Certain kits comprise a therapeutically effective amount of at least a frst WT1 peptide, antibody, antigen presenting cell, T cell, antigen binding fragment, or polynucleotide composition and instructions for administering the composition to an animal or subject having or at risk for developing mesothelioma, and in particular, malignant pleural mesothelioma.
Such kits may be combined with effective amounts of at least one diagnostic agent that detects a WT1 polypeptide or antibody, or at least one diagnostic agent that detects a mesothelioma cell; or with a therapeutically effective amount of at least one other anti-cancer, anti-mesothelioma or anti-WT1 polypeptide therapeutic agent.
Certain other therapeutic kits and uses of the compositions disclosed herein, may comprise an effective amount of at least a fixst WTI peptide, antibody, antigen presenting cell, T cell, antigen binding fragment, or polynucleotide and an effective amount of at least one diagnostic agent that detects detects a mesothelioma cell; or an effective amount of at least one, two, three, four or any number of other anti-cancer, anti-mesothelioma or anti-WT1 polypeptide therapeutic agents. Instructions may also be combined with these kits. Other biological agents or components may be included, such as those for making and using the drugs.
Exemplary diagnostic agents include molecular biological agents that detect at least a first WT1-encoding nucleic acid; at least a first WT1 peptide or polypeptide, at least a first antibody that detects at least a first WTl protein or peptide; and at least a first WTl protein or peptide that detects at least a first antibody that binds to a WT1 protein or peptide. The range of additional therapeutic agents will be known those of ordinary skill °in the art in light of the present disclosure, as exemplified by those described herein.
In such kits, the diagnostic agents are preferably disposed within a distinct container of the kit. The combined therapeutic agents, however, may be combined within a single container of the kit, i.e., in the same composition as the WT1 composition, such as in a "cocktail" or admixture. They may alternatively be maintained separately from the WT1 compound, in a distinct container.
The invention thus provides combination therapeutics comprising, in any pharmaceutically acceptable form, a therapeutically effective amount of a WTl compound in combination with a therapeutically effective amount of at least a second anti-WT1, anti-mesothelioma or anti-cancer therapeutic agent. Also provided are compositions for use in the manufacture of a medicament or medicinal cocktail, that comprise, in any pharmaceutically acceptable form, a therapeutically effective amount of at least a first WT1 composition.
Moreover, the invention provides compositions for use in the manufacture of a medicament or medicinal cocktail that comprise, in any pharmaceutically acceptable form, a first WTI
composition and a plurality of distinct anti-WTl, anti-mesothelioma or anti-cancer therapeutic agents. Combined uses and medicaments in which a WTl compound is one component of a therapeutic approach are also encompassed within the present invention.
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.

3. BRIEF DESCRIPTION OF THE DRAWINGS
The invention may be understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:
S FIG. 1 depicts a comparison of the mouse (MO) (SEQ ID N0:320) and human (HU) (SEQ ID N0:319) WT1 peptide sequences;
FIG. 2 depicts a histogram presenting the results of an ELISA assay to detect WTl-specific antibodies in malignant mesothelioma patients. WT180 and WTC19, as indicated, represent positive controls. D44 represents normal control serum, and the remaining samples were serum samples obtained from human patients afflicted with malignant mesothelioma;
FIG. 3A, FIG. 3B and FIG. 3C depict 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).
FIG. 4A and FIG. 48 show histograms illustrating the stimulation of pxolifexative 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 (B6lno antigen), B6 spleen cells pulsed with the peptides p6-22 (p6), p117-139 (p117), p244-262 (p244) (Vaccine A; FIG. 4A) or p287-301 (p287), p299-313 (p299), p421-43S (p421) (Vaccine B;
FIG. _4B) and spleen cells pulsed with an irrelevant control peptide (irrelevant peptide) at 2S ~,g/ml and were assayed after 96 hr for proliferation by (3H) thymidine incorporation.
2S 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);
FIG. 5A, FTG. 5B, FIG. SC, and FIG. SD 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 (B6lno antigen), B6 spleen cells pulsed with the peptides p6-22 (p6), p117-I39 (p117) or an irrelevant control peptide (irrelevant peptide) at 2S ~.glml and were assayed after 96 hr for proliferation by (3H) S 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);
FIG. 6A and FIG. 6B are graphs illustrating the elicitation of WTl peptide-specific CTL in mice immunized with WTl peptides. FIG. 6A illustrates the lysis of target cells by I O allogeneic cell lines and FIG. 6B 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 effectoraarget ratios. Results are provided for Lymphoma cells (LSTRA and E10), as well as EIO + p23S-243 (E10+P23S). E10 cells are also referred to herein as EL-4 cells;
FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D are graphs illustrating the elicitation of IS. WTl specific CTL, which kill WTl positive tumor cell lines but do not kill WTl negative cell lines, following vaccination of B6 mice with WT1 peptide P117. FIG. 7A
illustrates that T-cells of non-immunized B6 mice do not kill WTl positive tumor cell Lines. FIG. 7B
illustrates the lysis of the target cells by allogeneic cell lines. FIG. 7C
and FIG. 7D
demonstrate the lysis of WT1 positive tumor cell lines, as compared to WT1 negative cell 20 lines in two different studies. In addition, FIG. 7C and FIG. 7D show the Lysis of peptide-coated cell lines (WTl negative cell line E10 coated with the relevant WT1 peptide PI17).
In each case, the % lysis (as determined by standard chromium release assays) is shown at three indicated effectoraarget ratios. Results are provided for lymphoma cells (E10), prostate cancer cells (TRAMP-C), a transformed fibroblast cell line (BLIP-SV40), as well as 2S E10+p117;
FIG. 8A and FIG. 8B are histograms illustrating the ability of representative peptide PII7-139 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 p23S-243 or p117-139 were stimulated in vitro with the relevant peptide and tested for ability to Lyse 30 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. 8A shows the cytotoxic activity of the p235-243 specific T cell line against the WTl negative cell line EL-4 (EL-4, WTl negative); EL-4 pulsed with the relevant (used for immunization as well as for restimulation) peptide p235-5 (EL-4+p235); EL-4 pulsed with the irrelevant peptides p117-139 (EL-4+p117), pI26-I34 (EL-4+p126) or p130-138 (EL-4+p130) and the WT1 positive tumor cells BLK-SV40 (BLK-SV40, WTI positive) and TRAMP-C (TRAMP-C, WTl positive), as indicated.
FIG.
8B 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+pIl7) and EL-4 pulsed with the irrelevant 10 peptides p123-I3I (EL-4+p123), or p128-136 (EL-4+p128); BLK-SV40 and TRAMP-C, as indicated;
FIG. 9A and FIG. 9B are histograms illustrating the specificity of lysis of WTl 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.
IS FIG. 9A shows the cytotoxic activity of the p117-139 specific T Bell line against the WTl negative cell line EL-4 (EL-4, WTl negative); the WTl 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 p1 17-139 (TRAMP-C + p1 I7 cold target) without 5'Cr labeling and TRAMP-C cells incubated with EL-4 pulsed with an 20 irrelevant peptide without ''Cr labeling (TRAMP-C + irrelevant cold target), as indicated.
FIG. 9B shows the cytotoxic activity of the p117-139 specific T cell line against the WT1 negative cell line EL-4 (EL-4, WTl negative); the WTl positive tumor cell line (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;
FIG. 10A, FIG. 10B, and FIG. lOC are histograms depicting an evaluation of the nonapeptide CTL epitope within p117-I39. The p117-139 tumor specific CTL Iine was tested against peptides within aa117-139 containing or lacking an appropriate H-2b class I
binding motif and following restimulation with p126-134 or p130-I38. The bars represent the mean % specific lysis in chromium release assays performed in triplicate with an E:T

ratio of 25:1. FIG. 10A shows the cytotoxic activity of the p1 17-139 specific T cell Line against.the WTI negative cell line EL-4 (EL-4, WT1 negative) and EL-4 cells pulsed with the peptides p 117-13 9 (EL-4 + p 117), p 119-127 (EL-4 + p 119), p 120-128 (EL-4 + p 120), p123-131 (EL-4 + p123), p126-134 (EL-4 + p126), p128-136 (EL-4 + p128), and p130-138 (EL-4 + p130). FIG. lOS shows the cytotoxic activity of the CTL line after restimulation with p126-134 against the WTl negative cell Iine 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;
and FIG. 10C shows the cytotoxic activity of the CTL line after restimulation with p130-138 against EL-4, EL-4 cells pulsed with p I I 7-13 9 (EL-4 + p 117), p 13 0-13 8 (EL-4 + p 13 0) and the WT1 positive tumor cell line TRAMP-C.
4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
In order that the invention herein described may be more fully understood, the following description of various illustrative embodiments is set forth.
The present invention is generally directed to compositions and methods for the immunotherapy and diagnosis of WTI-associated diseases, such as malignant mesothelioma.
In particular WT1 expression, and immune responses to WT1 (e.g., the presence of WTl specific antibodies in patient sera), may be used as markers to identify patients with malig-nant mesothelioma and other WTI associated malignancies (such as leukemia (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) and childhood ALL), Myelodysplastic syndromes, myeloproliferative syndromes, prostate cancer, lung cancer, breast cancer, thyroid cancer, gastrointestinal cancer, kidney cancer, liver cancer, ovarian cancer, testicular cancer and melanoma). Such diagnostic methods (e.g., in high throughput assay format) may be used for early diagnosis of cancer, and permit screening of healthy individuals who have or might have been exposed to asbestos. Patients found to be afflicted with such malignancies may benefit from the WTl-based vaccine or T-cell therapeutic methods provided herein.
The compositions described herein generally comprise WTl peptides, WTl polynucleotides, antigen-presenting cells (APC; e.g., dendritic cells) that express a WT1 peptide, agents such as antibodies that specifically bind to a WT1 polypeptides and WT1-derived peptides; and/or immune system cells (e.g., T cells) specific for WTl.
WTl peptides of the present invention generally comprise at least a portion of a Wilms' tumor gene product (WTl) or a variant thereof. Nucleic acid sequences of the subject invention generally comprise a DNA, PNA, or RNA sequence that encodes all or a portion of such a peptide, 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 WTl peptide. T cells that may be employed within such compositions are generally cells (e.g., CD4~ and/or CD8+) that are specific for a WT1 peptide. Certain methods described herein further employ one or more antigen-presenting cells that express at least a first WTl peptide or polypeptide as provided herein.
4.1 WTI PEPTIDES
Within the context of the present invention, exemplary preferred WTl-derived antigenic peptides include those peptides of from 9 to about 100 amino acids in length, that comprises at least a first epitope, antigenic fragment, antibody binding site, or an immunogenic sequence that is selected from the group consisting of SEQ ID NO:1 to SEQ
ID N0:4, SEQ ID N0:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID NO:311, SEQ ID
N0:313, SEQ ID N0:314, SEQ ID NO:316 to SEQ ID N0:318, and SEQ ID N0:321 to SEQ ID N0:326.
The WTI-derived peptides may be of any intermediate length provided that it comprises at least a first immunogenic portion or epitope, or antibody binding site, of a native WT1 polypeptide or a variant thereof, and particularly those peptide sequences disclosed in any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID
NO:20, SEQ ID NO:28 to SEQ ID N0:311, SEQ ID N0:313, SEQ ID N0:314, SEQ ID NO:316 to SEQ ID NO:318, and SEQ ID N0:321 to SEQ ID N0:326. In other words, a WT1 peptide 2S may be an oligopeptide (i.e., those consisting of a relatively small number of amino acid residues, such as 9 to about 12 or 13 or so amino acid residues), larger oligopeptides (i.e., those consisting of a relatively larger number of amino acid residues, such as for example, about 14 to about 20 or so amino acid residues), still larger peptides (i.e., those consisting of a relatively larger number of amino acid residues, such as for example, about 21 to about 40 or so amino acid residues), and so forth, up to and including those peptides that consist of a significantly larger number of amino acid residues, such as for example, about 5 to about 90 or 100 or so amino acid residues, as well as aII peptides of intermediate sizes.
Within certain embodiments, the use of WTI peptides that contain a small number of consecutive amino acid residues of a native WT1 peptide is preferred. Such peptides axe preferred for certain uses in which the generation of a T cell response is desired. For example, such a WTl peptide preferably contain at least 9, or at least about 10, 11, 12, 13, 14, or 15 or more consecutive amino acid residues of the native WTl polypeptide.
Nonameric peptides (9-mers, or those comprising at least nine consecutive amino acid residues of a native WT1 polypeptide) are particularly contemplated to be useful in the methods disclosed herein. Additional sequences derived from the native Protein A and/or heterologous sequences may be present within any WT1 peptide, and such sequences may (but need not) possess further immunogenic or antigenic properties. Peptides as provided herein may further be associated (covalently or noncovalently) with other peptide or non-peptide compounds.
An "immunogenic portion," as used herein is a portion of a peptide 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 peptide to bind to MHC class I may be evaluated indirectly by monitoring the ability to promote incorporation of "-'I labeled (32-microglobulin (~32m) into MHC class I/~i2m/peptide heterotrimeric complexes (Parker et al., 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 2-14.
Exemplary immunogenic peptides of the present invention include, but are not limited to, those disclosed in the Examples illustrated in Table 2 through Table 49, and particularly, peptides that comprise at least a first amino acid sequence as defined in any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID NO:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID N0:31 l, SEQ ID N0:313, SEQ ID N0:314, SEQ ID NO:316 to SEQ ID N0:318, and SEQ ID N0:321 to SEQ ID N0:326.

