Compositions and methods for WT1 specific immunotherapy

Tag : dimethyl disulfide


Abstract
Compositions and methods for the therapy of malignant diseases,such as leukemia and cancer, are disclosed. The compositionscomprise one or more of a WT1 polynucleotide, a WT1 polypeptide, anantigen-presenting cell presenting a WT1 polypeptide, an antibodythat specifically binds to a WT1 polypeptide; or a T cell thatspecifically reacts with a WT1 polypeptide. Such compositions maybe used, for example, for the prevention and treatment ofmetastatic diseases.
Description of the Invention
The present invention is generally directed to compositions andmethods for the immunotherapy and diagnosis of malignant diseases.The compositions described herein may include WT1 polypeptides, WT1polynucleotides, antigen-presenting cells (APC, e.g., dendriticcells) that express a WT1 polypeptide, agents such as antibodiesthat bind to a WT1 polypeptide and/or immune system cells (e.g., Tcells) specific for WT1. WT1 Polypeptides of the present inventiongenerally comprise at least a portion of a Wilms Tumor gene product(WT1) or a variant thereof. Nucleic acid sequences of the subjectinvention generally comprise a DNA or RNA sequence that encodes allor a portion of such a polypeptide, or that is complementary tosuch a sequence. Antibodies are generally immune system proteins,or antigen-binding fragments thereof, that are capable of bindingto a portion of a WT1 polypeptide. T cells that may be employedwithin such compositions are generally T cells (e.g., CD4.sup.+and/or CD8.sup.+) that are specific for a WT1 polypeptide. Certainmethods described herein further employ antigen-presenting cellsthat express a WT1 polypeptide as provided herein.

The present invention is based on the discovery that an immuneresponse raised against a Wilms Tumor (WT) gene product (e.g., WT1)can provide prophylactic and/or therapeutic benefit for patientsafflicted with malignant diseases characterized by increased WT1gene expression. Such diseases include, but are not limited to,leukemias (e.g., acute myeloid leukemia (AML), chronic myeloidleukemia (CML), acute lymphocytic leukemia (ALL) and childhoodALL), as well as many cancers such as lung, breast, thyroid andgastrointestinal cancers and melanomas. The WT1 gene was originallyidentified and isolated on the basis of a cytogenetic deletion atchromosome 11p13 in patients with Wilms' tumor (see Call et al.,U.S. Pat. No. 5,350,840). The gene consists of 10 exons and encodesa zinc finger transcription factor, and sequences of mouse andhuman WT1 proteins are provided in FIG. 1 and SEQ ID NOs: 319 and320 (see Original Patent).

WT1 Polypeptides

Within the context of the present invention, a WT1 polypeptide is apolypeptide that comprises at least an immunogenic portion of anative WT1 (i.e., a WT1 protein expressed by an organism that isnot genetically modified), or a variant thereof, as describedherein. A WT1 polypeptide may be of any length, provided that itcomprises at least an immunogenic portion of a native protein or avariant thereof. In other words, a WT1 polypeptide may be anoligopeptide (i.e., consisting of a relatively small number ofamino acid residues, such as 8-10 residues, joined by peptidebonds), a full length WT1 protein (e.g., present within a human ornon-human animal, such as a mouse) or a polypeptide of intermediatesize. Within certain embodiments, the use of WT1 polypeptides thatcontain a small number of consecutive amino acid residues of anative WT1 polypeptide is preferred. Such polypeptides arepreferred for certain uses in which the generation of a T cellresponse is desired. For example, such a WT1 polypeptide maycontain less than 23, preferably no more than 18, and morepreferably no more than 15 consecutive amino acid residues, of anative WT1 polypeptide. Polypeptides comprising nine consecutiveamino acid residues of a native WT1 polypeptide are generallysuitable for such purposes. Additional sequences derived from thenative protein and/or heterologous sequences may be present withinany WT1 polypeptide, and such sequences may (but need not) possessfurther immunogenic or antigenic properties. Polypeptides asprovided herein may further be associated (covalently ornoncovalently) with other polypeptide or non-polypeptide compounds.

An "immunogenic portion," as used herein is a portion ofa polypeptide that is recognized (i.e., specifically bound) by aB-cell and/or T-cell surface antigen receptor. Certain preferredimmunogenic portions bind to an MHC class I or class II molecule.As used herein, an immunogenic portion is said to "bindto" an MHC class I or class II molecule if such binding isdetectable using any assay known in the art. For example, theability of a polypeptide to bind to MHC class I may be evaluatedindirectly by monitoring the ability to promote incorporation of.sup.125I labeled .beta.2-microglobulin (.beta.2m) into MHC classI/.beta.2m/peptide heterotrimeric complexes (see Parker et al., J.Immunol. 152:163, 1994). Alternatively, functional peptidecompetition assays that are known in the art may be employed.Certain immunogenic portions have one or more of the sequencesrecited within one or more of Tables II-XIV (see Original Patent).Representative immunogenic portions include, but are not limitedto, RDLNALLPAVPSLGGGG (human WT1 residues 6-22; SEQ ID NO:1),PSQASSGQARMFPNAPYLPSCLE (human and mouse WT1 residues 117-139; SEQID NOs: 2 and 3 respectively), GATLKGVAAGSSSSVKWTE (human WT1residues 244-262; SEQ ID NO:4), GATLKGVAA (human WT1 residues244-252; SEQ ID NO:88), CMTWNQMNL (human and mouse WT1 residues235-243; SEQ ID NOs: 49 and 258 respectively), SCLESQPTI (mouse WT1residues 136-144; SEQ ID NO:296), SCLESQPAI (human WT1 residues136-144; SEQ ID NO: 198), NLYQMTSQL (human and mouse WT1 residues225-233; SEQ ID NOs: 147 and 284 respectively); ALLPAVSSL (mouseWT1 residues 10-18; SEQ ID NO:255); or RMFPNAPYL (human and mouseWT1 residues 126-134; SEQ ID NOs: 185 and 293 respectively).Further immunogenic portions are provided herein, and others maygenerally be identified using well known techniques, such as thosesummarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (RavenPress, 1993) and references cited therein. Representativetechniques for identifying immunogenic portions include screeningpolypeptides for the ability to react with antigen-specificantisera and/or T-cell lines or clones. An immunogenic portion of anative WT1 polypeptide is a portion that reacts with such antiseraand/or T-cells at a level that is not substantially less than thereactivity of the full length WT1 (e.g., in an ELISA and/or T-cellreactivity assay). In other words, an immunogenic portion may reactwithin such assays at a level that is similar to or greater thanthe reactivity of the full length polypeptide. Such screens maygenerally be performed using methods well known to those ofordinary skill in the art, such as those described in Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988.

