WO2013106834A2 - Immunogenic wt-1 peptides and methods of use thereof - Google Patents

Immunogenic wt-1 peptides and methods of use thereof Download PDF

Info

Publication number
WO2013106834A2
WO2013106834A2 PCT/US2013/021448 US2013021448W WO2013106834A2 WO 2013106834 A2 WO2013106834 A2 WO 2013106834A2 US 2013021448 W US2013021448 W US 2013021448W WO 2013106834 A2 WO2013106834 A2 WO 2013106834A2
Authority
WO
WIPO (PCT)
Prior art keywords
another embodiment
seq
peptide
hla
cancer
Prior art date
Application number
PCT/US2013/021448
Other languages
French (fr)
Other versions
WO2013106834A3 (en
Inventor
Richard J. O'REILLY
Ekaterina DOUBROVINA
Annamalai SELVAKUMAR
Original Assignee
Memorial Sloan Kettering Cancer Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP13736176.2A priority Critical patent/EP2802347B1/en
Application filed by Memorial Sloan Kettering Cancer Center filed Critical Memorial Sloan Kettering Cancer Center
Priority to JP2014552368A priority patent/JP6282598B2/en
Priority to CA2861206A priority patent/CA2861206C/en
Priority to AU2013207669A priority patent/AU2013207669C1/en
Priority to CN201380014022.7A priority patent/CN104684577B/en
Priority to EP18215878.2A priority patent/EP3520810A3/en
Priority to US14/372,174 priority patent/US20150104413A1/en
Publication of WO2013106834A2 publication Critical patent/WO2013106834A2/en
Publication of WO2013106834A3 publication Critical patent/WO2013106834A3/en
Priority to HK15104710.4A priority patent/HK1204261A1/en
Priority to US15/608,964 priority patent/US10100087B2/en
Priority to AU2018201210A priority patent/AU2018201210B2/en
Priority to US16/125,213 priority patent/US10815274B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A61K39/001153—Wilms tumor 1 [WT1]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464452—Transcription factors, e.g. SOX or c-MYC
    • A61K39/464453—Wilms tumor 1 [WT1]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria

Definitions

  • This invention provides peptides, compositions and vaccines comprising same, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1- expressing cancer, comprising administering same.
  • Wilms tumor a pediatric nephroblastoma that occurs with a frequency of 1 in 10,000 births, has been the subject of intense clinical and basic research for several years.
  • the tumor is embryonic in origin; it is detected in children usually during the first 5 years of life and can occur unilaterally or bilaterally.
  • a WT arises when condensed metanephric mesenchymal cells of the developing kidney fail to properly differentiate.
  • WT-1 Wilms tumor 1
  • Wilms tumor protein I is a zinc finger transcription factor expressed during normal ontogenesis such as in fetal kidney, testis and ovary. In adults, WT-1 expression is limited to low levels on hematopoietic stem cells, myoepithelial progenitor cells, renal podocytes and some cells in testis and ovary. Recent demonstration that WT-1 is over expressed in several types of leukemia suggested that WT-1 would be an attractive target for immunotherapy for various cancers.
  • This invention provides peptides, compositions, and immunogenic compositions such as vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1 -expressing cancer, comprising administering immunogenic peptides.
  • the present invention provides an isolated WT-1 peptide having an amino acid (AA) sequence consisting ofany one of the sequences SEQ ID NO: 1-160, 162- 185, 190, 191 and 193.
  • the present invention provides an isolated HLA class I binding WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 190, 191 and 193.
  • the present invention provides an isolated HLA class II binding WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ
  • the present invention provides an isolated WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 1-160, 162-185, 190, 191 and 193, or a fragment of any of the foregoing.
  • the present invention provides an isolated HLA class I binding WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 190, 191 and 193.
  • the present invention provides an isolated HLA class II binding WT-1 peptide having an amino acid (AA) sequence consist
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) a peptide selected from SEQ ID NO: 1-160, 162-185, 190, 191 and 193.
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class I binding peptide selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class II binding peptide selected from SEQ ID NO: 149, 156, 173, 174 and 180. [09] In another embodiment, the present invention provides a vaccine comprising one or more peptides of SEQ ID NO: 1-160, 162-185, 190, 191 and 193.
  • the present invention provides a vaccine comprising one or more HLA class I binding peptides selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides a vaccine comprising one or more HLA class II binding peptides selected from SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, and one or more HLA class II binding peptides selected from SEQ ID NO: 149, 156, 173, 174 and 180.
  • the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a WT- lpeptide or vaccine of the present invention, thereby treating a subject with a WT-1- expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT-lpeptide or vaccine of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, orits relapse, in a subject.
  • the present invention provides a method of inducing formation and proliferation of a WT-1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT-1 protein-specific CTL.
  • This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
  • the present invention provides a method of inducing formation and proliferation of (a) a WT-1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT- 1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT-1 protein- specific CD8 lymphocyte; or (b) a CD4 + lymphocyte specific for the WT-1 protein; or a combination thereof.
  • This method can be conducted in vitro, ex vivo or in vivo.When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
  • the present invention provides a method of inducing an anticancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby inducing an anti-mesothelioma immune response in a subjectln one embodiment, the fragment of a WT-1 protein consists of a peptide or comprises a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 and 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.
  • the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby treating a subject with a mesothelioma.
  • the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.
  • the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the fragment of a WT- lprotein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.
  • the cancer is a WT-1 -expressing cancer.
  • the WT-1 -expressing cancer is an acute myelogenous leukemia (AML).
  • the WT-1 -expressing cancer is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the WT-1 -expressing cancer is an MDS.
  • the WT-1 -expressing cancer is a non-small cell lung cancer (NSCLC).
  • the WT-1 -expressing cancer is a Wilms' tumor.
  • the WT-1 -expressing cancer is a leukemia.
  • the WT-1 -expressing cancer is a hematological cancer.
  • the WT-1- expressing cancer is a lymphoma. In another embodiment, the WT-1 -expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT-1 -expressing cancer is a mesothelioma. In another embodiment, the WT-1 -expressing cancer is a malignant mesothelioma. In another embodiment, the WT-1 -expressing cancer is a gastric cancer. In another embodiment, the WT-1 -expressing cancer is a colon cancer. In another embodiment, the WT-1 -expressing cancer is a lung cancer. In another embodiment, the WT-1 -expressing cancer is abreast cancer. In another embodiment, the WT-1 -expressing cancer is a germ cell tumor.
  • the WT-1 -expressing cancer is an ovarian cancer. In another embodiment, the WT-1 -expressing cancer is a uterine cancer. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a liver cancer. In another embodiment, the WT-1- expressing cancer is a renal cancer. In another embodiment, the WT- 1 -expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT-1 -expressing cancer is a sarcoma.
  • the WT-1 -expressing cancer is any other carcinoma or sarcoma.
  • the WT-1 -expressing cancer is a solid tumor.
  • the solid tumor is associated with a WT-1 -expressing cancer.
  • the solid tumor is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • NSCLC non-small cell lung cancer
  • the solid tumor is associated with a lung cancer.
  • the solid tumor is associated with a breast cancer.
  • the solid tumor is associated with a colorectal cancer.