Illustrative WT1-derived peptide compositions include, but are not limited to, those that comprise at least a first amino acid sequence selected from the group consisting of SEQ
ID NO:1 to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID N0:20, SEQ ID NO:28 to SEQ ID
N0:311, SEQ ID N0:3I3, SEQ ID NO:314, and SEQ ID N0:316 to SEQ ID N0:318, and S particularly such sequences as disclosed in any one of the following:
RDLNALLPAVPSLGGGG (human WT1 residues 6-22; SEQ ID NO:1), PSQASSGQARMFPNAPYLPSCLE (human and mouse WT1 residues 117-139; SEQ ID
NO:2 and SEQ ID N0:3, respectively), GATLKGVAAGSSSSVKWTE (human WT1 residues 244-262; SEQ ID NO:4), GATLKGVAA (human WT1 residues 244-252; SEQ ID
N0:88), CMTWNQMNL (human and mouse WTl residues 23S-243; SEQ ID N0:49 and SEQ ID N0:2S8, respectively), SCLESQPTI (mouse WTl residues 136-144; SEQ ID
NO:296), SCLESQPAI (human WTl residues 136-144; SEQ ID N0:198), NLYQMTSQL
(human and mouse WT1 residues 22S-233; SEQ ID N0:147 and SEQ ID NO:284, respectively); ALLPAVSSL (mouse WTI residues 10-18; SEQ ID N0:2SS); or 1S RMFPNAPYL (human and mouse WT1 residues 126-134; SEQ ID NO:18S and SEQ ID
N0:293, respectively).
Further immunogenic fragments and peptides are provided herein, and others may generally be identified using well-known techniques (Paul, 1993).
Representative techniques for identifying immunogenic peptides, epitopes, and antibody binding motifs include, for example, screening peptides for the ability to react with antigen-specific antisera andlor 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 andlor T-cell reactivity assay). In other words, an immunogenic portion may react within such assays at a level that is similar to or 2S 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 (Harlow and Lane, 1988).
Alternatively, immunogenic portions may be identified using computer analysis, such as the Tsites program (Rothbard and Taylor, 1988; Deavin et al., 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., 1994) and other HLA peptide binding prediction analyses. To confznn immunogenicity, a peptide.may be tested using an HLA A2 transgenic mouse model and/or an in vitro stimulation assay using dendritic cells, fibroblasts or 5 peripheral blood cells.
As noted above, the peptides of the present invention may comprise one or more variants of the amino acid sequences as disclosed herein. A peptide "variant,"
as used herein, is a peptide that differs from a particular primary amino acid sequence in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the 10 peptide is substantially 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 peptide). 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 peptide from which the variant was derived.
15 Preferably, the biological activity of a peptide variant will not be diminished by more than 1 %, and preferably still will not be diminished by more than 2%, relative to the biological activity of the unmodified peptide. More preferably, the biological activity of a peptide variant will not be diminished by more than 3%, anal more preferably still will not be diminished by more than 4%, 5%, 6%, 7%, 8%, or 9%, relative to the biological activity of 20 the unmodified peptide. More preferably still, the biological activity of a peptide variant will not be diminished by more than 10%, and more preferably still, will not be diminished by more than II%, 12%, 13%, 14%, IS%, I6%, 17%, 18%, I9%, or 20% relative to the biological activity of the correseponding unmodified peptide.
Based upon % sequence homology, preferred peptide variant of the present invention 25 include those peptides that are from 9 to about 100 amino acids in length, and that comprise at least a first sequence region that is at least 75% identical to at least one of the amino acid sequences dislosed in any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID
N0:20, SEQ ID N0:28 to SEQ ID N0:311, SEQ ID N0:313, SEQ ID NO:314, SEQ ID
N0:316 to SEQ ID N0:318, and SEQ ID N0:321 to SEQ ID N0:326, and more preferably those that comprise at least a first sequence region that is at least 80%
identical to at least one of the amino acid sequences dislosed in any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID
N0:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID N0:311, SEQ ID N0:313, SEQ ID
N0:314, SEQ ID NO:316 to SEQ ID N0:3I8, and SEQ ID N0:321 to SEQ ID NO:326.
More preferably, based upon % sequence homology, preferred peptide variants of the present invention are those peptides that comprise at least a first sequence region that is at least 85%
identical to at least one of the amino acid sequences dislosed in any one of SEQ ID NO: l to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID NO:20, SEQ ID N0:28 to SEQ ID N0:31 l, SEQ
ID NO:313, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:3I8, and SEQ ID N0:32I to SEQ ID NO:326, and more preferably those that comprise at least a first sequence region that is at least 90% identical to at least one of the amino acid sequences dislosed in any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID NO:I3 to SEQ ID N0:20, SEQ ID N0:28 to SEQ
ID NO:31I, SEQ ID N0:313, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:318, and SEQ ID N0:321 to SEQ ID N0:326. Particularly preferred peptide variants of the present invention are those peptides that comprise at least a first sequence region that is at least 91 %, 92%, 93%, 94%, or 95% identical to at least one of the amino acid sequences dislosed in any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID N0:20, SEQ ID N0:28 to SEQ ID N0:311, SEQ ID NO:313, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID N0:3I8, and SEQ ID N0:321 to SEQ ID NO:326, with those peptides that comprise at least a first sequence region that is at least 96%, 97%, 98%, or 99% identical to at least one of the amino acid sequences dislosed in any one of SEQ ID NO:1 to SEQ ID N0:4, SEQ ID N0:13 to SEQ ID NO:20, SEQ ID N0:28 to SEQ ID N0:31I, SEQ ID N0:3I3, SEQ ID N0:314, SEQ ID N0:316 to SEQ ID NO:318, and SEQ ID N0:321 to SEQ ID N0:326.
Such peptide variants may tvpicallv be arenared by mc~ciifvina nne r,f the nPnfiirlP

ID NO:311, SEQ ID N0:313, SEQ ID NO:314, SEQ ID N0:316 to SEQ ID N0:318, and SEQ ID NO:321 to SEQ ID N0:326 by one or more conservative amino acid substitutions.
It has been found, within the context of the present invention, that a relatively small number of conservative or neutral substitutions (e.g., 1 or 2) may be made within the sequence of the nonameric peptide epitopes disclosed herein, without substantially altering the biological activity of the peptide. In some cases, the substitution of one or more amino acids in a particular peptide may in fact serve to enhance or otherwise improve the ability of the peptide to elicit an immune or T-cell response in an animal that has been provided with a composition that comprises the modified peptide, or a polynucleotide that encodes the IO peptide. Suitable substitutions may generally be identified by using computer programs, as described hereinbelow, and the effect of such substitutions may be confirmed based on the reactivity of the modified peptide with antisera and/or T-cells as described herein.
Accordingly, within certain preferred embodiments, a WTI peptide for use in the disclosed diagnostic and therapeutic methods may comprise a primary amino acid sequence in which one or more amino acid residues are substituted by one or more replacement amino acids, such that the ability of the modified peptide to react with antigen-specific antisera and/or T-cell lines or clones is not significantly less than that for the unmodified peptide. Exemplary such substitutions may preferably be located within one or more MHC binding sites on the peptide.
As described above, prefeiTed peptide variants are those that contain one or more 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 peptide 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. Examples of amino acid substitutions that represent a conservative change include: (1) replacement of one or more Ala, Pro, Gly, Glu, Asp, Gln, Asn, Sex, or Thr; residues with one or more residues from the same group; (2) replacement of one or more Cys, Ser, Tyr, or Thr residues with one or more residues from the same group; (3) replacement of one or more Val, Ile, Leu, Met, Ala, ox Phe residues with one S , or more residues from the same group; (4) replacement of one or more Lys, Arg, or His residues with one or more residues from the same group; and (5) replacement of one or more Phe, Tyr, Trp, or His residues with one or more residues from the same group.
A variant may also, or alternatively, contain nonconservative changes, for example, by substituting one of the amino acid residues from group (1) with an amino acid residue from group (2), group (3), group (4), or group (5). 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 peptide.
4.2 BIOLOGICAL FUNCTIONAL EQUIVALENTS
Modification and changes may be made in the structure of the polynucleotides and peptides of the present invention and still obtain a functional molecule that encodes a peptide with desirable characteristics, or still obtain a genetic construct with the desirable expression specificity and/or properties. As it is often desirable to introduce one or more mutations into a specific polynucleotide sequence, various means of introducing mutations into a polynucleotide or peptide sequence known to those of skill in the art may be employed for the preparation of heterologous sequences that may be introduced into the selected cell or animal species. In certain circumstances, the resulting encoded peptide sequence is altered by this mutation, or in other cases, the sequence of the peptide is unchanged by one or more mutations in the encoding polynucleotide. In other circumstances, one or more changes are introduced into the promoter and/or enhancer regions of the polynucleotide constructs to altar the activity, or specificity of the expression elements and thus alter the expression of the heterologous therapeutic nucleic acid segment operably positioned under the control of the elements.
When it is desirable to alter the amino acid sequence of one or more of the heterologous peptides encoded by the expression construct to create an equivalent, or even an improved, second-generation molecules, the amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 1.
For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA
sequences which encode said peptides without appreciable loss oftheir biological utility or activity.