Alternatively, immunogenic portions may be identified usingcomputer analysis, such as the Tsites program (see Rothbard andTaylor, EMBO J. 7:93-100, 1988; Deavin et al., Mol. Immunol.33:145-155, 1996), which searches for peptide motifs that have thepotential to elicit Th responses. CTL peptides with motifsappropriate for binding to murine and human class I or class II MHCmay be identified according to BIMAS (Parker et al., J. Immunol.152:163, 1994) and other HLA peptide binding prediction analyses.To confirm immunogenicity, a peptide may be tested using an HLA A2transgenic mouse model and/or an in vitro stimulation assay usingdendritic cells, fibroblasts or peripheral blood cells.

As noted above, a composition may comprise a variant of a nativeWT1 protein. A polypeptide "variant," as used herein, isa polypeptide that differs from a native polypeptide in one or moresubstitutions, deletions, additions and/or insertions, such thatthe immunogenicity of the polypeptide is retained (i.e., theability of the variant to react with antigen-specific antiseraand/or T-cell lines or clones is not substantially diminishedrelative to the native polypeptide). In other words, the ability ofa variant to react with antigen-specific antisera and/or T-celllines or clones may be enhanced or unchanged, relative to thenative polypeptide, or may be diminished by less than 50%, andpreferably less than 20%, relative to the native polypeptide. Suchvariants may generally be identified by modifying one of the abovepolypeptide sequences and evaluating the reactivity of the modifiedpolypeptide with antisera and/or T-cells as described herein. Ithas been found, within the context of the present invention, that arelatively small number of substitutions (e.g., 1 to 3) within animmunogenic portion of a WT1 polypeptide may serve to enhance theability of the polypeptide to elicit an immune response. Suitablesubstitutions may generally be identified by using computerprograms, as described above, and the effect confirmed based on thereactivity of the modified polypeptide with antisera and/or T-cellsas described herein. Accordingly, within certain preferredembodiments, a WT1 polypeptide comprises a variant in which 1 to 3amino acid resides within an immunogenic portion are substitutedsuch that the ability to react with antigen-specific antiseraand/or T-cell lines or clones is statistically greater than thatfor the unmodified polypeptide. Such substitutions are preferablylocated within an MHC binding site of the polypeptide, which may beidentified as described above. Preferred substitutions allowincreased binding to MHC class I or class II molecules.

Certain variants contain conservative substitutions. A"conservative substitution" is one in which an amino acidis substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expectthe secondary structure and hydropathic nature of the polypeptideto be substantially unchanged. Amino acid substitutions maygenerally be made on the basis of similarity in polarity, charge,solubility, hydrophobicity, hydrophilicity and/or the amphipathicnature of the residues. For example, negatively charged amino acidsinclude aspartic acid and glutamic acid; positively charged aminoacids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values includeleucine, isoleucine and valine; glycine and alanine; asparagine andglutamine; and serine, threonine, phenylalanine and tyrosine. Othergroups of amino acids that may represent conservative changesinclude: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys,ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his;and (5) phe, tyr, trp, his. A variant may also, or alternatively,contain nonconservative changes. Variants may also (oralternatively) be modified by, for example, the deletion oraddition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

In a preferred embodiment, a variant polypeptide of the WT1N-terminus (amino acids 1-249) is constructed, wherein the variantpolypeptide is capable of binding to an antibody that recognizesfull-length WT1 and/or WT1 N-terminus polypeptide. A non-limitingexample of an antibody is anti WT-1 antibody WT180 (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.).

As noted above, WT1 polypeptides may be conjugated to a signal (orleader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. A polypeptide may also, or alternatively, be conjugated toa linker or other sequence for ease of synthesis, purification oridentification of the polypeptide (e.g., poly-His), or to enhancebinding of the polypeptide to a solid support. For example, apolypeptide may be conjugated to an immunoglobulin Fc region.

WT1 polypeptides may be prepared using any of a variety of wellknown techniques. Recombinant polypeptides encoded by a WT1polynucleotide as described herein may be readily prepared from thepolynucleotide. In general, any of a variety of expression vectorsknown to those of ordinary skill in the art may be employed toexpress recombinant WT1 polypeptides. Expression may be achieved inany appropriate host cell that has been transformed or transfectedwith an expression vector containing a DNA molecule that encodes arecombinant polypeptide. Suitable host cells include prokaryotes,yeast and higher eukaryotic cells. Preferably, the host cellsemployed are E. coli, yeast or a mammalian cell line such as COS orCHO. Supernatants from suitable host/vector systems which secreterecombinant protein or polypeptide into culture media may be firstconcentrated using a commercially available filter. The concentratemay then be applied to a suitable purification matrix such as anaffinity matrix or an ion exchange resin. Finally, one or morereverse phase HPLC steps can be employed to further purify arecombinant polypeptide. Such techniques may be used to preparenative polypeptides or variants thereof. For example,polynucleotides that encode a variant of a native polypeptide maygenerally be prepared using standard mutagenesis techniques, suchas oligonucleotide-directed site-specific mutagenesis, and sectionsof the DNA sequence may be removed to permit preparation oftruncated polypeptides.

Certain portions and other variants may also be generated bysynthetic means, using techniques well known to those of ordinaryskill in the art. For example, polypeptides having fewer than about500 amino acids, preferably fewer than about 100 amino acids, andmore preferably fewer than about 50 amino acids, may besynthesized. Polypeptides may be synthesized using any of thecommercially available solid-phase techniques, such as theMerrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield,J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automatedsynthesis of polypeptides is commercially available from supplierssuch as Applied BioSystems, Inc. (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

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

Within further aspects, the present invention provides mimetics ofWT1 polypeptides. Such mimetics may comprise amino acids linked toone or more amino acid mimetics (i.e., one or more amino acidswithin the WT1 protein may be replaced by an amino acid mimetic) ormay be entirely nonpeptide mimetics. An amino acid mimetic is acompound that is conformationally similar to an amino acid suchthat it can be substituted for an amino acid within a WT1polypeptide without substantially diminishing the ability to reactwith antigen-specific antisera and/or T cell lines or clones. Anonpeptide mimetic is a compound that does not contain amino acids,and that has an overall conformation that is similar to a WT1polypeptide such that the ability of the mimetic to react withWT1-specific antisera and/or T cell lines or clones is notsubstantially diminished relative to the ability of a WT1polypeptide. Such mimetics may be designed based on standardtechniques (e.g., nuclear magnetic resonance and computationaltechniques) that evaluate the three dimensional structure of apeptide sequence. Mimetics may be designed where one or more of theside chain functionalities of the WT1 polypeptide are replaced bygroups that do not necessarily have the same size or volume, buthave similar chemical and/or physical properties which producesimilar biological responses. It should be understood that, withinembodiments described herein, a mimetic may be substituted for aWT1 polypeptide.