  • the solid tumor is associated with a prostate cancer.
  • the solid tumor is associated with an ovarian cancer.
  • the solid tumor is associated with a renal cancer. In another embodiment, the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
  • the present invention provides a composition comprising an isolated peptide of the invention in combination with at least ladditionalWT-1 peptide.
  • a composition comprising at least 2 different isolated peptides of the present invention is provided.
  • a composition comprising at least 3 or at least 4 different isolated peptides of the present invention is provided.
  • Each possibility represents a separate embodiment of the present invention.
  • the composition of the present invention is a vaccine.
  • the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a peptideor composition of the present invention, thereby treating a subject with a WT-1- expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a peptide or composition of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject.
  • the present invention provides a method of inducing formation and proliferation of a WT-1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptideor composition of the present invention, thereby inducing formation and proliferation of a WT-1 protein-specific CTL.
  • the present invention provides a method of inducing formation and proliferation of (a) a WT-1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT-1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT-1 protein- specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT-1 protein; or a combination thereof.
  • the invention is directed to a peptide of the invention with at least one amino acid change that increases the affinity of the peptide for binding to a HLA molecule.
  • Figure 2 A-E depicts the strategy for the generation of the total pool of overlapping pentadecapeptides spanning the whole sequence of the WT-1 protein and epitope mapping;
  • Figure 3 A-D shows that the combined HLA class I and II restricted WT-1 specific T cell response to the same immunodominant peptide sequence derived from WT-1 protein in the WT-1 CTL after 40 days of co-culture with the WT-1 total pool of overlapping 15-mers loaded on autologous CAMs;
  • Figure 5 depicts results using mixed A0201 epitopes loaded on A0201-AAPC in 8 normal A0201+ donors.
  • This invention provides immunogenic peptides, and compositions and vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1 -expressing cancer, comprising administering one or more immunogenic peptides.
  • This invention provides WT-1 peptides and methods of treating, reducing the incidence of, and inducing immune responses against a WT-1 -expressing cancer, comprising immunogenic peptides.
  • the WT-1 molecule from which the peptides of the present invention are derived has, in another embodiment, the sequence: 1 SRQRPHPGAL RNPTACPLPH FPPSLPPTHS PTHPPRAGTA AQAPGPRRLL
  • the foregoing sequence of the WT-1 protein is that published by Gessler et al. (37) which comprises 575 aminoacids and includes the first 126 aminoacids in the N-terminus missing in the (Exon 5+, KTS+) isoform of WT-116.
  • the WT-1 sequence is MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPA PPPAPP
  • the WT-1 molecule has the sequence:
  • the WT-1 molecule has the sequence: MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGR RSRGA
  • the WT-1 protein has the sequence set forth in GenBank Accession # NM_024426. In other embodiments, the WT-1 protein has or comprises one of the sequences set forth in one of the following sequence entries: NM_024425, NM_024424,
  • the WT-1 protein has any other WT-1 sequence known in the art.
  • This invention provides peptides, compositions, and immunogenic compositions such as vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1 -expressing cancer, comprising administering immunogenic peptides.
  • the present invention provides an isolated WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 1-160, 162- 185, 190, 191 and 193.
  • the present invention provides an isolated HLA class I binding WT- 1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides an isolated HLA class II binding WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151 , and the HLA class II peptide consists of or comprises SEQ ID NO: 149.
  • AA amino acid
  • the present invention provides an isolated WT-1 peptide having an amino acid (AA) sequence comprising any one of the sequences SEQ ID NO: 1-53 or 43- XXX, or a fragment thereof.
  • the present invention provides an isolated HLA class I binding WT-1 peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides an isolated HLA class II binding WT-1 peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO: 149.
  • AA amino acid
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) a peptide selected from SEQ ID NO: 1-160, 162-185, 190,
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class I binding peptide selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • an HLA class I binding peptide selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174,
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class II binding peptide selected from SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO: 149. [038] In another embodiment, the present invention provides a vaccine comprising one or more peptides of SEQ ID NO: 1-160, 162-185, 190, 191 and 193.
  • the present invention provides a vaccine comprising one or more HLA class I binding peptidesselected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, and 183.
  • the present invention provides a vaccine comprising one or more HLA class II binding peptides selected from SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176,
  • HLA class II binding peptides selected from SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class
  • I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO: 149.
  • the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a WT- lpeptide or vaccine of the present invention, thereby treating a subject with a WT-1- expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT- lpeptide or vaccine of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, orits relapse, in a subject.
  • the present invention provides a method of inducing an anticancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby inducing an anti-mesothelioma immune response in a subjectln one embodiment, the fragment of a WT-1 protein consists of a peptide or comprises a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 and 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177,
  • the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby treating a subject with a mesothelioma.
  • the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 and 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.
  • the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151
  • the HLA class II peptide consists of or comprises SEQ ID NO: 149.
  • the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147,
  • HLA class II peptide consists of or comprises SEQ ID NO: 149.
  • the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a WT- lpeptide or vaccine of the present invention, thereby treating a subject with a WT-1- expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT-lpeptide or vaccine of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, orits relapse, in a subject.
  • the present invention provides a method of inducing an anticancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby inducing an anti-mesothelioma immune response in a subject.
  • the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193.1n another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
  • the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby treating a subject with a mesothelioma.
  • the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193.
  • the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193.
  • the cancer is a WT-1 -expressing cancer.
  • the WT-1 -expressing cancer is an acute myelogenous leukemia (AML).
  • the WT-1 -expressing cancer is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the WT-1 -expressing cancer is an MDS.
  • the WT-1 -expressing cancer is a non-small cell lung cancer (NSCLC).
  • the WT-1 -expressing cancer is a Wilms' tumor.
  • the WT-1 -expressing cancer is a leukemia. In another embodiment, the WT-1 -expressing cancer is a hematological cancer. In another embodiment, the WT-1- expressing cancer is a lymphoma. In another embodiment, the WT-1 -expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT-1 -expressing cancer is a mesothelioma. In another embodiment, the WT-1 -expressing cancer is a malignant mesothelioma. In another embodiment, the WT-1 -expressing cancer is a gastric cancer. In another embodiment, the WT-1 -expressing cancer is a colon cancer. In another embodiment, the WT-1 -expressing cancer is a lung cancer.
  • the WT-1 -expressing cancer is abreast cancer. In another embodiment, the WT-1 -expressing cancer is a germ cell tumor. In another embodiment, the WT-1 -expressing cancer is an ovarian cancer. In another embodiment, the WT-1 -expressing cancer is a uterine cancer. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a liver cancer. In another embodiment, the WT-1- expressing cancer is a renal cancer.
  • the WT- 1 -expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT-1 -expressing cancer is a sarcoma. In another embodiment, the WT-1 -expressing cancer is any other carcinoma or sarcoma.
  • the WT-1 -expressing cancer is a solid tumor.
  • the solid tumor is associated with a WT-1 -expressing cancer.
  • the solid tumor is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • NSCLC non-small cell lung cancer
  • the solid tumor is associated with a lung cancer.
  • the solid tumor is associated with a breast cancer.
  • the solid tumor is associated with a colorectal cancer.