Amino Acids Codons Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acidAsp D GAC GAU

Glutamic acidGlu E GAA GAG

PhenylalaninePhe F UUC UUU

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine Ile I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutarnine Gln Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a 5 protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of 10 their hydrophobicity and charge characteristics (Kyte and Doolittle, I982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5);
asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i. e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ~2 is preferred, those that are within ~1 are particularly preferred, and those within ~0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U. S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U. S. Patent 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ~ 1); glutamate (+3.0 ~ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);
threonine (-0.4);
proline (-0.5 ~ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ~2 is preferred, those that are within ~1 are particularly preferred, and those within ~0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take several of the foregoing characteristics into consideration are well known to those of skill in the art and include: axginine and lysine; glutamate and aspartate; serine and threonine;
glutamine and asparagine; and valine, leucine and isoleucine.

The peptides and peptide variants of the present invention may be conjugated to a signal (or leader) sequence at the N-terminal end of the peptide, which co-translationally or post-translationally directs transfer of the peptide. The peptides may also, or alternatively, be conjugated to one or more linker sequences for ease of synthesis, purification or identification of the peptide (e.g., poly-His), or to enhance binding of the peptide to a solid support. For example, the peptides may be conjugated to an immunoglobulin Fc region.
The peptides and peptide variants of the present invention may be isolated and purified fxom native sources, such as for example, by isolating all or part of the primary amino acid sequence from a native WT1 peptide, or alternatively, may be chemically synthesized in whole or in part using any of a variety of well-known peptide synthesis techniques. For example, peptides having less than about 100 amino acids, preferably less than about 90 or 80 amino acids, and more preferably less than about 70, less than about 60, or less than about 50, about 40, about 30, or about 20 amino acids, 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 (Merrifield, 1963). Equipment for automated synthesis of peptides is commercially available from suppliers such as Applied BioSystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions.
The peptides and peptide variants as described herein may also be readily prepared from recombinant WT1 peptides, or may be prepared by translation of a polynucleotide sequence that encodes such a peptide. In general, any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant peptides.
Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a nucleic acid molecule that encodes the peptide. 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.
In general, peptides and polynucleotides as described herein are isolated. An "isolated" peptide or polynucleotide is one that is removed from its original environment.
For example, a naturally occurring peptide or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such peptides are at least about 80% or 85% pure, more preferably at least about 90% or 95% pure and most preferably at least about 96%, 97%, 98%, or 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.
Within further aspects, the present invention provides mimetics of WTI
peptides.
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 peptide 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 peptide 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 WTI peptide. Such IS mimetics may be designed based on standard techniques (e.g., nuelear 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 WTI peptide 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 peptide.
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 Iigated 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., 1985; Murphy et al.; 1986; U. S. Patent No. 4,935,233 and U. S. Patent No. 4,751,180. The linker sequence may generally be from 1 to about 10, about 20, about 30, about 40, or about 50 or so 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 axe 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 S recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al., 1997).
In one preferred embodiment, the immunological fusion partner is derived from a Mycobacterium sp., such as a Mycobactey~iurv~ tuberculosis-derived Ral2 fragment. Ral2 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 60/1S8,S8S, the disclosure of which is incorporated herein by reference in its entirety. Briefly, Ral2 refers to a polynucleotide region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is a sexine protease of 32 kDa molecular weight encoded by a gene in virulent and avirulent strains of M.
tuberculosis. The 1 S nucleotide sequence and amino acid sequence of MTB32A have been described (see for example, U. S. Patent Application 60/1S8,S8S; and Skeiky et al., 1999, each 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, Ral2 may enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One preferred Ral2 fusion polypeptide comprises a 14-kDa C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ral2 polynucleotides generally comprise at least about 1 S 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 Ral2 2S polypeptide. Ral2 polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Ral2 polypeptide or a portion thereof) or may comprise a variant of such a sequence. Ral2 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 Ral2 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 Ral2 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 Haefrcophilus influenza B
(Intl. Pat. Appl. Publ. No. WO 91/18926). Preferably, a Protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-I
10 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 fram influenzae virus, NS 1 (hemaglutinin). Typically, the N-terminal 8I
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 pheurrzoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene). 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 Within a preferred embodiment, a repeat portion of LYTA may be incozporated 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 I88-305.
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. Patent 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.
4.3 POLYNUCLEOTIDE COMPOSITIONS
Any polynucleotide that encodes a WTl peptide as described herein, or that is complementary to such a polynucleotide, is a WTl 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.
Addi-tional 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 WTl protein, or may encode a variant of WTI as described herein. Polynucleotide variants may contain one or more substitutions, additions, deletions andlor insertions such that the immunogenicity of the encoded peptide is not diminished, relative to a native WT1 protein. The effect on the immunogenicity of the encoded peptide may generally be assessed as described herein. Preferred peptide variants contain amino acid substitutions, deletions, insertions and/or additions at no more than about 20%, more preferably at no more than about I S%, and more preferably still, at no more than about IO% or 5% or Less of the amino acid positions relative to the corresponding native unmodified WT1 sequence.
Likewise, polynucleotides encoding such peptide variants should preferably contain nucleotide substitutions, deletions, insertions and/or additions at no more than about 20%;
more .preferably at no more than about 15%, and more preferably still, at no more than about 10% or 5% or less of the nucleotide positions relative to the corresponding polynucleotide sequence that encodes the native unmodified WT1 peptide sequence. Certain polynucleotide variants, of course, may be substantially homologous to, or substantially identical to the corresponding region of the nucleotide sequence encoding an unmodified peptide. Such polynucleotide variants are capable of hybridizing to a naturally occurring DNA sequence encoding a WT I peptide (or a complementary sequence) under moderately stringent, to highly stringent, to very highly stringent conditions.

3~S
Suitable moderately stringent conditions include prewashing in a solution containing about SX SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at a temperature of from about 50°C to about 60°C in SX SSC overnight; followed by washing twice at about 60 to 65°C for 20 min. with each of 2X, O.SX and 0.2X SSC containing 0.1%
SDS). Suitable highly stringent conditions include prewashing in a solution containing about SX SSC, 0.5%
SDS, 1.0 mM EDTA (pH 8.0); hybridizing at a temperature of from about 60°C to about 70°C in SX SSC overnight; followed by washing twice at about 65 to 70°C for 20 min. with each of 2X, O.SX and 0.2X SSC containing 0.I% SDS). Representative examples of very highly stringent hybridization conditions may include, for example, prewashing in a solution containing about SX SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at a temperature of from about 70°C to about 75°C in SX SSC overnight; followed by washing twice at about 70°C to about 75°C for 20 min. with each of2X, O.SX and 0.2X SSC
containing 0.1% SDS).
Such hybridizing DNA sequences are also within the scope of this invention.
It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a WTl peptide. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.
Once an immunogenic portion of 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 (PCRT"'~. 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 PCRTM amplification of a human WT1 gene include: first step - P 118: 1434-1414: 5'-GAGAGTCAGACTTGAAAGCAGT-3' (SEQ ID
NO:S) and P135: 5'-CTGAGCCTCAGCAAATGGGC-3' (SEQ ID N0:6); second step -P136: 5'-GAGCATGCATGGGCTCCGACGTGCGGG-3' (SEQ ID N0:7) and P137:
5'-GGGGTACCCACTGAACGGTCCCCGA-3' (SEQ ID N0:8). Primers for PCRTM ampli-fication of a mouse WT1 gene include: first step - P138: 5'-TCCGAGCCGCACCTCATG-3' (SEQ ID N0:9) and P139: 5'-GCCTGGGATGCTGGACTG-3' (SEQ ID NO:10), second step - P 140: 5'-GAGCATGCGATGGGTTCCGAGGTGCGG-3' (SEQ ID NO:11 ) and P 141:
5'-GGGGTACCTCAAAGCGCCACGTGGAGTTT-3' (SEQ ID NO:I2).
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 PCRTM
fragments. WTl polynucleotides may also be prepared by synthesizing oligonucleotide components, and ligating components together to generate the complete polynucleotide.
WTl 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 (Adelman et al., 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a WT1 peptide, 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 peptide, as described herein. In addition, or alternatively, a portion may be administered to a patient such that the encoded peptide is generated in vivo (e.g., by transfecting antigen-presenting cells such as dendritic cells with a cDNA construct encoding a WTl peptide, and administering the transfected cells to the patient).
Polynucleotides that encode a WT1 peptide may generally be used for production of the peptide, in vitf~o 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 WTl e:~pression. 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 nucleo tide sequences using established recombinant DNA techniques. For example, a polynucleo 5 tide 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 10 depend upon the desired use, and will be apparent to those of ordinary skill in the art.
Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those of ordinary skill in the art will appreciate that there axe many ways to achieve expression of a polynucleotide in a target cell, 15 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 poxvirus (e.g., avian poxvirus).
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 20 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 25 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
30 preferred colloidal system for use as a delivery vehicle i~c 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.
4.4 METHODS OF NUCLEIC ACID DELIVERY AND DNA TRANSFECTION
In certain embodiments, it is contemplated that one or more RNA or DNA and/or substituted polynucleotide compositions disclosed herein will be used to transfect an appropriate host cell. Technology for introduction of RNAs and DNAs, and vectors comprising them into suitable host cells is well known to those of skill in the art. In particular, such polynucleotides may be used to genetically transform one or more host cells, when therapeutic administration of one or more active peptides, compounds or vaccines is achieved through the expression of one or more polynucleotide constructs that encode one or more therapeutic compounds of interest.
A variety of means for introducing polynucleotides and/or polypeptides into suitable target cells is known to those of skill in the art. For example, when polynucleotides are contemplated for delivery to cells, several non-viral methods for the transfer of expression constructs into cultured mammalian cells are available to the skilled artisan for his use. These include, for example, calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); DEAE-dextran precipitation (copal, 1985);
electroporation(Wong and Neumann, 1982; Fromm et al., 1985; Tur-Kaspa et al., 1986; Potter et al., 1984; Suzuki et al., 1998; Vanbever et al., 1998), direct microinjection(Capecchi,1980;
Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;
Fraley et al., 1979; Takakura, 1998) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990; Klein et al., 1992), and receptor-mediated transfection (Curiel et al., 1991; Wagner et al., 1992; Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
A bacterial cell, a yeast cell, or an animal cell transformed with one or more of the disclosed expression vectors represent an important aspect of the present invention. Such transformed host cells are often desirable for use in the expression of the various DNA gene constructs disclosed herein. In some aspects of the invention, it is often desirable to modulate, regulate, or otherwise control the expression of the gene segments disclosed herein. Such methods are routine to those of skill in the molecular genetic arts.
Typically, when increased or over-expression of a particular gene is desired, various manipulations may be employed for enhancing the expression of the messenger RNA, particularly by using an active promoter, and in particular, a tissue-specific promoter such as those disclosed herein, as well as by employing sequences, which enhance the stability of the messenger RNA in the particular transformed host cell.
Typically, the initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal. In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double ' strand may be used by itself for transformation of a microorganism or eukaryotic host, but will usually be included with a DNA sequence involving a marker, where the second DNA
sequence may be joined to the expression construct during introduction of the DNA into the host.
Where no functional replication system is present, the construct will also preferably include a sequence of at least about 30 or about 40 or about 50 basepairs (bp) or so, preferably at least about 60, about 70, about 80, or about 90 to about 100 or so bp, and usually not more than about 500 to about 1000 or so by of a sequence homologous with a sequence in the host.
In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Desirably, the regulatory regions of the expression construct will be in close proximity to (and also operably positioned relative to) the selected therapeutic gene providing for complementation as well as the gene providing for the competitive advantage. Therefore, in the event that the therapeutic gene is lost, the resulting organism will be likely to also lose the gene providing for the competitive advantage, so that it will be unable to compete in the environmentwith the gene retaining the intact construct.