Within other illustrative embodiments, a polypeptide may be afusion polypeptide that comprises multiple polypeptides asdescribed herein, or that comprises at least one polypeptide asdescribed herein and an unrelated sequence, such as a known tumorprotein. A fusion partner may, for example, assist in providing Thelper epitopes (an immunological fusion partner), preferably Thelper epitopes recognized by humans, or may assist in expressingthe protein (an expression enhancer) at higher yields than thenative recombinant protein. Certain preferred fusion partners areboth immunological and expression enhancing fusion partners. Otherfusion partners may be selected so as to increase the solubility ofthe polypeptide or to enable the polypeptide to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of thepolypeptide.

Fusion polypeptides may generally be prepared using standardtechniques, including chemical conjugation. Preferably, a fusionpolypeptide is expressed as a recombinant polypeptide, allowing theproduction of increased levels, relative to a non-fusedpolypeptide, in an expression system. Briefly, DNA sequencesencoding the polypeptide components may be assembled separately,and ligated into an appropriate expression vector. The 3' end ofthe DNA sequence encoding one polypeptide component is ligated,with or without a peptide linker, to the 5' end of a DNA sequenceencoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation intoa single fusion polypeptide that retains the biological activity ofboth component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its secondary and tertiarystructures. Such a peptide linker sequence is incorporated into thefusion polypeptide using standard techniques well known in the art.Suitable peptide linker sequences may be chosen based on thefollowing factors: (1) their ability to adopt a flexible extendedconformation; (2) their inability to adopt a secondary structurethat could interact with functional epitopes on the first andsecond polypeptides; and (3) the lack of hydrophobic or chargedresidues that might react with the polypeptide functional epitopes.Preferred peptide linker sequences contain Gly, Asn and Serresidues. Other near neutral amino acids, such as Thr and Ala mayalso be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Marateaet al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci.USA 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. Thelinker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions thatcan be used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. Theregulatory elements responsible for expression of DNA are locatedonly 5' to the DNA sequence encoding the first polypeptides.Similarly, stop codons required to end translation andtranscription termination signals are only present 3' to the DNAsequence encoding the second polypeptide.

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

In one preferred embodiment, the immunological fusion partner isderived from a Mycobacterium sp., such as a Mycobacteriumtuberculosis-derived Ra12 fragment. Ra12 compositions and methodsfor their use in enhancing the expression and/or immunogenicity ofheterologous polynucleotide/polypeptide sequences is described inU.S. patent application Ser. No. 60/158,585, now lapsed, thedisclosure of which is incorporated herein by reference in itsentirety. Briefly, Ra12 refers to a polynucleotide region that is asubsequence of a Mycobacterium tuberculosis MTB32A nucleic acid.MTB32A is a seine protease of 32 KD molecular weight encoded by agene in virulent and avirulent strains of M. tuberculosis. Thenucleotide sequence and amino acid sequence of MTB32A have beendescribed (for example, U.S. Patent Application 60/158,585, nowlapsed; see also, Skeiky et al., Infection and Immun. (1999)67:3998-4007, incorporated herein by reference). C-terminalfragments of the MTB32A coding sequence express at high levels andremain as soluble polypeptides throughout the purification process.Moreover, Ra12 may enhance the immunogenicity of heterologousimmunogenic polypeptides with which it is fused. One preferred Ra12fusion polypeptide comprises a 14 KD C-terminal fragmentcorresponding to amino acid residues 192 to 323 of MTB32A. Otherpreferred Ra12 polynucleotides generally comprise at least about 15consecutive nucleotides, at least about 30 nucleotides, at leastabout 60 nucleotides, at least about 100 nucleotides, at leastabout 200 nucleotides, or at least about 300 nucleotides thatencode a portion of a Ra12 polypeptide. Ra12 polynucleotides maycomprise a native sequence (i.e., an endogenous sequence thatencodes a Ra12 polypeptide or a portion thereof) or may comprise avariant of such a sequence. Ra12 polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not substantially diminished, relative to a fusionpolypeptide comprising a native Ra12 polypeptide. Variantspreferably exhibit at least about 70% identity, more preferably atleast about 80% identity and most preferably at least about 90%identity to a polynucleotide sequence that encodes a native Ra12polypeptide or a portion thereof.

Within other preferred embodiments, an immunological fusion partneris derived from protein D, a surface protein of the gram-negativebacterium Haemophilus influenza B (WO 91/18926). Preferably, aprotein D derivative comprises approximately the first third of theprotein (e.g., the first N-terminal 100-110 amino acids), and aprotein D derivative may be lipidated. Within certain preferredembodiments, the first 109 residues of a Lipoprotein D fusionpartner is included on the N-terminus to provide the polypeptidewith additional exogenous T-cell epitopes and to increase theexpression level in E. coli (thus functioning as an expressionenhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners includethe non-structural protein from influenzae virus, NS1(hemaglutinin). Typically, the N-terminal 81 amino acids are used,although different fragments that include T-helper epitopes may beused.

In another embodiment, the immunological fusion partner is theprotein known as LYTA, or a portion thereof (preferably aC-terminal portion). LYTA is derived from Streptococcus pneumoniae,which synthesizes an N-acetyl-L-alanine amidase known as amidaseLYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is anautolysin that specifically degrades certain bonds in thepeptidoglycan backbone. The C-terminal domain of the LYTA proteinis responsible for the affinity to the choline or to some cholineanalogues such as DEAE. This property has been exploited for thedevelopment of E. coli C-LYTA expressing plasmids useful forexpression of fusion proteins. Purification of hybrid proteinscontaining the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into afusion polypeptide. A repeat portion is found in the C-terminalregion starting at residue 178. A particularly preferred repeatportion incorporates residues 188-305.