  • the solid tumor is associated with a prostate cancer.
  • the solid tumor is associated with an ovarian cancer.
  • the solid tumor is associated with a renal cancer.
  • the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
  • the present invention provides a composition comprising an isolated peptide of the invention in combination with at least 1 additional WT-1 peptide.
  • a composition comprising at least 2 different isolated peptides of the present invention is provided.
  • a composition comprising at least 3 or at least 4 different isolated peptides of the present invention is provided.
  • Each possibility represents a separate embodiment of the present invention.
  • the composition of the present invention is a vaccine.
  • the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a peptide or composition of the present invention, thereby treating a subject with a WT-1 -expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a peptide or composition of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject.
  • the present invention provides a method of inducing formation and proliferation of a WT-1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT-1 protein-specific CTL.
  • the present invention provides a method of inducing formation and proliferation of (a) a WT-1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT- 1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT-1 protein- specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT-1 protein; or a combination thereof.
  • the invention is directed to a peptide of the invention with at least one amino acid change that increases the affinity of the peptide for binding to a HLA molecule.
  • Peptide in another embodiment of methods and compositions of the present invention, refers to a compound of subunit A A connected by peptide bonds.
  • the peptide comprises an AA analogue.
  • the peptide comprises a peptidomimetic.
  • the different AA analogues and peptidomimetics that can be included in the peptides of methods and compositions of the present invention are enumerated hereinbelow.
  • the subunits are, in another embodiment, linked by peptide bonds.
  • the subunit is linked by another type of bond, e.g. ester, ether, etc. Each possibility represents a separate embodiment of the present invention.
  • WT- 1 peptides The unaltered peptides of the present invention (as described both above and below) are referred to collectively herein as " WT- 1 peptides. " Each of the embodiments enumerated below for "WT-1 peptides” applies to unaltered WT-1 peptides and HLA class I and class II heteroclitic peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
  • a WT-1 peptide of the present invention binds to an HLA class I molecule or a class II molecule. In another embodiment the peptide binds to both a class I and a class II molecule.
  • the HLA class II molecule is an HLA- DRB molecule.
  • the HLA class Il-molecule is an HLA-DRA molecule.
  • the HLA molecule is an HLA-DQAl molecule.
  • the HLA molecule is an HLA-DQB 1 molecule.
  • the HLA molecule is an HLA-DPA1 molecule.
  • the HLA molecule is an HLA-DPB 1 molecule.
  • the HLA molecule is an HLA-DMA molecule. In another embodiment, the HLA molecule is an HLA-DMB molecule. In another embodiment, the HLA molecule is an HLA-DOA molecule. In another embodiment, the HLA molecule is an HLA-DOB molecule. In another embodiment, the HLA molecule is any other HLA class Il-molecule known in the art. Each possibility represents a separate embodiment of the present invention.
  • the HLA class I molecule whose binding motif is contained in or comprising a peptide of the present invention is, in another embodiment, an HLA-A molecule. In another embodiment, the HLA class I molecule is an HLA-B molecule.
  • the HLA class I molecule is an HLA-C molecule. In another embodiment, the HLA class I molecule is an HLA-A0201 molecule. In another embodiment, the molecule is HLA Al. In another embodiment, the HLA class I molecule is HLA A2. In another embodiment, the HLA class I molecule is HLA A2.1. In another embodiment, the HLA class I molecule is HLA A3. In another embodiment, the HLA class I molecule is HLA A3.2. In another embodiment, the HLA class I molecule is HLA Al l. In another embodiment, the HLA class I molecule is HLA A24. In another embodiment, the HLA class I molecule is HLA B7. In another embodiment, the HLA class I molecule is HLA B27. In another embodiment, the HLA class I molecule is HLA B8.Each possibility represents a separate embodiment of the present invention.
  • the HLA class I molecule-binding WT-1 peptide of methods and compositions of the present invention binds to a superfamily of HLA class I molecules.
  • the superfamily is the A2 superfamily.
  • the superfamily is the A3 superfamily.
  • the superfamily is the A24 superfamily.
  • the superfamily is the B7 superfamily.
  • the superfamily is the B27 superfamily.
  • the superfamily is the B44 superfamily.
  • the superfamily is the CI superfamily.
  • the superfamily is the C4 superfamily.
  • the superfamily is any other superfamily known in the art. Each possibility represents a separate embodiment of the present invention.
  • the HLA molecule is a AO 101, A0201, A0203, A2402, A6901, B0702, A3101, B3501, B3503, B3508, B3802, B3801, B3901, B4001, B4402, B4701, B5701, C0401, C1701, DRBjOlOl, DRBJ0402, DRBJ0402, DRBi0401 or DRBJ1104 molecule.
  • the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151
  • the HLA class II peptide consists of or comprises SEQ ID NO: 149, and bind to the corresponding HLA molecule or molecules indicated for each peptide in Table 1 or Table 2.1n
  • certain peptides can bind to more than one HLA allele.
  • a modification of a peptide of the invention comprises at least one heteroclitic amino acid change, also referred to as a mutation or mutated, or an anchor residue mutation (see below).
  • An HLA class I molecule binding motif of a modified peptide of the present invention exhibits an increased affinity for the HLA class I molecule, relative to the unmutated counterpart of the peptide.
  • the point mutation increases the affinity of the isolated, mutated WT- 1 peptide for the HLA class I molecule.
  • the increase in affinity is relative to the affinity (for the same HLA class I molecule) of the isolated, unmutated WT-1 peptide wherefrom the isolated, mutated WT-1 peptide was derived.
  • a WT- 1 peptide of methods and compositions of the present invention is so designed as to exhibit affinity for an HLA molecule.
  • the affinity is a high affinity, as described herein.
  • HLA molecules known in another embodiment as major histocompatibility complex (MHC) molecules, bind peptides and present them to immune cells.
  • MHC major histocompatibility complex
  • the immunogenicity of a peptide is partially determined by its affinity for HLA molecules.
  • HLA class I molecules interact with CD8 molecules, which are generally present on cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • HLA class II molecules interact with CD4 molecules, which are generally present on helper T lymphocytes.
  • a peptide of the present invention is immunogenic.
  • immunogenic refers to an ability to stimulate, elicit or participate in an immune response.
  • the immune response elicited is a cell-mediated immune response.
  • the immune response is a combination of cell- mediated and humoral responses.
  • T cells that bind to the MHC molecule-peptide complex become activated and induced to proliferate and lyse cells expressing a protein comprising the peptide.
  • T cells are typically initially activated by "professional" antigen presenting cells ("APC”; e.g. dendritic cells, monocytes, and macrophages), which present costimulatory molecules that encourage T cell activation as opposed to anergy or apoptosis.
  • APC antigen presenting cells
  • the response is heteroclitic, as described herein, such that the CTL lyses a neoplastic cell expressing a protein which has an AA sequence homologous to a peptide of this invention, or a different peptide than that used to first stimulate the T cell.
  • an encounter of a T cell with a peptide of this invention induces its differentiation into an effector and/or memory T cell. Subsequent encounters between the effector or memory T cell and the same peptide, or, in another embodiment, with a related peptide of this invention, leads to a faster and more intense immune response. Such responses are gauged, in another embodiment, by measuring the degree of proliferation of the T cell population exposed to the peptide. In another embodiment, such responses are gauged by any of the methods enumerated hereinbelow.