The selected therapeutic gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This construct may be included in a plasmid, which will include at least one replication system, but rnay include more than one, where one replication system is employed fox cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host, in this case, a mammalian host cell. In addition, one or more markers may be present, which have been described previously. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome.
Genes or other nucleic acid segments, as disclosed herein, can be inserted into host cells using a variety of techniques that are well known in the art. Five general methods fox delivering a nucleic segment into cells have been described: (1) chemical methods (Graham and VanDerEb, 1973); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (U. S. Patent x,472,869; Wong and Neumann, 1982; Fromm et al., 1985), microprojectile bombardment (U. S. Patent 5,874,265, specifically incorporated herein by reference in its entirety), "gene gun" (Yang et al., 1990); (3) viral vectors (Eglitis and Anderson, 1988); (4) receptor-mediated mechanisms (Curiel et al., 1991; Wagner et al., 1992);
and (5) bacterial-mediated transformation.
4.5 WT1-SPECIFIC ANTIBODIES AND ANTIGEN-BINDING FRAGMENTS THEREOF
The present invention further provides antibodies and antigen-binding fragments thereof, that specifically bind to (or are immunospecific for) at least a first peptide or peptide variant as disclosed herein. As used herein, an antibody or an antigen-binding fragment is said to "specifically bind" to a peptide if it reacts at a detectable level (within, for example, an ELISA) with the peptide, and does not react detestably with unrelated peptides or proteins under similar conditions. As used herein, "binding" refers to a noncovalent association between two separate molecules such that a "complex" is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex.
The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations. In the context of the present invention, in general, two compounds are said to "bind" when the binding constant for complex formation exceeds about 10' L/mol. The binding constant maybe determined using methods well known in the art.
Any agent that satisfies the above requirements may be a binding agent. In illustrative embodiments, a binding agent is an antibody or an antigen-binding fragment thereof. Such antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (Harlow and Lane, 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 imtnunogen comprising the peptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the peptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short peptides, a superior immune response may be elicited if the peptide 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 peptide may then be purified from such antisera by, for example, affinity chromatography using the peptide coupled to a suitable solid support.
Monoclonal antibodies specific for the antigenic peptide of interest may be prepared, for example, using the technique of Kohler and Milstein (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 peptide 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 peptide. Hybridomas having high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of growing hybridoma 5 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 peptides of this 10 invention may be used in the purification process in, for example, an affinity chromatography step.
Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, irnmunoglobulins may be purified from rabbit serum by affinity 15 chromatography on Protein A bead columns (Harlow and Lane, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on Protein A bead columns.
Monoclonal antibodies and fragments thereof may be coupled to one or more thera peutic agents. Suitable agents in this regard include radioactive tracers and chemotherapeutic 20 agents, which may be used, for example, to purge autologous bone marrow in vitro). Repre sentative therapeutic agents include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include 9°Y, 'z3I, 'zSI, '3'I, 'BgRe, '$BRe, zl'At, and zizBi. ~ Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include 25 ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein. For diagnostic purposes, coupling of radioactive agents may be used to facilitate tracing of metastases or to determine the location of WT1-positive tumors.
A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable 30 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 S 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 polyfunc-tional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, IL), may be employed as the linker group.
Coupling 1 S may be affected, for example, through amino groups, carboxyl groups, and sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such method-ology, e.g., U. S. Patent No. 4,671,958.
Where a therapeutic agent is more potent when free from the antibody portion of the immunaconjugates of the present invention, it may be desirable to use a linker group that 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 (L1. S.
Patent No.4,489,710), by irradiation of a photolabile bond (U. S. Patent No.4,625,014), by hydrolysis of derivatized amino acid side chains (U. S. Patent No. 4,638,045), by serum 2S complement-mediated hydrolysis (U. S. Patent No. 4,671,958), and acid-catalyzed hydrolysis (U. S. Patent No. 4,569,789).
It may be desirable to couple more than one agent to an antibody. In one embodi-ment, 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 (U. S. Patent No. 4,507,234), peptides and polysaccharides such as aminodextran (U. S. Patent No. 4,699,784). A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (U. S. Patent No. 4,429,008 and U. S. Patent No. 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. Fox example, U. S. Patent No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. A
radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
For example, U. S. Patent No. 4,673,562 discloses representative chelating compounds and their synthesis.
A variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/ immuno conjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody.
Also provided herein are anti-idiotypic antibodies that mimic an immunogenic portion of WT1. Such antibodies may be raised against an antibody, or an antigen-binding fragment thereof, that specifically binds to an immunogenic portion of WT1, using well-known techniques. Anti-idiotypic antibodies that mimic an immunogenic portion of WT1 are those antibodies that bind to an antibody, or antigen-binding fragment thereof, that specifically binds to an immunogenic portion of WT1, as described herein.
Irrespective of the source of the original WT1 peptide-specific antibody, the intact antibody, antibody multimers, or any one of a variety of functional, antigen-binding regions of the antibody may be used in the present invention. Exemplary functional regions include scFv, Fv, Fab', Fab and F(ab')2 fragments of the WT1 peptide-specific antibodies.
Techniques for preparing such constructs are well known to those in the art and axe further exemplified herein.