Yet another illustrative embodiment involves fusion polypeptides,and the polynucleotides encoding them, wherein the fusion partnercomprises a targeting signal capable of directing a polypeptide tothe endosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fusedwith this targeting signal, will associate more efficiently withMHC class II molecules and thereby provide enhanced in vivostimulation of CD4.sup.+ T-cells specific for the polypeptide.

WT1 Polynucleotides

Any polynucleotide that encodes a WT1 polypeptide as describedherein is a WT1 polynucleotide encompassed by the presentinvention. Such polynucleotides may be single-stranded (coding orantisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. Additional coding or non-codingsequences may, but need not, be present within a polynucleotide ofthe present invention, and a polynucleotide may, but need not, belinked to other molecules and/or support materials.

WT1 polynucleotides may encode a native WT1 protein, or may encodea variant of WT1 as described herein. Polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the immunogenicity of the encoded polypeptideis not diminished, relative to a native WT1 protein. The effect onthe immunogenicity of the encoded polypeptide may generally beassessed as described herein. Preferred variants contain nucleotidesubstitutions, deletions, insertions and/or additions at no morethan 20%, preferably at no more than 10%, of the nucleotidepositions that encode an immunogenic portion of a native WT1sequence. Certain variants are substantially homologous to a nativegene, or a portion thereof. Such polynucleotide variants arecapable of hybridizing under moderately stringent conditions to anaturally occurring DNA sequence encoding a WT1 polypeptide (or acomplementary sequence). Suitable moderately stringent conditionsinclude prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mMEDTA (pH 8.0); hybridizing at 50.degree. C.-65.degree. C.,5.times.SSC, overnight; followed by washing twice at 65.degree. C.for 20 minutes with each of 2.times., 0.5.times. and 0.2.times.SSCcontaining 0.1% SDS). Such hybridizing DNA sequences are alsowithin 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 manynucleotide sequences that encode a WT1 polypeptide. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence ofany native gene. Nonetheless, polynucleotides that vary due todifferences in codon usage are specifically contemplated by thepresent invention.

Once an immunogenic portion of WT1 is identified, as describedabove, a WT1 polynucleotide may be prepared using any of a varietyof techniques. For example, a WT1 polynucleotide may be amplifiedfrom cDNA prepared from cells that express WT1. Suchpolynucleotides may be amplified via polymerase chain reaction(PCR). For this approach, sequence-specific primers may be designedbased on the sequence of the immunogenic portion and may bepurchased or synthesized. For example, suitable primers for PCRamplification of a human WT1 gene include: first step--P118:1434-1414: 5' GAG AGT CAG ACT TGA AAG CAGT 3' (SEQ ID NO:5) andP135: 5'CTG AGC CTC AGC AAA TGG GC 3' (SEQ ID NO:6); secondstep--P136: 5' GAG CAT GCA TGG GCT CCG ACG TGC GGG 3' (SEQ ID NO:7)and P137: 5' GGG GTA CCC ACT GAA CGG TCC CCG A 3' (SEQ ID NO:8).Primers for PCR amplification of a mouse WT1 gene include: firststep--P138: 5' TCC GAG CCG CAC CTC ATG 3' (SEQ ID NO:9) and P139:5' GCC TGG GAT GCT GGA CTG 3' (SEQ ID NO:10), second step--P140: 5'GAG CAT GCG ATG GGT TCC GAC GTG CGG 3' (SEQ ID NO:11) and P141: 5'GGG GTA CCT CAA AGC GCC ACG TGG AGT TT 3' (SEQ ID NO:12).

An amplified portion may then be used to isolate a full length genefrom a human genomic DNA library or from a suitable cDNA library,using well known techniques. Alternatively, a full length gene canbe constructed from multiple PCR fragments. WT1 polynucleotides mayalso be prepared by synthesizing oligonucleotide components, andligating components together to generate the completepolynucleotide.

WT1 polynucleotides may also be synthesized by any method known inthe art, including chemical synthesis (e.g., solid phasephosphoramidite chemical synthesis). Modifications in apolynucleotide sequence may also be introduced using standardmutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis (see Adelman et al., DNA 2:183, 1983).Alternatively, RNA molecules may be generated by in vitro or invivo transcription of DNA sequences encoding a WT1 polypeptide,provided that the DNA is incorporated into a vector with a suitableRNA polymerase promoter (such as T7 or SP6). Certain portions maybe used to prepare an encoded polypeptide, as described herein. Inaddition, or alternatively, a portion may be administered to apatient such that the encoded polypeptide is generated in vivo(e.g., by transfecting antigen-presenting cells such as dendriticcells with a cDNA construct encoding a WT1 polypeptide, andadministering the transfected cells to the patient).

Polynucleotides that encode a WT1 polypeptide may generally be usedfor production of the polypeptide, in vitro or in vivo. WT1polynucleotides that are complementary to a coding sequence (i.e.,antisense polynucleotides) may also be used as a probe or toinhibit WT1 expression. cDNA constructs that can be transcribedinto antisense RNA may also be introduced into cells of tissues tofacilitate the production of antisense RNA.

Any polynucleotide may be further modified to increase stability invivo. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends; the use ofphosphorothioate or 2' O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditionalbases such as inosine, queosine and wybutosine, as well asacetyl-methyl-, thio- and other modified forms of adenine,cytidine, guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a varietyof other nucleotide sequences using established recombinant DNAtechniques. For example, a polynucleotide may be cloned into any ofa variety of cloning vectors, including plasmids, phagemids, lambdaphage derivatives and cosmids. Vectors of particular interestinclude expression vectors, replication vectors, probe generationvectors and sequencing vectors. In general, a vector will containan origin of replication functional in at least one organism,convenient restriction endonuclease sites and one or moreselectable markers. Other elements will depend upon the desireduse, and will be apparent to those of ordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so asto 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 willappreciate that there are many ways to achieve expression of apolynucleotide in a target cell, and any suitable method may beemployed. For example, a polynucleotide may be incorporated into aviral vector such as, but not limited to, adenovirus,adeno-associated virus, retrovirus, or vaccinia or other pox virus(e.g., avian pox virus). Techniques for incorporating DNA into suchvectors are well known to those of ordinary skill in the art. Aretroviral vector may additionally transfer or incorporate a genefor a selectable marker (to aid in the identification or selectionof transduced cells) and/or a targeting moiety, such as a gene thatencodes a ligand for a receptor on a specific target cell, torender the vector target specific. Targeting may also beaccomplished using an antibody, by methods known to those ofordinary skill in the art. cDNA constructs within such a vector maybe used, for example, to transfect human or animal cell lines foruse in establishing WT1 positive tumor models which may be used toperform tumor protection and adoptive immunotherapy experiments todemonstrate tumor or leukemia-growth inhibition or lysis of suchcells.