  • the peptides of methods and compositions of the present invention bind an HLA class II molecule with high affinity.
  • the HLA class II molecule is any HLA class II molecule enumerated herein.
  • each possibility represents a separate embodiment of the present invention.
  • derivatives of peptides of methods and compositions of the present invention bind an HLA class I molecule with high affinity.
  • the MHC class I molecule is any MHC class I molecule enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention binds an HLA class II molecule with significant affinity, while a peptide derived from the original peptide binds an HLA class I molecule with significant affinity.
  • affinity refers to the concentration of peptide necessary for inhibiting binding of a standard peptide to the indicated MHC molecule by 50%.
  • high affinity refers to an affinity is such that a concentration of about 500 nanomolar (nM) or less of the peptide is required for 50% inhibition of binding of a standard peptide. In another embodiment, a concentration of about 400 nM or less of the peptide is required.
  • the binding affinity is 300 nM. In another embodiment, the binding affinity is 200 nM. In another embodiment, the binding affinity is 150 nM. In another embodiment, the binding affinity is 100 nM. In another embodiment, the binding affinity is 80 nM.
  • the binding affinity is 60 nM. In another embodiment, the binding affinity is 40 nM. In another embodiment, the binding affinity is 30 nM. In another embodiment, the binding affinity is 20 nM. In another embodiment, the binding affinity is 15 nM. In another embodiment, the binding affinity is 10 nM. In another embodiment, the binding affinity is 8 nM. In another embodiment, the binding affinity is 6 nM. In another embodiment, the binding affinity is 4 nM. In another embodiment, the binding affinity is 3 nM. In another embodiment, the binding affinity is 2 nM. In another embodiment, the binding affinity is 1.5 nM. In another embodiment, the binding affinity is 1 nM. In another embodiment, the binding affinity is 0.8 nM.
  • the binding affinity is 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. In another embodiment, the binding affinity is 0.4 nM. In another embodiment, the binding affinity is 0.3 nM. In another embodiment, the binding affinity is less than 0.3 nM.
  • affinity refers to a measure of binding strength to the MHC molecule.
  • affinity is measured using a method known in the art to measure competitive binding affinities.
  • affinity is measured using a method known in the art to measure relative binding affinities.
  • the method is a competitive binding assay.
  • the method is radioimmunoassay or RIA.
  • the method is BiaCore analyses.
  • the method is any other method known in the art.
  • the method yields an IC50 in relation to an IC50 of a reference peptide of known affinity.
  • "high affinity" refers to an IC50 of 0.5-500 nM.
  • the IC50 is 1-300 nM. In another embodiment, the IC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. In another embodiment, the IC50 is 3-100 nM. In another embodiment, the IC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. In another embodiment, the IC50 is 10-100 nM. In another embodiment, the IC50 is 30- 100 nM. In another embodiment, the IC50 is 3-80 nM. In another embodiment, the IC50 is 4- 60 nM.
  • the IC50 is 5-50 nM. In another embodiment, the IC50 is 6- 50 nM. In another embodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is 10- 50 nM. In another embodiment, the IC50 is 20-50 nM. In another embodiment, the IC50 is 6- 40 nM. In another embodiment, the IC50 is 8-30 nM. In another embodiment, the IC50 is 10- 25 nM. In another embodiment, the IC50 is 15-25 nM.
  • Each affinity and range of affinities represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention binds to a superfamily of HLA molecules.
  • Superfamilies of HLA molecules share very similar or identical binding motifs.
  • the superfamily is a HLA class I superfamily.
  • the superfamily is a HLA class II superfamily. Each possibility represents a separate embodiment of the present invention.
  • HLA-binding peptide refers, in another embodiment, to a peptide that binds an HLA molecule with measurable affinity.
  • the terms refer to a peptide that binds an HLA molecule with high affinity.
  • the terms refer to a peptide that binds an HLA molecule with sufficient affinity to activate a T cell precursor.
  • the terms refer to a peptide that binds an HLA molecule with sufficient affinity to mediate recognition by a T cell.
  • the HLA molecule is, in other embodiments, any of the HLA molecules enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • Heteroclitic refers, in another embodiment, to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived (e.g. the peptide not containing the anchor residue mutations).
  • original peptide refers to a peptide of the present invention.
  • heteroclitic refers to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide.
  • a “heteroclitic” immune response refers to an immune response that recognizes the original peptide from which the improved peptide was derived (e.g. the peptide not containing the anchor residue mutations).
  • a “heteroclitic” immune response refers to an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide.
  • the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response substantially equal to the response to vaccination with the original peptide.
  • a heteroclitic peptide of the present invention is an HLA class I heteroclitic peptide.
  • a heteroclitic peptide of the present invention induces an immune response that is increased at least 2-fold relative to the WT- 1 peptide from which the heteroclitic peptide was derived ("native peptide").
  • the increase is 3- fold relative to the native peptide.
  • the increase is 5 -fold relative to the native peptide.
  • the increase is 7-fold relative to the native peptide.
  • the increase is 10-fold relative to the native peptide.
  • the increase is 15 -fold relative to the native peptide.
  • the increase is 20-fold relative to the native peptide.
  • the increase is 30-fold relative to the native peptide.
  • the increase is 50- fold relative to the native peptide. In another embodiment, the increase is 100-fold relative to the native peptide. In another embodiment, the increase is 150-fold relative to the native peptide. In another embodiment, the increase is 200-fold relative to the native peptide. In another embodiment, the increase is 300-fold relative to the native peptide. In another embodiment, the increase is 500-fold relative to the native peptide. In another embodiment, the increase is 1000-fold relative to the native peptide. In another embodiment, the increase is more than 1000-fold relative to the native peptide. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a HLA class II heteroclitic peptide derived from an isolated WT-1 peptide of the present invention.
  • the process of deriving comprises introducing a mutation that enhances a binding of the peptide to an HLA class II molecule.
  • the process of deriving consists of introducing a mutation that enhances a binding of the peptide to an HLA class I molecule.
  • the mutation is in an HLA class II anchor residue.
  • a heteroclitic class II peptide of the present invention is identified and tested in a manner analogous to identification and testing of HLA class I heteroclitic peptides, as exemplified herein.
  • Each possibility represents a separate embodiment of the present invention.
  • the HLA class II binding site in a peptide of the present invention is created or improved by mutation of an HLA class II motif anchor residue.
  • theanchor residue that is modified is in the PI position.
  • the anchor residue is at the P2 position.
  • the anchor residue is at the P6 position.
  • the anchor residue is at the P9 position.
  • the anchor residue is selected from the PI, P2, P6, and P9 positions.
  • the anchor residue is at the P3 position.
  • the anchor residue is at the P4 position.
  • the anchor residue is at the P5 position.
  • the anchor residue is at the P6 position.