The choice of antibody construct may be irdluenced by various factors. For example, prolonged half life can result from the active readsorption of intact antibodies within the kidney, a property of the Fc piece of immunoglobulin. IgG based antibodies, therefore, are expected to exhibit slower blood clearance than their Fab' counterparts.
However, Faf fragment-based compositions will generally exhibit better tissue penetrating capability.
Antibody fragments can be obtained by proteolysis of the whole immunoglobulin by the non-specific thiol protease, papain. Papain digestion yields two identical antigen-binding fragments, termed "Fab fragments," each with a single antigen-binding site, and a residual "Fc fragment."
Papain should first be activated by reducing the sulphydryl group in the active site with cysteine, 2-mercaptoethanol or dithiothreitol. Heavy metals in the stock enzyme should be removed by chelation with EDTA (2 mM) to ensure maximum enzyme activity.
Enzyme and substrate are normally mixed together in the ratio of 1:100 by weight. After incubation, the reaction can be stopped by irreversible alkylation of the thiol group with iodoacetamide or simply by dialysis. The completeness of the digestion should be monitored by SDS-PAGE and the various fractions separated by Protein A-Sepharose or ion exchange chromatography.
The usual procedure for preparation of F(ab')~ fragments from IgG of rabbit and human origin is limited proteolysis by the enzyme pepsin. The conditions, 100x antibody excess wt./wt. in acetate buffer at pH 4.5, 37°C, suggest that antibody is cleaved at the C-terminal side of the inter-heavy-chain disulfide bond. Rates of digestion of mouse IgG may vary with subclass and it may be difficult to obtain high yields of active F(ab')2 fragments without some 'undigested or completely degraded IgG. In particular, IgG~b is highly susceptible to complete degradation. The other subclasses require different incubation conditions to produce optimal results, all of which is known in the art.
Pepsin treatment of intact antibodies yields an F(af), fragment that has two antigen-combining sites and is still capable of cross-linking antigen. Digestion of rat IgG by pepsin requires conditions including dialysis in 0.1 M acetate buffer, pH 4.5, and then incubation for four hrs with 1 % wt./wt. pepsin; IgG, and IgG~a digestion is improved if first dialyzed against 0.1 M formate buffer, pH 2.8, at 4°C, for 16 hrs followed by acetate buffer. IgG26 gives more consistent results with incubation in staphylococcal V 8 protease (3% wt./wt.) in 0.1 M sodium phosphate buffer, pH 7.8, for four hrs at 37°C.
A Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHl domain including one or more cysteine(s) from the antibody hinge region. F(ab')Z antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The teen "variable," as used herein in reference to antibodies, means that certain portions of the variable domains differ extensively in sequence among antibodies, and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments termed "hypervariable regions," both in the light chain and the heavy chain variable domains.
The more highly conserved portions of variable domains are called the framework region (FR). The variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a (3-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases, forming part of, the (3-sheet structure.
The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (Kabat et al., 1991, specifically incorporated herein by reference). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular toxicity.
The term "hypervariable region," as used herein, refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i. e.
residues 24-34 (L 1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-56 (H2) and 95-102 (H3) in the heavy chain variable domain (Kabat et al., 1991, specifically incorporated herein by reference) and/or those residues from a "hypervariable loop" (i.e., residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-1 Ol (H3) in the heavy chain variable domain). "Framework" or "FR"
residues are those variable domain residues other than the hypervariable region residues as herein defined.
5 An "Fv" fragment is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, con-covalent association. It is in this configuration that three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH VL dimer. Collectively, six hypervariable regions confer antigen-binding 10 specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
Generally, the Fv 15 polypeptide further comprises a polypeptide linker between the VH and VL
domains that enables the sFv to form the desired structure for antigen binding.
"Diabodies" are small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V~ connected to a light chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to 20 allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described in European Pat. Appl. No. EP 404,097 and Intl. Pat. Appl. Publ.
No. WO
93/11.161, each specifically incorporated herein by reference. "Linear antibodies", which can be bispecific or monospecific, comprise a pair of tandem Fd segments (VH-CH1-VH CH1) that 25 form a pair of antigen binding regions, as described in Zapata et al.
(1995), specifically incorporatedherein by reference.
Other types of variants are antibodies with improved biological properties relative to the parent antibody from which they are generated. Such variants, or second-generation compounds, are typically substitutional variants involving one or more substituted hypervariable region residues of a parent antibody: A convenient way for generating such substitutional variants is affinity maturation using phage display.
In affinity maturation using phage display, several hypervariable region sites (e.g., 6 to 7 sites) are mutated to generate aII possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.
In order to identify candidate hypervariable region sites for modification, alanine-scanning mutagenesis can be performed on hypervariable region residues identified as contributing significantly to antigen binding.
Alternatively, or in addition, the crystal structure of the antigen-antibody complex be delineated and analyzed to identify contact points between the antibody and 'target. Such contact residues and neighboring residues are candidates for substitution.
Once such variants are generated, the panel of variants is subjected to screening, and antibodies with analogues but different or even superior properties in one or more relevant assays are selected for further development.
In using a Faf or antigen binding fragment of an antibody, with the attendant benefits on tissue penetration, one may derive additional advantages from modifying the fragment to increase its half life. A variety of techniques may be employed, such as manipulation or modification of the antibody molecule itself, and also conjugation to inert carriers. Any conjugation for the sole purpose of increasing half life, rather than, to deliver an agent to a target, should be approached carefully in that Faf and other fragments are chosen to penetrate tissues. Nonetheless, conjugation to non-protein polymers, such PEG and the like, is contemplated.
Modifications other than conjugation are therefore based upon modifying the structure of the antibody fragment to render it more stable, andlor to reduce the rate of catabolism in the body. One mechanism for such modifications is the use of D-amino acids in place of L-amino acids. Those of ordinary skill in the art will understand that the introduction of such modifications needs to be followed by rigorous testing of the resultant molecule to ensure that it still retains the desired biological properties. Further stabilizing modifications include the use of the addition of stabilizing moieties to either the N-terminal or the C-terminal, or both, which is generally used to prolong the half life of biological molecules. By way of example only, one may wish to modify the termini by acylation or amination.
Moderate conjugation-type modifications for use with the present invention include S incorporating a salvage receptor binding epitope into the antibody fragment.
Techniques for achieving this include mutation of the appropriate region of the antibody fragment or incorporating the epitope as a peptide tag that is attached to the antibody fxagment. Intl. Pat.
Appl. Publ. No. WO 96/32478 is specifically incorporated herein by reference for the purposes of further exemplifying such technology. Salvage receptor binding epitopes are typically regions of three or more amino acids from one or two lops of the Fc domain that are transferred to the analogous position on the antibody fragment. The salvage receptor-binding epitopes disclosed in Intl. Pat. Appl. Publ. No. WO 98/45331 are incorporated herein by reference for use with the present invention.
1 S 4.fi T CELL COMPOSITIONS SPECIFIC FOR WTl PEPTIDES
Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for WTl. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be present within (or isolated from) bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood of a mammal, such as a patient, using a commercially available cell separation system, such as the IsolexTM System, available from Nexell Therapeutics, Inc. (Irvine, CA; see also U. S. Patent No. 5,240,856; U.
S. Patent No. 5,215,926; Intl. Pat. Appl. Publ. No. WO 89/06280; Intl. Pat.
Appl. Publ. No.
WO 91/16116 and Intl. Pat. Appl. Publ. No. WO 92/07243). Alternatively, T
cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
2S T cells may be stimulated with WT1 peptide, polynucleotide encoding a WT1 peptide and/or an antigen-presenting cell (APC) that expresses a WTl peptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the WTI peptide. Preferably, a WT1 peptide 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), axe incubated with WTl peptide. For example, T cells may be incubated ifz vitro for 2-9 days (typically 4 days) at 37°C with WT1 peptide (e.g., 5 to 25 ~g/ml) or cells synthesizing a comparable amount of WT1 peptide. It may be desirable to incubate a separate aliquot of a T cell sample in the absence of WT1 peptide to serve as a control.
T cells are considered to be specific for a WT1 peptide if the T cells kill target cells coated with a WT1 peptide or expressing a gene encoding such a peptide. 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. (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'~ flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yI)-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 WTl peptide may be quantified. Contact with a WT1 peptide (200 ng/ml - 100 ~,g/ml, preferably 100 ng/m1 - 25 ~.g/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 hrs should result in activation of the T cells, as measured using standard cytokine assays in which a two-fold increase in the level of cytokine release (e.g., TNF or IFN-y) is indicative of T cell activation (Coligan et al., 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 peptide, polynucleotide or WTl-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 WTI).
Fox CD4+ T cells, a preferred method for detecting specific T cell activation is the detection of the proliferation of T cells. For CD8~ T cells, a preferred method for detecting specific T cell activation is the detection of the generation of cytolytic activity.
For therapeutic purposes, CD4+ or CD8~ T cells that proliferate in response to the WTI peptide, polynucleotide or APC can be expanded in number either ih vitf°o ox irz 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 peptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a WT1 peptide. The addition of stimulator cells is preferred where generating CD~+ T cell responses. T cells can be grown to large numbers ija vitro with retention of specificity in response to intermittent restimulation with WT1 peptide. Briefly, for the primary in vita°o stimulation (IVS), large numbers of lymphocytes (e.g., greater than 4 x 10') may be placed in flasks with media containing human serum. WT1 peptide (e.g., peptide at IO p,g/ml) may be added directly, along with tetanus toxoid (e.g., 5 ~,g/ml). The flasks may then be incubated (e.g., 37°C fox 7 days). For a second IVS, T cells are then harvested and placed in new flasks with 2-3 x 10' irradiated peripheral blood mononuclear cells. WT1 peptide (e.g., 10 p,g/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-S units of interleukin-2 (IL-2) may be added. For a third IVS, the T
cells may be placed in wells and stimulated with the individual's own EBV transformed B
cells coated with the peptide. IL-2 may be added on days 2 and 4 of each cycle. As soon as the cells are shown to be specific cytotoxic T cells, they may be expanded using a 10-day stimulation cycle with higher IL-2 (20 units) on days 2, 4 and 6.
2S Alternatively, one or more T cells that proliferate in the presence of WT1 peptide 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 peptide is used as the antigenic stimulus and autologous peripheral blood lymphocytes (PBL) or Iymphoblastoid cell lines (LCL) immortalized by infection with Epstein Barr virus are used as antigen-presenting cells. In order to generate CD~+ T cell lines, autologous antigen-presenting cells transfected with an expression vector that produces WT1 peptide may be used as stimulator 5 cells. Established T cell lines may be cloned 2-4 days following antigen stimulation by plating stimulated T cells at a frequency of 0.5 cells per well in 96-well flat-bottom plates with 1 x 106 irradiated PBL or LCL cells and recombinant interleukin-2 (rIL2) (50 U/ml).
Wells with established clonal growth may be identified at approximately 2-3 weeks after initial plating and restimulated with appropriate antigen in the presence of autologous 10 antigen-presenting cells, then subsequently expanded by the addition of low doses of rIL2 (10 Ulml) 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.
Cloned and/or expanded cells may be administered back to the patient as described, for example, by Chang et al., (1996).
15 Within certain embodiments, allogeneic T-cells may be primed (i.e., sensitized to WTl) in vivo and/or ira vitro. Such priming may be achieved by contacting T
cells with a WT1 peptide, a polynucleotide encoding such a peptide or a cell producing such a peptide 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 WTl peptide results in 20 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 ira vitro may be employed, for example, within bone marrow transplantation or as donor 25 lymphocyte infusion.
T cells specific for WTl 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 WTl bearing leukemia and cancer cells.
Vaccines to increase the number of T cells that can react to WTl positive cells are one method of 30 targeting WTl bearing cells. T cell therapy with T cells specific for WT1 is another method.