Other therapeutic formulations for polynucleotides includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system for use as a deliveryvehicle in vitro and in vivo is a liposome (i.e., an artificialmembrane vesicle). The preparation and use of such systems is wellknown in the art.

Antibodies and Fragments Thereof

The present invention further provides binding agents, such asantibodies and antigen-binding fragments thereof, that specificallybind to a WT1 polypeptide. As used herein, an agent is said to"specifically bind" to a WT1 polypeptide if it reacts ata detectable level (within, for example, an ELISA) with a WT1polypeptide, and does not react detectably with unrelated proteinsunder similar conditions. As used herein, "binding"refers to a noncovalent association between two separate moleculessuch that a "complex" is formed. The ability to bind maybe evaluated by, for example, determining a binding constant forthe formation of the complex. The binding constant is the valueobtained when the concentration of the complex is divided by theproduct of the component concentrations. In general, two compoundsare said to "bind," in the context of the presentinvention, when the binding constant for complex formation exceedsabout 10.sup.3 L/mol. The binding constant maybe determined usingmethods well known in the art.

Any agent that satisfies the above requirements may be a bindingagent. In a preferred embodiment, a binding agent is an antibody oran antigen-binding fragment thereof. Certain antibodies arecommercially available from, for example, Santa Cruz Biotechnology(Santa Cruz, Calif.). Alternatively, antibodies may be prepared byany of a variety of techniques known to those of ordinary skill inthe art. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988. In general, antibodiescan be produced by cell culture techniques, including thegeneration of monoclonal antibodies as described herein, or viatransfection of antibody genes into suitable bacterial or mammaliancell hosts, in order to allow for the production of recombinantantibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety ofmammals (e.g., mice, rats, rabbits, sheep or goats). In this step,the polypeptides of this invention may serve as the immunogenwithout modification. Alternatively, particularly for relativelyshort polypeptides, a superior immune response may be elicited ifthe polypeptide is joined to a carrier protein, such as bovineserum albumin or keyhole limpet hemocyanin. The immunogen isinjected into the animal host, preferably according to apredetermined schedule incorporating one or more boosterimmunizations, and the animals are bled periodically. Polyclonalantibodies specific for the polypeptide may then be purified fromsuch antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide ofinterest may be prepared, for example, using the technique ofKohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, andimprovements thereto. Briefly, these methods involve thepreparation of immortal cell lines capable of producing antibodieshaving the desired specificity (i.e., reactivity with thepolypeptide of interest). Such cell lines may be produced, forexample, from spleen cells obtained from an animal immunized asdescribed above. The spleen cells are then immortalized by, forexample, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells andmyeloma cells may be combined with a nonionic detergent for a fewminutes and then plated at low density on a selective medium thatsupports the growth of hybrid cells, but not myeloma cells. Apreferred selection technique uses HAT (hypoxanthine, aminopterin,thymidine) selection. After a sufficient time, usually about 1 to 2weeks, colonies of hybrids are observed. Single colonies areselected and their culture supernatants tested for binding activityagainst the polypeptide. Hybridomas having high reactivity andspecificity are preferred.

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

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

Monoclonal antibodies and fragments thereof may be coupled to oneor more therapeutic agents. Suitable agents in this regard includeradioactive tracers and chemotherapeutic agents, which may be used,for example, to purge autologous bone marrow in vitro).Representative therapeutic agents include radionuclides,differentiation inducers, drugs, toxins, and derivatives thereof.Preferred radionuclides include .sup.90Y, .sup.123I, .sup.125I,.sup.131I, .sup.186Re, .sup.188Re, .sup.211At, and .sup.212Bi.Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol estersand butyric acid. Preferred toxins include ricin, abrin, diptheriatoxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigellatoxin, and pokeweed antiviral protein. For diagnostic purposes,coupling of radioactive agents may be used to facilitate tracing ofmetastases or to determine the location of WT1-positive tumors.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g.,via a linker group). A direct reaction between an agent and anantibody is possible when each possesses a substituent capable ofreacting with the other. For example, a nucleophilic group, such asan amino or sulfhydryl group, on one may be capable of reactingwith a carbonyl-containing group, such as an anhydride or an acidhalide, or with an alkyl group containing a good leaving group(e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agentand an antibody via a linker group. A linker group can function asa spacer to distance an antibody from an agent in order to avoidinterference with binding capabilities. A linker group can alsoserve to increase the chemical reactivity of a substituent on anagent or an antibody, and thus increase the coupling efficiency. Anincrease in chemical reactivity may also facilitate the use ofagents, or functional groups on agents, which otherwise would notbe possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of thePierce Chemical Co., Rockford, Ill.), may be employed as the linkergroup. Coupling may be effected, for example, through amino groups,carboxyl groups, sulfhydryl groups or oxidized carbohydrateresidues. There are numerous references describing suchmethodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from theantibody portion of the immunoconjugates of the present invention,it may be desirable to use a linker group which is cleavable duringor upon internalization into a cell. A number of differentcleavable linker groups have been described. The mechanisms for theintracellular release of an agent from these linker groups includecleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.4,489,710, to Spitler), by irradiation of a photolabile bond (e.g.,U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis ofderivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045,to Kohn et al.), by serum complement-mediated hydrolysis (e.g.,U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzedhydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody.In one embodiment, multiple molecules of an agent are coupled toone antibody molecule. In another embodiment, more than one type ofagent may be coupled to one antibody. Regardless of the particularembodiment, immunoconjugates with more than one agent may beprepared in a variety of ways. For example, more than one agent maybe coupled directly to an antibody molecule, or linkers whichprovide multiple sites for attachment can be used. Alternatively, acarrier can be used. A carrier may bear the agents in a variety ofways, including covalent bonding either directly or via a linkergroup. Suitable carriers include proteins such as albumins (e.g.,U.S. Pat. No. 4,507,234, to Kato et al.), peptides andpolysaccharides such as aminodextran (e.g., U.S. Pat. No.4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No.4,735,792 discloses representative radiohalogenated small moleculesand their synthesis. A radionuclide chelate may be formed fromchelating compounds that include those containing nitrogen andsulfur atoms as the donor atoms for binding the metal, or metaloxide, radionuclide. For example, U.S. Pat. No. 4,673,562, toDavison et al. discloses representative chelating compounds andtheir synthesis.