  • the anchor residue is at the P8 position. In another embodiment, the anchor residue is at the P10 position. In another embodiment, the anchor residue is at the PI 1 position. In another embodiment, the anchor residue is at the PI 2 position. In another embodiment, the anchor residue is at the PI 3 position. In another embodiment, the anchor residue is at any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than PI, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding. Each possibility represents a separate embodiment of the present invention. [082] In another embodiment, a heteroclitic peptide is generated by introduction of a mutation that creates an anchor motif. "Anchor motifs" or “anchor residues" refers, in another embodiment, to 1 or a set of preferred residues at particular positions in an HLA-binding sequence. In another embodiment, the
  • HLA-binding sequence is an HLA class Il-binding sequence.
  • the HLA-binding sequence is an HLA class I-binding sequence.
  • the positions corresponding to the anchor motifs are those that play a significant role in binding the HLA molecule.
  • the anchor residue is a primary anchor motif.
  • the anchor residue is a secondary anchor motif. Each possibility represents a separate embodiment of the present invention.
  • Methods for predicting MHC class H epitopes are well known in the art.
  • the MHC class II epitope is predicted using TEPITOPE (Meister GE, Roberts CG et al, Vaccine 1995 13: 581-91).
  • the MHC class II epitope is predicted using EpiMatrix (De Groot AS, Jesdale BM et al, AIDS Res Hum Retroviruses 1997 13: 529-31). In another embodiment, the MHC class II epitope is predicted using the Predict Method (Yu K, Petrovsky N et al, Mol Med. 2002 8: 137- 48). In another embodiment, the MHC class II epitope is predicted using the SYFPEITHI epitope prediction algorithm (Examples). In another embodiment, the MHC class II epitope is predicted using Rankpep. In another embodiment, the MHC class II epitope is predicted using any other method known in the art. Each possibility represents a separate embodiment of the present invention.
  • the anchor residue that is modified is in the PI position.
  • the anchor residue is in the P2 position.
  • the anchor residue is in the P6 position.
  • the anchor residue is in the P9 position.
  • the anchor residue is the P3, P4, P5, P6, P8, P10, Pl l, P12, or P13 position.
  • the anchor residue is any other anchor residue of an HLA class II molecule that is known in the art.
  • a WT-1 peptide of the present invention binds to 2 distinct HLA class II molecules.
  • the peptide binds to three distinct HLA class II molecules.
  • the peptide binds to four distinct HLA class II molecules.
  • the peptide binds to five distinct HLA class II molecules.
  • the peptide binds to six distinct HLA class II molecules.
  • the peptide binds to more than six distinct HLA class II molecules.
  • the HLA class II molecules that are bound by a WT-1 peptide of the present invention are encoded by two or more distinct alleles at a given HLA class II locus.
  • the HLA class II molecules are encoded by 3 distinct alleles at a locus.
  • the HLA class II molecules are encoded by 4 distinct alleles at a locus.
  • the HLA class II molecules are encoded by 5 distinct alleles at a locus.
  • the HLA class II molecules are encoded by 6 distinct alleles at a locus.
  • the HLA class II molecules are encoded by more than six distinct alleles at a locus.
  • the HLA class II molecules bound by the WT-1 peptide are encoded by HLA class II genes at 2 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 2 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at more than 4 distinct loci.
  • the loci are selected from HLA-DRB loci.
  • the HLA class Il-binding peptide is an HLA-DRA binding peptide.
  • the peptide is an HLA- DQA1 binding peptide.
  • the peptide is an HLA-DQB 1 binding peptide.
  • the peptide is an HLA-DPAl binding peptide.
  • the peptide is an HLA-DPB 1 binding peptide.
  • the peptide is an HLA-DMA binding peptide.
  • the peptide is an HLA- DMB binding peptide.
  • the peptide is an HLA-DOA binding peptide.
  • the peptide is an HLA-DOB binding peptide.
  • the peptide binds to any other HLA class II molecule known in the art. Each possibility represents a separate embodiment of the present invention.
  • a WT-1 peptide of the present invention binds to 2 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 3 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 4 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 5 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 6 distinct HLA- DRB molecules. In another embodiment, the peptide binds to more than 6 distinct HLA-DRB molecules.
  • a WT-1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by 3 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by 4 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by 5 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by 6 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by more than 6 distinct HLA- DRB alleles. Each possibility represents a separate embodiment of the present invention.
  • a WT-1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501.
  • the WT-1 peptide binds to HLA-DRB molecules encoded by 3 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501.
  • the WT-1 peptide binds to HLA-DRB molecules encoded by 4 distinct HLA- DRB alleles selected from DRB 101 , DRB 301, DRB 401 , DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT-1 peptide binds to HLA-DRB molecules encoded by 5 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101 , DRB 1104 and DRB 1501.
  • the WT-1 peptide binds to HLA- DRB molecules encoded by each of the following HLA-DRB alleles: DRB 101, DRB 301, DRB 401 , DRB 701, DRB 1101 , and DRB 1501.
  • HLA-DRB alleles DRB 101, DRB 301, DRB 401 , DRB 701, DRB 1101 , and DRB 1501.
  • the present invention provides a composition comprising 2 distinct WT-1 peptides of the present invention.
  • the 2 distinct WT-1 peptides are both unaltered.
  • 1 of the WT-1 peptides is unaltered, while the other is heteroclitic.
  • both of the WT-1 peptides are heteroclitic.
  • the composition comprises 3 distinct WT-1 peptides of the present invention. In another embodiment, the composition comprises 4 distinct WT-1 peptides of the present invention. In another embodiment, the composition comprises 5 distinct WT-1 peptides of the present invention. In another embodiment, the composition comprises more than 5 distinct isolated WT-1 peptides of the present invention.
  • 2 of the WT-1 peptides in the composition are unaltered. In another embodiment, 2 of the WT-1 peptides in the composition are heteroclitic. In another embodiment, 2 of the WT-1 peptides in the composition are unaltered, and 2 are heteroclitic. In another embodiment, more than 2 of the WT-1 peptides in the composition are unaltered.
  • more than 2 of the WT-1 peptides in the composition are heteroclitic. In another embodiment, more than 2 of the WT-1 peptides in the composition are unaltered, and more than 2 are heteroclitic. Each possibility represents a separate embodiment of the present invention.
  • 1 of the additional WT-1 peptides in a composition of the present invention has a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193.
  • 2 of the additional WT-1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193.
  • 3 of the additional WT-1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193.
  • any other immunogenic WT-1 peptide known in the art is utilized as an additional WT-1 peptide.
  • any combination of immunogenic WT-1 peptides known in the art is utilized.
  • Non-limiting sources of other WT peptides include WO2005053618, WO2007047764 and WO2007120673.
  • a composition of the present invention contains 2 HLA class II heteroclitic peptides that are derived from the same isolated WT-1 peptide of the present invention.
  • the 2 HLA class II heteroclitic peptides contain mutations in different HLA class II molecule anchor residues.
  • the 2 HLA class II heteroclitic peptides contain different mutations in the same anchor residues.
  • 2 of the HLA class II heteroclitic peptides are derived from different isolated WT-1 peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
  • 2 WT-1 peptides of the present invention, or the WT-1 peptides that correspond to two HLA class II heteroclitic peptides of the present invention overlap with one another.
  • the overlap between the peptides is at least 7 amino acids (AA).
  • the overlap is at least 8 AA.
  • the overlap is at least 9 AA.