An alternative method is to introduce the TCR chains specific fox WTl into T
cells or other cells with lytic potential. In a suitable embodiment, the TCR alpha and beta chains are cloned out from a WTI specific T cell line and used for adoptive T cell therapy, such as described in W096/305I 6, incorporated herein by reference.
4.7 PHARMACEUTICAL COMPOSITIONS AND VACCINE FORMULATIONS
Within certain aspects, peptides, polynucleotides, antibodies and/or T cells may be incorporated into pharmaceutical compositions or immunogenic compositions (i.e., vaccines). Alternatively, a pharmaceutical composition may comprise an antigen-presenting cell (e.g., a dendritic cell) transfected with a WTl polynucleotide such that the antigen-presenting cell expresses a WTI peptide. Pharmaceutical compositions comprise one or more such compounds or cells and a physiologically acceptable carrier or excipient.
Vaccines may comprise one or more such compounds or cells and an immunostimulant, such as an adjuvant or a liposome (into which the compound is incorporated). An immunostimu-lant may be any substance that enhances or potentiates an immune response (antibody-and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated) (U. S. Patent No. 4,235,877). Vaccine preparation is generally described in, for example, Powell and Newman (1995). Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be present, either incorporated into a fusion peptide or as a separate compound, within the composition or vaccine.
Within certain embodiments, pharmaceutical compositions and vaccines are designed to elicit T cell responses specific for a WTI peptide in a patient, such as a human. In general, T cell responses may be favored through the use of relatively short peptides (e.g., comprising less than 23 consecutive amino acid residues of a native WT1 peptide, preferably 4-16 consecutive residues, more preferably 8-16 consecutive residues and still more preferably 8-10 consecutive residues). Alternatively, or in addition, a vaccine may comprise an immunostimulant that preferentially enhances a T cell response. In other words, the immunostimulant may enhance the level of a T cell response to a WTl peptide by an amount that is proportionally greater than the amount by which an antibody response is enhanced.
For example, when compared to a standard oil based adjuvant, such as CFA, an immunostimulant that preferentially enhances a T cell response may enhance a proliferative T cell response by at least two fold, a lytic response by at least 10%, and/or T cell activation by at least two fold compared to WTl-negative control cell lines, while not detectably enhancing an antibody response. The amount by which a T cell or antibody response to a WT1 peptide is enhanced may generally be determined using any representative technique known in the art, such as the techniques provided herein.
A pharmaceutical composition or vaccine may contain DNA encoding one or more of the peptides as described above, such that the peptide is generated in situ.
As noted above, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expres-sion systems and mammalian expression systems. Numerous gene delivery techniques are well known in the art (Rolland, 1998, and references cited therein).
Appropriate nucleic acid expression systems contain the necessary DNA, cDNA or RNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-CalrrZette-Gue~rin) that expresses an immunogenic portion of the peptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus (Fisher-Hoch et al., 1989;
Flexner et al., 1989; Flexner et al., 1990; U. S. Patent No. 4,603,112, U. S. Patent No.
4,769,330, U. S.
Patent No. 5,017,487; Intl. Pat. Appl. Publ. No. WO 89/01973; U. S. Patent No.
4,777,127;
Great Britain Patent No. GB 2,200,651; European Patent No. EP 0,345,242; Intl.
Pat. Appl.
Publ. No. WO 91/02805; Berkner, 1988; Rosenfeld et al., 1991; Kolls et al., 1994;
Kass-Eisler et al., 1993; Guzman et al., 1993a; and Guzman et al., 1993).
Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al. (1993) and reviewed by Cohen (1993). The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which axe efficiently transported into the cells. It will be apparent that a vaccine may comprise both a polynucleotide and a peptide component. Such vaccines may provide for an enhanced immune response.
As noted above, a pharmaceutical composition or vaccine may comprise an antigen presenting cell that expresses a WTl peptide. For therapeutic purposes, as described herein, the antigen-presenting cell is preferably an autologous dendritic cell. Such cells may be prepared and transfected using standard techniques (Reeves et al., 1996;
Tuting et al., 1998;
and Nair et al., 1998). Expression of a WTl peptide on the surface of an antigen-presenting cell may be confirmed by ifz vitro stimulation and standard proliferation as well as chromium release assays, as described herein.
It will be apparent to those of ordinary skill in the art having the benefit of the present teachings that a vaccine may contain pharmaceutically acceptable salts of the polynucleotides and peptides provided herein. Such salts may be prepared 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). The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other significant untoward reaction when administered to an animal, or a human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents 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.
For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the Food and Drug Administration Office of Biologics standards. Supplementaryactive ingredients can also be incorporatedinto the compositions.
While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions of this invention.
Suitable biodegradable microspheres axe disclosed, for example, in U. S. Patent Nos.
4,897,268;
5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252. For certain topical applications, formulation as a cream or lotion, using well-known components, is preferred.
Such compositions may also comprise buffers (e.g., neutral buffered saline or phos-phate buffered saline), carbohydrates (e.g., glucose, manriose, sucrose or dextrans), mannitol, proteins, peptides 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, or formulated with one or more liposomes, microspheres, nanoparticles, or micronized delivery systems using well-known technology.
Any of a variety of immunostimulants, such as adjuvants, may be employed in the preparation of vaccine compositions of this invention. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mvcobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, alum-based adjuvants (e.g., Alhydrogel, Rehydragel, aluminum phosphate, Algammulin, aluminum hydroxide); oil based adjuvants (Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI), Specol, RIBI, TiterMax, Montanide ISA50 or Seppic MONTANIDE ISA 720); nonionic block copolymer-based adjuvants, cytokines (e.g., GM-CSF or Flat3-ligand); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); salts of calcium, iron or zinc;

an insoluble suspension of acylated tyrosine; acylated sugars; canonically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres;
monophosphoryl lipid A and Quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
5 Hemocyanins and hemoerythrins may also be used in the invention. The use of hemocyanin from keyhole limpet (I~LH) is particularly preferred, although other molluscan and arthropod hemocyanins and hemoerythrins may be employed. Various polysaccharide adjuvants may also be used. Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin.
10 A further preferred group of adjuvants are the muramyl dipeptide (MDP, N-acetylmuramyl-~.-alanyl-n-isoglutarnine) group of bacterial peptidoglycans.
Derivatives of muramyl dipeptide, such as the amino acid derivative threonyl-MDP, and the fatty acid derivative MTPPE, axe also contemplated.
U. S. Patent No. 4,950,645 describes a lipophilic disacchaxide-tripeptide derivative of 15 muramyl dipeptide that is proposed for use in artificial liposomes formed from phosphatidyl choline and phosphatidyl glycerol. It is said to be effective in activating human monocytes and destroying tumor cells, but is non-toxic in generally high doses. The compounds of U. S.
Patent No. 4,950,645, and Intl. Pat. Appl. Publ. No. WO 91/16347 are also proposed for use in achieving particular aspects of the present invention.
20 BCG and BCG-cell wall skeleton (CWS) may also be used as adjuvants in the invention, with or without trehalose dimycolate. Trehalose dirnycolate may be used itself.
Azuma et al. (1988) show that trehalose dimycolate administration correlates with augmented resistance to influenza virus infection in mice. Trehalose dimycolate may be prepared as described in U. S. Patent No. 4,579,945.
25 Amphipathic and surface-active agents, e.g., saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of preferred adjuvants for use with the immunogens of the present invention. Nonionic block copolymer surfactants (Rabinovich et al., 1994; Hunter et al., 1991) may also be employed. Oligonucleotides, as described by Yamamoto et al. (1988) are another useful group of adjuvants. Quil A and lentinen are also 30 preferred adjuvants.

Superantigens are also contemplated for use as adjuvants in the present invention.
"Superantigens" are generally bacterial products that stimulate a greater proportion of T
lymphocytes than peptide antigens without a requirement for antigen processing (Mooney et. al., 1994). Superantigens include Staphylococcus exoproteins, such as the a, (3, y and 8 enterotoxins from S. au~eus and S. epidet~midis, and the a, (3, y and ~ E.
coli exotoxins.
Common Staphylococcus enterotoxins are known as staphylococcal enterotoxin A
(SEA) and staphylococcal enterotoxin B (SEB), with enterotoxins through E
(SEE) being described (Rott et. al., 1992). Streptococcus pyogenes B (SEB), Clostridium perfi°i~agens enterotoxin (Bowness et. al., 1992), cytoplasmic membrane-associated protein (CAP) from S
pyogenes (Sato et. al., 1994) and toxic shock syndrome toxin-1 (TSST-1) from S. auf~eus (Schwab et. al., 1993) are further useful superantigens.
One group of adjuvants particularly preferred for use in the invention are the detoxified endotoxins, such as the refined detoxified endotoxin of U. S.
Patent No.
4,866,034. These refined detoxified endotoxins are effective in producing.
adjuvant responses in mammals.
The detoxified endotoxins may be combined with other adjuvants. Combination of detoxified endotoxins with trehalose dimycolate is contemplated, as described in U. S. Patent No. 4,435,386. Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U. S. Patent No. 4,505,899), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U. S. Patent Nos. 4,436,727, 4,436,728 and 4,505,900.
Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins are also envisioned to be useful, as described in U. S. Patent No. 4,520,019.
MPL is currently one preferred immunopotentiating agent for use herein.
References that concern the uses of MPL include Tomai et al. (1987), Chen et al. (1991) and Garg and Subbarao (1992), that each concern certain roles of MPL in the reactions of aging mice;
Elliott et al. ( 1991 ), that concerns the D-galactosamine loaded mouse and its enhanced sensitivity to lipopolysaccharide and MPL; Chase et al. (1986), that relates to bacterial infections; and Masihi et al. (1988), that describes the effects of MPL and endotoxin on resistance of mice to Toxoplasma gondii. Fitzgerald (199I) also reported on the use of MPL