A variety of routes of administration for the antibodies andimmunoconjugates may be used. Typically, administration will beintravenous, intramuscular, subcutaneous or in the bed of aresected tumor. It will be evident that the precise dose of theantibody/immunoconjugate will vary depending upon the antibodyused, the antigen density on the tumor, and the rate of clearanceof the antibody.

Also provided herein are anti-idiotypic antibodies that mimic animmunogenic portion of WT1. Such antibodies may be raised againstan antibody, or antigen-binding fragment thereof, that specificallybinds to an immunogenic portion of WT1, using well knowntechniques. Anti-idiotypic antibodies that mimic an immunogenicportion of WT1 are those antibodies that bind to an antibody, orantigen-binding fragment thereof, that specifically binds to animmunogenic portion of WT1, as described herein.

T Cells

Immunotherapeutic compositions may also, or alternatively, compriseT cells specific for WT1. Such cells may generally be prepared invitro or ex vivo, using standard procedures. For example, T cellsmay be present within (or isolated from) bone marrow, peripheralblood or a fraction of bone marrow or peripheral blood of a mammal,such as a patient, using a commercially available cell separationsystem, such as the CEPRATE.TM. system, available from CellProInc., Bothell Wash. (see also U.S. Pat. No. 5,240,856; U.S. Pat.No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243).Alternatively, T cells may be derived from related or unrelatedhumans, non-human animals, cell lines or cultures.

T cells may be stimulated with WT1 polypeptide, polynucleotideencoding a WT1 polypeptide and/or an antigen presenting cell (APC)that expresses a WT1 polypeptide. Such stimulation is performedunder conditions and for a time sufficient to permit the generationof T cells that are specific for the WT1 polypeptide. Preferably, aWT1 polypeptide or polynucleotide is present within a deliveryvehicle, such as a microsphere, to facilitate the generation ofantigen-specific T cells. Briefly, T cells, which may be isolatedfrom a patient or a related or unrelated donor by routinetechniques (such as by FICOLL.RTM./HYPAQUE.RTM. density gradientcentrifugation of peripheral blood lymphocytes), are incubated withWT1 polypeptide. For example, T cells may be incubated in vitro for2-9 days (typically 4 days) at 37.degree. C. with WT1 polypeptide(e.g., 5 to 25 .mu.g/ml) or cells synthesizing a comparable amountof WT1 polypeptide. It may be desirable to incubate a separatealiquot of a T cell sample in the absence of WT1 polypeptide toserve as a control.

T cells are considered to be specific for a WT1 polypeptide if theT cells kill target cells coated with a WT1 polypeptide orexpressing a gene encoding such a polypeptide. T cell specificitymay be evaluated using any of a variety of standard techniques. Forexample, within a chromium release assay or proliferation assay, astimulation index of more than two fold increase in lysis and/orproliferation, compared to negative controls, indicates T cellspecificity. Such assays may be performed, for example, asdescribed in Chen et al., Cancer Res. 54:1065-1070, 1994.Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells withtritiated thymidine and measuring the amount of tritiated thymidineincorporated into DNA). Other ways to detect T cell proliferationinclude measuring increases in interleukin-2 (IL-2) production,Ca.sup.2+ flux, or dye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium.Alternatively, synthesis of lymphokines (such as interferon-gamma)can be measured or the relative number of T cells that can respondto a WT1 polypeptide may be quantified. Contact with a WT1polypeptide (200 ng/ml-100 .mu.g/ml, preferably 100 ng/ml-25.mu.g/ml) for 3-7 days should result in at least a two foldincrease in proliferation of the T cells and/or contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a twofold increase in the level of cytokine release (e.g., TNF orIFN-.gamma.) is indicative of T cell activation (see Coligan etal., Current Protocols in Immunology, vol. 1, Wiley Interscience(Greene 1998). WT1 specific T cells may be expanded using standardtechniques. Within preferred embodiments, the T cells are derivedfrom a patient or a related or unrelated donor and are administeredto the patient following stimulation and expansion.

T cells that have been activated in response to a WT1 polypeptide,polynucleotide or WT1-expressing APC may be CD4.sup.+ and/orCD8.sup.+. Specific activation of CD4.sup.+ or CD8.sup.+ T cellsmay be detected in a variety of ways. Methods for detectingspecific T cell activation include detecting the proliferation of Tcells, the production of cytokines (e.g., lymphokines), or thegeneration of cytolytic activity (i.e., generation of cytotoxic Tcells specific for WT1). For CD4.sup.+ T cells, a preferred methodfor detecting specific T cell activation is the detection of theproliferation of T cells. For CD8.sup.+ T cells, a preferred methodfor detecting specific T cell activation is the detection of thegeneration of cytolytic activity.

For therapeutic purposes, CD4.sup.+ or CD8.sup.+ T cells thatproliferate in response to the WT1 polypeptide, polynucleotide orAPC can be expanded in number either in vitro or in vivo.Proliferation of such T cells in vitro may be accomplished in avariety of ways. For example, the T cells can be re-exposed to WT1polypeptide, with or without the addition of T cell growth factors,such as interleukin-2, and/or stimulator cells that synthesize aWT1 polypeptide. The addition of stimulator cells is preferredwhere generating CD8.sup.+ T cell responses. T cells can be grownto large numbers in vitro with retention of specificity in responseto intermittent restimulation with WT1 polypeptide. Briefly, forthe primary in vitro stimulation (IVS), large numbers oflymphocytes (e.g., greater than 4.times.10.sup.7) may be placed inflasks with media containing human serum. WT1 polypeptide (e.g.,peptide at 10 .mu.g/ml) may be added directly, along with tetanustoxoid (e.g., 5 .mu.g/ml). The flasks may then be incubated (e.g.,37.degree. C. for 7 days). For a second IVS, T cells are thenharvested and placed in new flasks with 2-3.times.10.sup.7irradiated peripheral blood mononuclear cells. WT1 polypeptide(e.g., 10 .mu.g/ml) is added directly. The flasks are incubated at37.degree. C. for 7 days. On day 2 and day 4 after the second IVS,2-5 units of interleukin-2 (IL-2) may be added. For a third IVS,the T cells may be placed in wells and stimulated with theindividual'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 thecells are shown to be specific cytotoxic T cells, they may beexpanded using a 10 day stimulation cycle with higher IL-2 (20units) on days 2, 4 and 6.