  • the overlap is 7 AA.
  • the overlap is 8 AA.
  • the overlap is 9 AA.
  • the overlap is 10 AA.
  • the overlap is 11 AA.
  • the overlap is 12 AA.
  • the overlap is 13 AA.
  • the overlap is 14 AA.
  • the overlap is 15 AA.
  • the overlap is 16 AA.
  • the overlap is more than 16 AA.
  • the peptides in a composition of the present invention bind to 2 distinct HLA class II molecules. In another embodiment, the peptides bind to 3 distinct HLA class II molecules. In another embodiment, the peptides bind to 4 distinct HLA class II molecules. In another embodiment, the peptides bind to 5 distinct HLA class II molecules. In another embodiment, the peptides bind to more than 5 distinct HLA class II molecules. In another embodiment, the peptides in the composition bind to the same HLA class II molecules.
  • each of the WT 1 peptides in a composition of the present invention binds to a set of HLA class II molecules. In another embodiment, each of the WT-1 peptides binds to a distinct set of HLA class II molecules. In another embodiment, the WT-1 peptides in the composition bind to the same set of HLA class II molecules. In another embodiment, 2 of the WT- 1 peptides bind to a distinct but overlapping set of HLA class II molecules. In another embodiment, 2 or more of the WT-1 peptides bind to the same set of HLA class II molecules, while another of the WT-1 peptides binds to a distinct set.
  • 2 or more of the WT-1 peptides bind to an overlapping set of HLA class II molecules, while another of the WT-1 peptides binds to a distinct set.
  • 2 or more of the WT-1 peptides in a composition of the present invention each binds to more than 1 HLA-DRB molecule.
  • the 4 or more HLA-DRB molecules bound by the peptides in the composition are distinct from one another.
  • the HLA-DRB molecules are encoded by different HLA-DRB alleles. Each possibility represents a separate embodiment of the present invention.
  • WT-1 peptides in a composition of the present invention are HLA-DRB molecules.
  • 3 or more of the HLA class II molecules that are bound are HLA-DRB molecules.
  • the HLA class II molecules that are bound can be any of the HLA class II molecules enumerated herein.
  • the HLA class II molecules that are bound are encoded by 2 or more distinct HLA class II alleles at a given locus.
  • the HLA class II molecules that are bound are encoded by HLA class II genes at 2 or more distinct loci.
  • a "set of HLA class II molecules" refers to the HLA class II molecules encoded by different alleles at a particular locus.
  • the term refers to HLA class II molecules with a particular binding specificity.
  • the term refers to HLA class II molecules with a particular peptide consensus sequence.
  • the term refers to a superfamily of HLA class II molecules.
  • the present invention provides a composition comprising an unaltered HLA class II molecule-binding WT-1 peptide of the present invention and a second, HLA class I molecule-binding WT-1 peptide.
  • the composition comprises more than 1 HLA class II molecule-binding WT-1 peptide of the present invention, in addition to the HLA class I molecule- binding WT-1 peptide.
  • the composition comprises more than 1 HLA class I molecule-binding WT-1 peptide, in addition to the HLA class II molecule-binding WT-1 peptide.
  • the AA sequence of the HLA class I molecule-binding WT-1 peptide comprises a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or
  • the AA sequence of the HLA class I molecule-binding WT-1 peptide is selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. Each possibility represents a separate embodiment of the present invention.
  • the HLA class I molecule-binding WT-1 peptide is an HLA class I heteroclitic peptide.
  • the HLA class I molecule-binding WT-1 peptide contains a mutation in an HLA class I molecule anchor residue thereof, as described further herein.
  • WT- 1 -derived peptides were modified in HLA anchor residues to generate heteroclitic peptides with increased predicted binding to HLA- A0201 and HLA-A0301. Peptides with increased predicted binding also exhibited enhanced ability to bind HLA class I molecules and increased immunogenicity.
  • the mutation that enhances MHC binding is in the residue at position 1 of the HLA class I heteroclitic peptide.
  • the residue is changed to tyrosine. In another embodiment, the residue is changed to glycine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to phenylalanine. In another embodiment, the residue is changed to any other residue known in the art. In another embodiment, a substitution in position 1 (e.g. to tyrosine) stabilizes the binding of the position 2 anchor residue.
  • the mutation is in position 2 of the HLA class I heteroclitic peptide.
  • the residue is changed to leucine.
  • the residue is changed to valine.
  • the residue is changed to isoleucine.
  • the residue is changed to methionine.
  • the residue is changed to any other residue known in the art.
  • the mutation is in position 6 of the HLA class I heteroclitic peptide.
  • the residue is changed to valine.
  • the residue is changed to cysteine.
  • the residue is changed to glutamine.
  • the residue is changed to histidine.
  • the residue is changed to any other residue known in the art.
  • the mutation is in position 9 of the HLA class I heteroclitic peptide.
  • the mutation changes the residue at the C- terminal position thereof.
  • the residue is changed to valine.
  • the residue is changed to threonine.
  • the residue is changed to isoleucine.
  • the residue is changed to leucine.
  • the residue is changed to alanine.
  • the residue is changed to cysteine.
  • the residue is changed to any other residue known in the art.
  • the point mutation is in a primary anchor residue.
  • the HLA class I primary anchor residues are positions 2 and 9.
  • the point mutation is in a secondary anchor residue.
  • the HLA class I secondary anchor residues are positions 1 and 8.
  • the HLA class I secondary anchor residues are positions 1, 3, 6, 7, and 8.
  • the point mutation is in a position selected from positions 4, 5, and 8. Each possibility represents a separate embodiment of the present invention.
  • the point mutation is in 1 or more residues in positions selected from positions 1 , 2, 8, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1 , 2, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions selected from positions 2 and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions selected from positions 6 and 9. Each possibility represents a separate embodiment of the present invention. [0115] Each of the above anchor residues and substitutions represents a separate embodiment of the present invention.
  • the HLA class I molecule-binding WT peptide has length of 9 AA. In another embodiment, the peptide has length of 10 AA. As provided herein, native and heteroclitic peptides of 9- 10 AA exhibited substantial binding to HLA class I molecules and ability to elicit cytokine secretion and cytolysis by CTL.
  • the HLA class I molecule that is bound by the HLA class I molecule- binding WT- 1 peptide is an HLA- A molecule.
  • the HLA class I-molecule is an HLA-A2 molecule.
  • the HLA class I-molecule is an HLA-A3 molecule.
  • the HLA class I-molecule is an HLA-A1 1 molecule.
  • the HLA class I-molecule is an HLA-B 8 molecule.
  • the HLA class I-molecule is an HLA-0201 molecule.
  • the HLA class I-molecule binds any other HLA class I molecule known in the art. Each possibility represents a separate embodiment of the present invention.
  • a WT-1 peptide of methods and compositions of the present invention has a length of 8-30 amino acids.
  • the peptide has a length of 9-11 AA.
  • the peptide ranges in size from 7-25 AA, or in another embodiment, 8-11, or in another embodiment, 8-15, or in another embodiment, 9-20, or in another embodiment, 9-18, or in another embodiment, 9-15, or in another embodiment, 8-12, or in another embodiment, 9-11 AA in length.