to up-regulate the immunogenicty of a syphilis vaccine and to confer significant protection against challenge infection in rabbits.
Thus MPL is known to be safe for use, as shown in the above model systems.
Phase-I clinical trials have also shown MPL to be safe for use (Vosika et al., 1984). Indeed, I00 ~g/mz is known to be safe for human use, even on an outpatient basis (Vosika et al., 1984).
MPL generally induces polyclonal B cell activation (Baker et al., 1994), and has been shown to augment antibody production in many systems, for example, in immunologically immature mice (Baker et al., 1988); in aging mice (Tomai and Johnson; 1989);
and in nude and Xid mice (Madonna and Vogel, 1986; Myers et al., 1995). Antibody production has been shown against erythrocytes (Hraba et al., 1993); T cell dependent and independent antigens; Pnu-immune vaccine (Garg and Subbarao, 1992); isolated tumor-associated antigens (U. S. Patent 4,877,611); against syngeneic tumor cells (Livingston et al., 1985;
Ravindranath et al., 1994a;b); and against tumor-associated gangliosides (Ravindranath et al., 1994a;b).
Another useful attribute of MPL is that is augments IgM. responses, as shown by Baker et al. (1988a), who describe the ability of MPL to increase antibody responses in young mice. This is a particularly useful feature of an adjuvant for use in certain embodiments of the present invention. Myers et al. (1995) recently reported on the ability of MPL to induce IgM antibodies, by virtue T cell-independent antibody production.
In the Myers et al. (I995) studies, MPL was conjugated to the hapten, TNP. MPL
was proposed for use as a carrier for other haptens, such as peptides.
MPL also activates and recruits macrophages (Verma et al., 1992). Tomai and Johnson (1989) showed that MPL-stimulated T cells enhance IL-1 secretion by macrophages.
MPL is also known to activate superoxide production, lysozyme activity, phagocytosis, and killing of Candida in murine peritoneal macrophages (Chen et al., 1991).
The effects of MPL on T cells include the endogenous production of cytotoxic factors, such as TNF, in serum of BCG-primed mice by MPL (Bennett et al., 1988). Kovach et al. (1990) and Elliot et al. (1991) also show that MPL induces TNF
activity. MPL is known to act with TNF-a to induce release of IFN-y by NK cells. IFN-'y production by T cells in response to MPL was also documented by Tomai and Johnson (1989), and Odean et al. (1990).
MPL is also known to be a potent T cell adjuvant. For example, MPL stimulates proliferation of melanoma-antigen specific CTLs (Mitchell et al., 1988, 1993).
Further, Baker et al. (1988b) showed that nontoxic MPL inactivated suppressor T cell activity.
Naturally, in the physiological environment, the inactivation of T suppressor cells allows for increased benefit for the animal, as realized by, e.g., increased antibody production. Johnson and Tomai (I988) have reported on the possible cellular and molecular mediators of the adjuvant action of MPL.
MPL is also known to induce aggregation of platelets and to phosphorylate a platelet protein prior to induction of serotonin secretion (Grabarek et al., 1990).
This study shows that MPL is involved in protein kinase C activation and signal transduction.
Many articles concern the structure and function of MPL include. These include Johnson et al. (1990), that describes the structural characterization of MPL
homologs obtained from Salmonella minnesota Re595 lipopolysaccharide. The work of Johnson et al.
(1990), in common with Grabarek et al. (1990), shows that the fatty acid moieties of MPL
can vary, even in commercial species. In separating MPL into eight fractions by thin layer chromatography, Johnson et al. (I990) found that three were particularly active, as assessed using human platelet responses. The chemical components of the various MPL
species were characterized by Johnson et al. (1990).
Baker et al. (1992) further analyzed the structural features that influence the ability of lipid A and its analogs to abolish expression of suppressor T cell activity.
They reported that decreasing the number of phosphate groups in lipid A from two to one (i.e., creating monophosphoryl lipid A, MPL) as well as decreasing the fatty acyl content, primarily by removing the residue at the 3 position, resulted in a progressive reduction in toxicity;
however, these structural modifications did not influence its ability to abolish the expression of Ts function (Baker et al., 1992). These types of MPL are ideal for use in the present invention.
Baker et al. (1992) also showed that reducing the fatty acyl content from five to four (lipid A precursor IVA or Ia) eliminated the capacity to influence Ts function but not to induce polyclonal activation of B cells. These studies show that in order to be able to abolish the expression of Ts function, lipid A must be a glucosamine disaccharide; may have either one or two phosphate groups; and must have at least ftve fatty acyl groups. Also, the chain length of the nonhydroxylated fatty acid, as well as the Location of acyloxyacyl groups (2' versus 3' position), may play an important role (Baker et al., 1992).
In examining the relationship between chain length and position of fatty acyl groups on the ability of lipid A to abolish the expression of suppressor T-cell (Ts) activity, Baker et al. (1994) found that fatty acyl chain lengths of C,Z to C,4 appeared to be optimal for bioactivity. Therefore, although their use is still possible, lipid A
preparations with fatty acyl groups of relatively short chain length (C,o to C,Z from Pseudomohas aef°ugif~osa and Clar~omobacte~ium violaceum) or predominantly long chain length (C18 from Helicobacter pylori) are less preferred for use in this invention.
Baker et al. (1994) also showed that the lipid A proximal inner core region oligosaccharides of some bacterial lipopolysaccharides increase the expression of Ts activity;
due mainly to the capacity of such oligosaccharides, which are relatively conserved in structure among gram-negative bacterial, to enlarge or expand upon the population of CD8+
Ts generated during the course of a normal antibody response to unrelated microbial antigens. The minimal structure required for the expression of the added immunosuppression observed was reported to be a hexasaccharide containing one 2-keto-3-deoxyoctonate residue, two glucose residues, and three heptose residues to which are attached two pyrophosphorylethanolamine groups (Baker et al., 1994). This information may be considered in utilizing or even designing further adjuvants for use in the invention.
In a generally related line of work, Tanamoto et al. (1994a;b; 1995) described the dissociation of endotoxic activities in a chemically synthesized Lipid A
precursor after acetylation or succinylation. Thus, compounds such as "acetyl 406" and "succinyl 516"
(Tanamoto et al., 1994a;b; 1995) are also contemplated for use in the invention.
Synthetic MPLs form a particularly preferred group of antigens. For example, Brade et al. (1993) described an artificial glycoconjugate containing the bisphosphorylated glucosamine disaccharide backbone of lipid A that binds to anti-Lipid A MAbs.
This is one candidate for use in certain aspects of the invention.

The MPL derivatives described in U. S. Patent No. 4,987,237 are particularly contemplated for use in the present invention. U. S. Patent No. 4,987,237 describes MPL
derivatives that contain one or more free groups, such as amines, on a side chain attached to the primary hydroxyl groups of the monophosphoryl lipid A nucleus through an ester group.
5 The derivatives provide a convenient method for coupling the lipid A through coupling agents to various biologically active materials. The immunostimulant properties of lipid A
are maintained. A11~ MPL derivatives in accordance with U. S. Patent No.
4,987,237 are envisioned for use in the MPL adjuvant-incorporated cells of this invention.
Various adjuvants, even those that are not commonly used in humans, may still be 10 employed in animals, where, for example, one desires to raise antibodies or to subsequently obtain activated T cells. The toxicity or other adverse effects that may result from either the adjuvant or the cells, e.g., as may occur using non-irradiated tumor cells, is irrelevant in such circumstances.
Within the vaccines provided herein, the adjuvant composition is preferably designed 15 to induce an immune response predominantly of the Thl type. High levels of Thl-type cytokines (e.g., IFN-y, TNFa, 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 20 response that includes Thl- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Thl-type, the level of Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines see e.g., Mosmann and Coffman (1989).
25 Preferred adjuvants for use in eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated mono-phosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, WA; see e.g., U. S. Patent Nos. 4,436,727;
4,877,611;
4,866,034 and 4,912,094, each of which is specifically incorporated herein by reference in its 30 entirety). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Thl response. Such oligonucleotides are well known and are described, for example, in Intl. Pat. Appl. Publ. No. WO 96/02555 and Intl.
Pat. Appl. Publ.
No. WO 99/33488. Immunostimulatory DNA sequences are also described, for example, by Sato et al. (1996). Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, MA), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL (see e.g., Intl. Pat. Appl. Publ. No. WO 94/00153), or a less reactogenic composition where the QS21 is quenched with cholesterol (see e.g., Intl. Pat. Appl. Publ.
No.
WO 96/33739). Other preferred formulations comprise an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL
and tocopherol in an oil-in-water emulsion has also been described (see e.g., Intl. Pat. Appl. Publ.
No. WO 95/17210).
Other preferred adjuvants include Montanide ISA 720 (Seppic), SAF (Chiron), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa Corporation), RC-529 (Corixa Corporation) and aminoalkyl glucosaminide 4-phosphates (AGPs).
Any vaccine provided herein may be prepared using well-known methods that result in a combination of one or more antigens, one or more immunostimulants or adjuvants and one or more suitable carriers, excipients, or pharmaceutically acceptable buffers. The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel [composed of polysaccharides, for example] that effects a slow release of compound following administration).
Such formula-dons may generally be prepared using well-known technology (Coombes et al., 1996) and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a peptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate-controlling membrane.
Carriers for use within such formulations are preferably biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. Such carriers include microparticles of poly(lactide-co-glycolide), as well as polyacrylate, latex, starch, cellulose and dextran. Other 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 (U. S. Patent No. 5,151,254;
Tntl. Pat.
Appl. Publ. No. WO 94/20078; Intl. Pat. Appl. Publ. No. W0/94/23701; and Intl.
Pat. Appl.
Publ. No. 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.
I0 Any of a variety of delivery vehicles may be employed within pharmaceutical compo-sitions and vaccines to facilitate production of an antigen-specific immune response that targets tumor cells. Delivery vehicles include antigen-presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (Timmerman and Levy, I999). In general, dendritic cells may be identified based on their typical shape (stellate ih situ, with marked cytoplasmic processes (dendrites) visible in vitf~o), their ability to take up, process and present antigens with high efficiency and their ability to activate naive 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 (Zitvogel et al., 1998).
Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFa to cultures of monocytes harvested from peripheral blood.
Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFa, CD40 ligand, LPS, flt3 ligand and/or other compounds) that induce differentia-tion, 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 I5 differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcy receptor and mamiose 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 activa tion such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD1I) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).
APCs may generally be transfected with a polynucleotide encoding a WT1 peptide, such that the peptide, or an immunogenic portion thereof, is expressed on the cell surface.
Such .transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alter-natively, 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 Intl. Pat. Appl. Publ. No. WO
97/24447, or the gene gun approach described by Mahvi et al. (1997). Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the WTI
peptide, 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 peptide may be covalently conjugated to an immunoIogicaI partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a S non-conjugated immunological partner, separately or in the presence of the peptide.
Combined therapeutics is also contemplated, and the same type of underlying pharmaceutical compositions may be employed for both single and combined medicaments.
Vaccines and pharmaceutical compositions may be presented in unit-dose or mufti-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles.
Alternatively, a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
I S 4.S METHODS FOR THE THERAPY OF MALIGNANT DISEASE
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 WTI 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., 2S tumor cells) at some time during the course of the disease generate detectably higher levels of a WT1 peptide 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 expres-sion may subsequently be lost. Alternatively, a malignant disease that is not characterized by an increase in WTl expression may, at a Iater time, progress to a disease that is characterized by increased WTl expression. Accordingly, any malignant disease in which diseased cells formerly expressed, currently express or are expected to subsequently express increased levels of WTI is considered to be "associated with WTI expression." Within certain embodiments, the therapies provided herein are administered to a patient afflicted with, or considered at risk for, malignant mesothelioma.
5 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 WTI ar a cell expressing WT1. Alternatively, WTl-expressing cells may be removed ex vivo (e.g., by treatment of autologous bone marrow, peripheral blood or a 10 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 adminis-tered 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 15 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 20 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 malignant mesothelioma.
Other. WT1-associated conditions include leukemia (e.g., AML, CML, ALL or childhood ALL), a myelodysplastic syndrome (MDS) and cancer (e.g., gastrointestinal, lung, thyroid or 25 breast cancer or a melanoma), where the cancer or leukemia is WT1 positive (i.e., reacts detectably with an anti-WTI antibody, as provided herein or expresses WT1 mRNA
at a level detectable by RT-PCRTM, as described herein), as well as autoimmune diseases directed against WTl-expressing cells.
Other diseases associated with WT1 overexpression include kidney cancer (such as 30 renal cell carcinoma, or Wilms tumor), as described in Satoh et al. (2000), and Campbell et al. (1998j; and mesothelioma, as described in Amin et al., (1995). Harada et al. (1999) describe WTl gene expression in human testicular germ-cell tumors. Nonomura et al.
Hinyokika (1999) describe molecular staging of testicular cancer using polymerase chain reaction of the testicular cancer-specific genes. Shimizu et al. (2000) describe the immunohistochemical detection of the Wilms' tumor gene (WTl) in epithelial ovarian tumors.
WT1 overexpression was also described in desmoplastic small round cell tumors, by Barnoud, et al., (2000). WT1 overexpression in glioblastoma and other cancer was described by Menssen et al., (2000), "Wilms' 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's lymphoma and nasopharyngeal cancer (Spinsanti et al., 2000), "Wilms' tumor gene expression by normal and malignant human B lymphocytes."
Pan et al. (2000) describe ifs 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 WTl gene expression. Patmasiriwat et al. (1999) reported WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia.
Tamaki et al. (1999) reported that the Wilms tumor gene WTl is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Expression of the Wilms tumor gene WTl 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.
(1999).
The compositions provided herein may be used alone or in combination with conven-tional 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 ih vivo. Such WTl-specific T cells may be used, for example, within donor lymphocyte infusions.
Routes and frequency of administration, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracu-taneous, 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 I and I O 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'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 peptides, the amount of each peptide present in a dose ranges from about 100 ~.g 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 compounds) 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 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 WTI peptide 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 devel-opment of a malignant disease. Typically, about 1 x 10~ to I x I O" T cells/MZ
are adminis-tered 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 depend-ent upon the response of the patient.
Within certain embodiments, T cells may be stimulated prior to autologous bone marrow transplantation. Such stimulation may take place i~ vivo or iya vitro.
For iyz vitf~o 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 WTl peptide, a polynucleotide encoding a WT1 peptide and/or an APC that expresses a WT1 peptide under conditions and for a time sufficient to permit the stimulation of T cells as described above.
Bone marrow, peripheral blood stem cells and/or WTl-specific T cells may then be administered to a patient using standard techniques.
Within related embodiments, T cells of a related or unrelated donor may be stimu-lated prior to 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 peptide, WTI
polynucleotide and/or APC that expresses a WT1 peptide 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 IO%, preferably less than 5% and more preferably Iess 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 PCRTM
analysis, morphology, immunohistochemistry and FACS analysis. Bone marrow or PB (or a fraction thereof) may then be administered to a patient using standard techniques.
I O 4.~ DIAGNOSTIC AND PROG