Alternatively, one or more T cells that proliferate in the presenceof WT1 polypeptide can be expanded in number by cloning. Methodsfor cloning cells are well known in the art, and include limitingdilution. Responder T cells may be purified from the peripheralblood of sensitized patients by density gradient centrifugation andsheep red cell rosetting and established in culture by stimulatingwith the nominal antigen in the presence of irradiated autologousfiller cells. In order to generate CD4.sup.+ T cell lines, WT1polypeptide is used as the antigenic stimulus and autologousperipheral blood lymphocytes (PBL) or lymphoblastoid cell lines(LCL) immortalized by infection with Epstein Barr virus are used asantigen presenting cells. In order to generate CD8.sup.+ T celllines, autologous antigen-presenting cells transfected with anexpression vector which produces WT1 polypeptide may be used asstimulator cells. Established T cell lines may be cloned 2-4 daysfollowing antigen stimulation by plating stimulated T cells at afrequency of 0.5 cells per well in 96-well flat-bottom plates with1.times.10.sup.6 irradiated PBL or LCL cells and recombinantinterleukin-2 (rIL2) (50 U/ml). Wells with established clonalgrowth may be identified at approximately 2-3 weeks after initialplating and restimulated with appropriate antigen in the presenceof autologous antigen-presenting cells, then subsequently expandedby the addition of low doses of rIL2 (10 U/ml) 2-3 days followingantigen stimulation. T cell clones may be maintained in 24-wellplates by periodic restimulation with antigen and rIL2approximately every two weeks.

Within certain embodiments, allogeneic T-cells may be primed (i.e.,sensitized to WT1) in vivo and/or in vitro. Such priming may beachieved by contacting T cells with a WT1 polypeptide, apolynucleotide encoding such a polypeptide or a cell producing sucha polypeptide under conditions and for a time sufficient to permitthe priming of T cells. In general, T cells are considered to beprimed if, for example, contact with a WT1 polypeptide results inproliferation and/or activation of the T cells, as measured bystandard proliferation, chromium release and/or cytokine releaseassays as described herein. A stimulation index of more than twofold increase in proliferation or lysis, and more than three foldincrease in the level of cytokine, compared to negative controls,indicates T-cell specificity. Cells primed in vitro may beemployed, for example, within a bone marrow transplantation or asdonor lymphocyte infusion.

Pharmaceutical Compositions and Vaccines

Within certain aspects, polypeptides, polynucleotides, antibodiesand/or T cells may be incorporated into pharmaceutical compositionsor vaccines. Alternatively, a pharmaceutical composition maycomprise an antigen-presenting cell (e.g., a dendritic cell)transfected with a WT1 polynucleotide such that the antigenpresenting cell expresses a WT1 polypeptide. Pharmaceuticalcompositions comprise one or more such compounds or cells and aphysiologically acceptable carrier or excipient. Certain vaccinesmay comprise one or more such compounds or cells and a non-specificimmune response enhancer, such as an adjuvant or a liposome (intowhich the compound is incorporated). Pharmaceutical compositionsand vaccines may additionally contain a delivery system, such asbiodegradable microspheres which are disclosed, for example, inU.S. Pat. Nos. 4,897,268 and 5,075,109. Pharmaceutical compositionsand vaccines within the scope of the present invention may alsocontain other compounds, which may be biologically active orinactive.

Within certain embodiments, pharmaceutical compositions andvaccines are designed to elicit T cell responses specific for a WT1polypeptide in a patient, such as a human. In general, T cellresponses may be favored through the use of relatively shortpolypeptides (e.g., comprising less than 23 consecutive amino acidresidues of a native WT1 polypeptide, preferably 4-16 consecutiveresidues, more preferably 8-16 consecutive residues and still morepreferably 8-10 consecutive residues. Alternatively, or inaddition, a vaccine may comprise a non-specific immune responseenhancer that preferentially enhances a T cell response. In otherwords, the immune response enhancer may enhance the level of a Tcell response to a WT1 polypeptide by an amount that isproportionally greater than the amount by which an antibodyresponse is enhanced. For example, when compared to a standard oilbased adjuvant, such as CFA, an immune response enhancer thatpreferentially enhances a T cell response may enhance aproliferative T cell response by at least two fold, a lyticresponse by at least 10%, and/or T cell activation by at least twofold compared to WT1-megative control cell lines, while notdetectably enhancing an antibody response. The amount by which a Tcell or antibody response to a WT1 polypeptide is enhanced maygenerally be determined using any representative technique known inthe art, such as the techniques provided herein.

A pharmaceutical composition or vaccine may contain DNA encodingone or more of the polypeptides as described above, such that thepolypeptide is generated in situ. As noted above, the DNA may bepresent within any of a variety of delivery systems known to thoseof ordinary skill in the art, including nucleic acid expressionsystems, bacterial and viral expression systems and mammalianexpression systems. Appropriate nucleic acid expression systemscontain the necessary DNA, cDNA or RNA sequences for expression inthe patient (such as a suitable promoter and terminating signal).Bacterial delivery systems involve the administration of abacterium (such as Bacillus-Calmette-Guerrin) that expresses animmunogenic portion of the polypeptide on its cell surface. In apreferred embodiment, the DNA may be introduced using a viralexpression system (e.g., vaccinia or other pox virus, retrovirus,or adenovirus), which may involve the use of a non-pathogenic(defective), replication competent virus. Techniques forincorporating DNA into such expression systems are well known tothose of ordinary skill in the art. The DNA may also be"naked," as described, for example, in Ulmer et al.,Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased bycoating the DNA onto biodegradable beads, which are efficientlytransported into the cells.