  • the peptide is 8 AA in length, or in another embodiment, 9 AA or in another embodiment, 10 AA or in another embodiment, 12 AA or in another embodiment, 25 AA in length, or in another embodiment, any length therebetween.
  • the peptide is of greater length, for example 50, or 100, or more.
  • the cell processes the peptide to a length of 7 and 25 AA in length.
  • the cell processes the peptide to a length of 9-11 AA Each possibility represents a separate embodiment of the present invention.
  • the peptide is 15-23 AA in length. In another embodiment, the length is 15-24 AA. In another embodiment, the length is 15-25 AA. In another embodiment, the length is 15-26 AA. In another embodiment, the length is 15-27 AA. In another embodiment, the length is 15-28 AA. In another embodiment, the length is 14-30 AA. In another embodiment, the length is 14-29 AA. In another embodiment, the length is 14-28 AA. In another embodiment, the length is 14-26 AA. In another embodiment, the length is 14-24 AA. In another embodiment, the length is 14-22 AA. In another embodiment, the length is 14-20 AA. In another embodiment, the length is 16-30 AA.
  • the length is 16-28 AA. In another embodiment, the length is 16-26 AA. In another embodiment, the length is 16-24 AA. In another embodiment, the length is 16-22 AA. In another embodiment, the length is 18-30 AA. In another embodiment, the length is 18-28 AA. In another embodiment, the length is 18-26 AA. In another embodiment, the length is 18-24 AA. In another embodiment, the length is 18-22 AA. In another embodiment, the length is 18-20 AA. In another embodiment, the length is 20-30 AA. In another embodiment, the length is 20-28 AA. In another embodiment, the length is 20-26 AA. In another embodiment, the length is 20-24 AA. In another embodiment, the length is 22-30 AA.
  • the length is 22-28 AA. In another embodiment, the length is 22-26 AA. In another embodiment, the length is 24-30 AA. In another embodiment, the length is 24-28 AA. In another embodiment, the length is 24-26 AA.
  • minor modifications are made to peptides of the present invention without decreasing their affinity for HLA molecules or changing their TCR specificity, utilizing principles well known in the art. In the case of HLA class I-binding peptides, "minor modifications" refers, in another embodiment, to e.g.
  • peptides of the present invention are not limited to peptides predicated by the algorithms to exhibit strong MHC class I-binding affinity.
  • the types are modifications that can be made are listed below. Each modification represents a separate embodiment of the present invention.
  • a peptide enumerated in the Examples of the present invention is further modified by mutating an anchor residue to an MHC class I preferred anchor residue, which can be, in other embodiments, any of the anchor residues enumerated herein.
  • a peptide of the present invention containing an MHC class I preferred anchor residue is further modified by mutating the anchor residue to a different MHC class I preferred residue for that location.
  • the different preferred residue can be, in other embodiments, any of the preferred residues enumerated herein.
  • the anchor residue that is further modified is in the 1 position. In another embodiment, the anchor residue is in the 2 position. In another embodiment, the anchor residue is in the 3 position.
  • the anchor residue is in the 4 position. In another embodiment, the anchor residue is in the 5 position. In another embodiment, the anchor residue is in the 6 position. In another embodiment, the anchor residue is in the 7 position. In another embodiment, the anchor residue is in the 8 position. In another embodiment, the anchor residue is in the 9 position.
  • residues other than 2 and 9 can serve as secondary anchor residues; therefore, mutating them can improve MHC class I binding. Each possibility represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention is a length variant of a peptide enumerated in the Examples.
  • the length variant is one amino acid (AA) shorter than the peptide from the Examples. In another embodiment, the length variant is two AA shorter than the peptide from the Examples. In another embodiment, the length variant is more than two A A shorter than the peptide from the Examples. In another embodiment, the shorter peptide is truncated on the N-terminal end. In another embodiment, the shorter peptide is truncated on the C- terminal end. In another embodiment, the truncated peptide is truncated on both the N- terminal and C- terminal ends. Peptides are, in another embodiment, amenable to truncation without changing affinity for HLA molecules, as is well known in the art.
  • each of the above truncated peptides represents a separate embodiment of the present invention.
  • the length variant is longer than a peptide enumerated in the Examples of the present invention.
  • the longer peptide is extended on the N-terminal end in accordance with the surrounding WT- 1 sequence.
  • Peptides are, in another embodiment, amenable to extension on the N-terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples.
  • the N-terminal extended peptide is extended by one residue.
  • the N- terminal extended peptide is extended by two residues.
  • the N-terminal extended peptide is extended by three residues. In another embodiment, the N-terminal extended peptide is extended by more than three residues. [0127] In another embodiment, the longer peptide is extended on the C terminal end in accordance with the surrounding WT-1 sequence. Peptides are, in another embodiment, amenable to extension on the C- terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention. In another embodiment, the C -terminal extended peptide is extended by one residue. In another embodiment, the C- terminal extended peptide is extended by two residues. In another embodiment, the C-terminal extended peptide is extended by three residues. In another embodiment, the C-terminal extended peptide is extended by more than three residues.
  • the extended peptide is extended on both the N- terminal and C-terminal ends in accordance with the surrounding WT- 1 sequence. [0129] Each of the above extended peptides represents a separate embodiment of the present invention.
  • a truncated peptide of the present invention retains the
  • HLA anchor residues e.g. the HLA class I anchor residues
  • C- terminal residue with a smaller number of intervening residues (e.g.5) than a peptide enumerated in the Examples of the present invention.
  • Peptides are, in another embodiment, amenable to such mutation without changing affinity for
  • HLA molecules In another embodiment, such a truncated peptide is designed by removing one of the intervening residues of one of the above sequences.
  • the HLA anchor residues are retained on the second and eighth residues.
  • the HLA anchor residues are retained on the first and eighth residues.
  • an extended peptide of the present invention retains the HLA anchor residues (e.g. the HLA class I anchor residues) on the second residue and the C-terminal residue, with a larger number of intervening residues (e.g. 7 or 8) than a peptide enumerated in the Examples of the present invention.
  • such an extended peptide is designed by adding one or more residues between two of the intervening residues of one of the above sequences. It is well known in the art that residues can be removed from or added between the intervening sequences of HLA-binding peptides without changing affinity for HLA. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention.
  • the HLA anchor residues are retained on the second and ninth residues. In another embodiment, the HLA anchor residues are retained on the first and eighth residues. In another embodiment, the HLA anchor residues are retained on the two residues separated by six intervening residues. Each possibility represents a separate embodiment of the present invention.
  • “Fragment,” in another embodiment, refers to a peptide of 11 or more AA in length. In another embodiment, a peptide fragment of the present invention is 16 or more AA long. In another embodiment, the fragment is 12 or more AA long. In another embodiment, the fragment is 13 or more AA. In another embodiment, the fragment is 14 or more AA. In another embodiment, the fragment is 15 or more AA. In another embodiment, the fragment is
  • the fragment is 18 or more AA. In another embodiment, the fragment is 19 or more AA. In another embodiment, the fragment is 22 or more AA. In another embodiment, the fragment is 8-12 AA. In another embodiment, the fragment is about 8-12 AA. In another embodiment, the fragment is 16- 19 AA. In another embodiment, the fragment is about 16-19 AA. In another embodiment, the fragment 10-25 AA. In another embodiment, the fragment is about 10-25 AA. In another embodiment, the fragment has any other length. Each possibility represents a separate embodiment of the present invention.