Claims

CLAIMS:

1. Use of a composition comprising at least a first isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide, in the manufacture of a medicament for treating or preventing mesothelioma;

wherein said peptide comprises a first contiguous amino acid sequence according to any one of SEQ ID NO:1 to SEQ ID NO:4, SEQ ID NO:13 to SEQ ID NO:20, SEQ
ID NO:28 to SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:316 to SEQ ID NO:318, and SEQ ID NO:321 to SEQ ID NO:326.

2. Use according to claim 1, wherein said composition comprises at least a first isolated peptide of from 9 to about 35 amino acids in length, or at least a first nucleic acid segment that encodes said peptide.

3. Use according to claim 1 or claim 2, wherein said composition comprises at least a first isolated peptide of from 9 to about 30 amino acids in length, or at least a first nucleic acid segment that encodes said peptide.

4. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 25 amino acids in length, or at least a first nucleic acid segment that encodes said peptide.

5. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 20 amino acids in length, or at least a first nucleic acid segment that encodes said peptide.

6. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 15 amino acids in length, or at least a first nucleic acid segment that encodes said peptide.

7. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 13 amino acids in length, or at least a first nucleic acid segment that encodes said peptide.

8. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 11 amino acids in length, or at least a first nucleic acid segment that encodes said peptide.

9. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said peptide comprising a first contiguous amino acid sequence according to any one of SEQ ID NO:13 to SEQ ID
NO:20, SEQ ID NO:28 to SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:316 to SEQ ID NO:318.

10. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said peptide comprising a first contiguous amino acid sequence according to any one of SEQ ID NO:28 to SEQ ID
NO:311, SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:316 to SEQ ID
NO:318.

11. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said peptide comprising at least a first contiguous amino acid sequence selected from the group consisting of ALLPAVPSL

(SEQ ID NO:34), ALLPAVSSL (SEQ ID NO:35), CMTWNQMNL (SEQ ID
NO:49), GATLKGVAA (SEQ ID NO:88), NLYQMTSQL (SEQ ID NO:147), RMFPNAPYL (SEQ ID NO:185), SCLESQPAI (SEQ ID NO:198), and SCLESQPTI
(SEQ ID NO:199).

12. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 11 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said peptide consisting essentially of the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO:4, SEQ ID
NO:13 to SEQ ID NO:20, SEQ ID NO:28 to SEQ ID NO:311, SEQ ID NO:313, SEQ
ID NO:314, SEQ ID NO:316 to SEQ ID NO:318, and SEQ ID NO:321 to SEQ ID
NO:326.

13. Use according to any preceding claim, wherein said composition comprises at least a .
first isolated peptide of from 9 to about 11 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said peptide consisting essentially of the amino acid sequence of any one of SEQ ID NO:13 to SEQ ID NO:20, SEQ ID
NO:28 to SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:316 to SEQ ID NO:318.

14. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide of from 9 to about 11 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said peptide consisting essentially of the amino acid sequence ALLPAVPSL (SEQ ID NO:34), ALLPAVSSL (SEQ ID
NO:35), CMTWNQMNL (SEQ ID NO:49), GATLKGVAA (SEQ ID NO:88), NLYQMTSQL (SEQ ID NO:147), RMFPNAPYL (SEQ ID NO:185), SCLESQPAI
(SEQ ID NO:198), or SCLESQPTI (SEQ ID NO:199).

15. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide that consists of the amino acid sequence according to any one of SEQ ID NO:1 to SEQ ID NO:4, SEQ ID NO:13 to SEQ ID NO:20, SEQ ID NO:28 to SEQ ID NO:3I1, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:316 to SEQ ID
NO:318, and SEQ ID NO:321 to SEQ ID NO:326, or at least a first nucleic acid segment that encodes said peptide.

16. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide that consists of the amino acid sequence according to any one of SEQ ID NO:28 to SEQ ID NO:311, or at least a first nucleic acid segment that encodes said peptide.

17. Use according to any preceding claim, wherein said composition comprises at least a first isolated peptide that consists of the amino acid sequence ALLPAVPSL (SEQ
ID
NO:34), ALLPAVSSL (SEQ ID NO:35), CMTWNQMNL (SEQ ID NO:49), GATLKGVAA (SEQ ID NO:88), NLYQMTSQL (SEQ ID NO:147), RMFPNAPYL
(SEQ ID NO:185), SCLESQPAI (SEQ ID NO:198), or SCLESQPTI (SEQ ID
NO:199); or at least a first nucleic acid segment that encodes said peptide.

18. Use according to any preceding claim, wherein said composition further comprises at least a second isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said second peptide comprising at least a first contiguous amino acid sequence according to any one of SEQ ID
NO:1 to SEQ ID NO:4, SEQ ID NO:13 to SEQ ID NO:20, SEQ ID NO:28 to SEQ ID
NO:311, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:316 to SEQ ID NO:318, and SEQ ID NO:321 to SEQ ID NO:326.

19. Use according to any preceding claim, wherein said composition further comprises at least a second isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said second peptide comprising at least a first contiguous amino acid sequence according to any one of SEQ ID

NO:13 to SEQ ID NO:20, SEQ ID NO:28 to SEQ ID NO:311, SEQ ID NO:313, SEQ
ID NO:314, and SEQ ID NO:316 to SEQ ID NO:318.

20. Use according to any preceding claim, wherein said composition further comprises at least a second isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said second peptide comprising at least a first contiguous amino acid selected from the group consisting of ALLPAVPSL (SEQ ID NO:34), ALLPAVSSL (SEQ ID NO:35), CMTWNQMNL
(SEQ ID NO:49), GATLIKGVAA (SEQ ID NO:88), NLYQMTSQL (SEQ ID
NO:147), RMFPNAPYL (SEQ ID NO:18S), SCLESQPAI (SEQ ID NO:198), and SCLESQPTI (SEQ ID NO:199).

21. Use according to any one of claims 18 to 20, wherein said composition further comprises at least a third isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said third peptide comprising at least a first contiguous amino acid sequence according to any one of SEQ ID NO:1 to SEQ ID NO:4, SEQ ID NO:13 to SEQ ID NO:20, SEQ ID NO:28 to SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:316 to SEQ ID
NO:318, and SEQ ID NO:321 to SEQ ID NO:326.

22. Use according to any one of claims 18 to 21, wherein said composition further comprises at least a third isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said third peptide comprising at least a first contiguous amino acid sequence according to any one of SEQ ID NO:28 to SEQ ID NO:311.

23. Use according to any one of claims 18 to 22, wherein said composition further comprises at least a third isolated peptide of from 9 to about 40 amino acids in length, or at least a first nucleic acid segment that encodes said peptide; said third peptide comprising at least a first contiguous amino acid selected from the group consisting of ALLPAVPSL (SEQ ID NO:34), ALLPAVSSL (SEQ ID NO:35), CMTWNQMNL
(SEQ ID NO:49), GATLKGVAA (SEQ ID NO:88), NLYQMTSQL (SEQ ID
NO:147), RMFPNAPYL (SEQ ID NO:185), SCLESQPAI (SEQ ID NO:198), and SCLESQPTI (SEQ ID NO:199).

24. Use according to any preceding claim, wherein said composition comprises at least a first nucleic acid segment of from 27 to about 5000 nucleotides in length.

25. Use according to any preceding claim, wherein said composition comprises at least a first nucleic acid segment of from 27 to about 3000 nucleotides in length.

26. Use according to any preceding claim, wherein said composition comprises at least a first nucleic acid segment of from 27 to about 1000 nucleotides in length.

27. Use according to any preceding claim, wherein said composition comprises at least a first nucleic acid segment of from 27 to about 500 nucleotides in length.

28. Use according to any preceding claim, wherein said composition comprises at least a first nucleic acid segment operably positioned under the control of at least a first heterologous promoter.

29. Use according to any preceding claim, wherein said composition comprises at least a first nucleic acid segment that is comprised within a vector.

30. Use according to any preceding claim, wherein said composition comprises at least a first nucleic acid segment that is comprised within a plasmid or viral vector.

31. Use according to any preceding claim, wherein said composition further comprises at least a first pharmaceutically acceptable excipient.

32. Use according to any preceding claim, wherein said composition further comprises at least a first immunostimulant or at least a first adjuvant.

33. Use according to any preceding claim, wherein said composition further comprises at least a first immunostimulant or at least a first adjuvant that preferentially enhances a T-cell response in a human.

34. Use according to any preceding claim, wherein said composition further comprises at least a first immunostimulant or at least a first adjuvant selected from the group consisting of Montanide ISA50, Seppic Montanide ISA720, a cytokine, a microsphere, a dimethyl dioctadecyl ammonium bromide adjuvant, AS-1, AS-2, Ribi Adjuvant, QS21, saponin, microfluidized Syntex adjuvant, MV, ddMV, an immune stimulating complex and an inactivated toxin.

35. Use according to any preceding claim, wherein said medicament is intended for generating a T cell response in a patient with mesothelioma.

36. Use according to any preceding claim, wherein said medicament is intended for administration to a patient with malignant pleural mesothelioma.

37. Use according to any preceding claim, wherein said medicament is formulated for parenteral, intravenous, intraperitoneal, subcutaneous, intranasal, transdermal, or oral administration.

38. Use according to any preceding claim, wherein said composition further comprises at least a first detection reagent.

39. Use according to any preceding claim, wherein said composition further comprises at least a first detection reagent that specifically binds to a WT1 peptide or polypeptide.

40. Use according to any preceding claim, wherein said composition further comprises at least a second therapeutic agent for treating or preventing mesothelioma.