As noted above, a pharmaceutical composition or vaccine maycomprise an antigen-presenting cell that expresses a WT1polypeptide. For therapeutic purposes, as described herein, theantigen presenting cell is preferably an autologous dendritic cell.Such cells may be prepared and transfected using standardtechniques, such as those described by Reeves et al., Cancer Res.56:5672-5677, 1996; Tuting et al., J. Immunol. 160:1139-1147, 1998;and Nair et al., Nature Biotechnol. 16:364-369, 1998). Expressionof a WT1 polypeptide on the surface of an antigen-presenting cellmay be confirmed by in vitro stimulation and standard proliferationas well as chromium release assays, as described herein.

While any suitable carrier known to those of ordinary skill in theart may be employed in the pharmaceutical compositions of thisinvention, the type of carrier will vary depending on the mode ofadministration. Compositions of the present invention may beformulated for any appropriate manner of administration, includingfor example, topical, oral, nasal, intravenous, intracranial,intraperitoneal, subcutaneous or intramuscular administration. Forparenteral administration, such as subcutaneous injection, thecarrier preferably comprises water, saline, alcohol, a fat, a waxor a buffer. For oral administration, any of the above carriers ora solid carrier, such as mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, glucose, sucrose,and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employedas carriers for the pharmaceutical compositions of this invention.For certain topical applications, formulation as a cream or lotion,using well known components, is preferred.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides oramino acids such as glycine, antioxidants, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/orpreservatives. Alternatively, compositions of the present inventionmay be formulated as a lyophilizate. Compounds may also beencapsulated within liposomes using well known technology.

Any of a variety of non-specific immune response enhancers, such asadjuvants, may be employed in the vaccines of this invention. Mostadjuvants contain a substance designed to protect the antigen fromrapid catabolism, such as aluminum hydroxide or mineral oil, and astimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Suitablenon-specific immune response enhancers include alum-based adjuvants(e.g., Alhydrogel, Rehydragel, aluminum phosphate, Algammulin,aluminum hydroxide); oil based adjuvants (Freund's adjuvant (FA),Specol, RIBI, TiterMax, MONTANIDE.RTM. ISA50 or SeppicMONTANIDE.RTM. ISA 720; cytokines (e.g., GM-CSF or Fl3-ligand);microspheres; nonionic block copolymer-based adjuvants; dimethyldioctadecyl ammoniumbromide (DDA) based adjuvants AS-1, AS-2 (SmithKline Beecham); Ribi Adjuvant system based adjuvants; QS21(Aquila); saponin based adjuvants (crude saponin, the saponin QuilA); muramyl dipeptide (MDP) based adjuvants such as SAF (Syntexadjuvant in its microfluidized form (SAF-m)); dimethyl-dioctadecylammonium bromide (DDA); human complement based adjuvants m. vaccaeand derivatives; immune stimulating complex (ISCOM.RTM.) basedadjuvants; inactivated toxins; and attenuated infectious agents(such as M tuberculosis).

As noted above, within certain embodiments, immune responseenhancers are chosen for their ability to preferentially elicit orenhance a T cell response (e.g., CD4.sup.+ and/or CD8.sup.+) to aWT1 polypeptide. Such immune response enhancers are well known inthe art, and include (but are not limited to) MONTANIDE.RTM. ISA50,Seppic MONTANIDE.RTM. ISA 720, cytokines (e.g., GM-CSF,Fl3-ligand), microspheres, dimethyl dioctadecyl ammoniumbromide(DDA) based adjuvants, AS-1 (Smith Kline Beecham), AS-2 (SmithKline Beecham), Ribi Adjuvant system based adjuvants, QS21(Aquila), saponin based adjuvants (crude saponin, the saponin QuilA), Syntex adjuvant in its microfluidized form (SAF-m), MV, ddMV(Genesis), immune stimulating complex (iscom) based adjuvants andinactivated toxins.

The compositions and vaccines described herein may be administeredas part of a sustained release formulation (i.e., a formulationsuch as a capsule or sponge that effects a slow release of compoundfollowing administration). Such formulations may generally beprepared using well known technology and administered by, forexample, oral, rectal or subcutaneous implantation, or byimplantation at the desired target site. Sustained-releaseformulations may contain a polypeptide, polynucleotide, antibody orcell dispersed in a carrier matrix and/or contained within areservoir surrounded by a rate controlling membrane. Carriers foruse within such formulations are biocompatible, and may also bebiodegradable; preferably the formulation provides a relativelyconstant level of active component release. The amount of activecompound contained within a sustained release formulation dependsupon the site of implantation, the rate and expected duration ofrelease and the nature of the condition to be treated or prevented.

Therapy of Malignant Diseases

In further aspects of the present invention, the compositions andvaccines described herein may be used to inhibit the development ofmalignant diseases (e.g., progressive or metastatic diseases ordiseases characterized by small tumor burden such as minimalresidual disease). In general, such methods may be used to prevent,delay or treat a disease associated with WT1 expression. In otherwords, therapeutic methods provided herein may be used to treat anexisting WT1-associated disease, or may be used to prevent or delaythe onset of such a disease in a patient who is free of disease orwho is afflicted with a disease that is not yet associated with WT1expression.

As used herein, a disease is "associated with WT1expression" if diseased cells (e.g., tumor cells) at some timeduring the course of the disease generate detectably higher levelsof a WT1 polypeptide than normal cells of the same tissue.Association of WT1 expression with a malignant disease does notrequire that WT1 be present on a tumor. For example, overexpressionof WT1 may be involved with initiation of a tumor, but the proteinexpression may subsequently be lost. Alternatively, a malignantdisease that is not characterized by an increase in WT1 expressionmay, at a later time, progress to a disease that is characterizedby increased WT1 expression. Accordingly, any malignant disease inwhich diseased cells formerly expressed, currently express or areexpected to subsequently express increased levels of WT1 isconsidered to be "associated with WT1 expression."

Immunotherapy may be performed using any of a variety oftechniques, in which compounds or cells provided herein function toremove WT1-expressing cells from a patient. Such removal may takeplace as a result of enhancing or inducing an immune response in apatient specific for WT1 or a cell expressing WT1. Alternatively,WT1-expressing cells may be removed ex vivo (e.g., by treatment ofautologous bone marrow, peripheral blood or a fraction of bonemarrow or peripheral blood). Fractions of bone marrow or peripheralblood may be obtained using any standard technique in the art.