  • a peptide of the present invention is homologous to a peptide enumerated in the Examples.
  • Homology is, in another embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art.
  • computer algorithm analysis of nucleic acid sequence homology includes the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.
  • homology refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 70%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 75%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 78%.
  • “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 82%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 85%.
  • “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 87%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than[0128] 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 92%.
  • “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 95%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 97%.
  • homology refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of 100%. Each possibility represents a separate embodiment of the present invention. [00114] In another embodiment, homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, "Nucleic AcidHybridization” Hames, B.
  • methods of hybridization are carried out under moderate to stringent conditions, to the complement of aDNA encoding a native caspase peptide.
  • Hybridization conditions being, for example, overnight incubation at 42 °C in a solution comprising: 10-20 % formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate ( H 7.6), 5 X Denhardt's solution, 10 % dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • the present invention provides a composition comprising a peptide of this invention.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the composition further comprises an adjuvant.
  • the composition comprises 2 or more peptides of the present invention.
  • the composition further comprises any of the additives, compounds, or excipients set forth hereinbelow.
  • the adjuvant is KLH, QS21 , Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG or alum.
  • the carrier is any carrier enumerated herein.
  • the adjuvant is any adjuvant enumerated herein.
  • this invention provides a vaccine comprising a peptide of this invention.
  • this invention provides a vaccine comprising an antigen-presenting cell (APC) and a peptide of this invention.
  • the vaccine further comprises a carrier.
  • the vaccine further comprises an adjuvant.
  • the vaccine further comprises an APC.
  • the vaccine further comprises a combination of more than 1 of an antigen, carrier, and/or APC.
  • the vaccine is a cell-based composition. Each possibility represents a separate embodiment of the present invention.
  • the term "vaccine” refers to a material or composition that, when introduced into a subject, provides a prophylactic or therapeutic response for a particular disease, condition, or symptom of same.
  • this invention comprises peptide-based vaccines, wherein the peptide comprises any embodiment listed herein, including immunomodulating compounds such as cytokines, adjuvants, etc.
  • a vaccine of methods and compositions of the present invention further comprises an adjuvant.
  • the adjuvant is a Montanide.
  • the adjuvant is Montanide ISA 51.
  • Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier.
  • the adjuvant is GM-CSF.
  • Recombinant GM-CSF is a human protein grown, in another embodiment, in a yeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, APC, and dendritic cells and T cells.
  • the adjuvant is a cytokine. In another embodiment, the adjuvant is a growth factor. In another embodiment, the adjuvant is a cell population. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is Freund's incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. In another embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is BCG. In another embodiment, the adjuvant is alum. [0145] In another embodiment, the adjuvant is an interleukin. In another embodiment, the adjuvant is a chemokine. In another embodiment, the adjuvant is any other type of adjuvant known in the art.
  • the WT-1 vaccine comprises two the above adjuvants. In another embodiment, the WT-1 vaccine comprises more than two the above adjuvants. Each possibility represents a separate embodiment of the present invention.
  • a vaccine or composition of the present invention can comprise any of the embodiments of WT-1 peptides of the present invention and combinations thereof. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby treating a subject with a WT-1 -expressing cancer.
  • the present invention provides a method of treating a subject with an MDS, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby treating a subject with an MDS.
  • the present invention provides a method of suppressing or halting the progression of a WT-1 -expressing cancer in a subject, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby suppressing or halting the progression of a WT-1- expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer in a subject, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer in a subject.
  • the present invention provides a method of reducing the incidence of an AML in a subject, the method comprising administering to the subject a WT- 1 vaccine of the present invention, thereby reducing the incidence of an AML.
  • the present invention provides a method of reducing the incidence of relapse of a WT-1 -expressing cancer in a subject, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby reducing the incidence of relapse of a WT-1 -expressing cancer in a subject.
  • the present invention provides a method of reducing the incidence ofrelapse of an AML in a subject, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby reducing the incidence of relapse of an AML in a subject.
  • the present invention provides a method of breaking a
  • T cell tolerance of a subject to a WT-1 -expressing cancer comprising administering to the subject a WT-1 vaccine of the present invention, thereby breaking a T cell tolerance to a WT-1 -expressing cancer.
  • the present invention provides a method of treating a subject having aWT-1 -expressing cancer, comprising (a) inducing in a donor formation and proliferation of human cytotoxic T lymphocytes (CTL) that recognize a malignant cell of the cancer by a method of the present invention; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer.
  • CTL cytotoxic T lymphocytes
  • the present invention provides a method of treating a subject having a WT 1 -expressing cancer, comprising (a) inducing ex vivo formation and proliferation of human CTL that recognize a malignant cell of the cancer by a method of the present invention, wherein the human immune cells are obtained from a donor; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer.
  • the present invention provides a method of inducing formation and proliferation of (a) a WT-1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT- 1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT-1 protein- specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT-1 protein; or a combination thereof.
  • This method can be conducted in vitro, ex vivo or in vivo.When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
  • the present invention provides a method of inducing the formation and proliferation of CTL specific for cells of a WT- 1 -expressing cancer, the method comprising contacting a lymphocyte population with a vaccine of the present invention.
  • the vaccine is an APC associated with a peptide of the present invention.
  • the vaccine is an APC associated with a mixture of peptides of the present invention.
  • this invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against a WT-1 -expressing cancer, the method comprising administering to the subject a vaccine of the present invention, thereby generating a heteroclitic immune response.
  • the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT-1 protein; or (b) a fragment of a WT protein, thereby inducing an anti-mesothelioma immune response in a subject.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising a nucleotide molecule encoding (a) a WT-1 protein; or (b) a fragment of a WT-1 protein, thereby inducing an anti- mesothelioma immune response in a subject.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; or (b) a fragment of a WT protein, thereby treating a subject with a mesothelioma.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT-1 protein; or (b) a fragment of a WT-1 protein, thereby treating a subject with a mesothelioma.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; or (b) a fragment of a WT protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT-1 protein; or (b) a fragment of a WT-1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the mesothelioma is a malignant mesothelioma.
  • a target cell of an immune response elicited by a method of the present invention presents the WT-1 peptide of the present invention, or a corresponding WT-1 fragment, on an HLA molecule.
  • the HLA molecule is an HLA class I molecule.
  • the HLA molecule is any HLA class I subtype or HLA class I molecule known in the art.
  • the immune response against the WT-1 peptide or fragment is a heteroclitic immune response. Each possibility represents a separate embodiment of the present invention.
  • the WT-1 -expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT-1 -expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT-1 -expressing cancer is an MDS. In another embodiment, the WT-1- expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT-1 -expressing cancer is a Wilms' tumor. In another embodiment, the WT-1 -expressing cancer is a leukemia. In another embodiment, the WT-1 -expressing cancer is a hematological cancer. In another embodiment, the WT-1- expressing cancer is a lymphoma.
  • AML acute myelogenous leukemia
  • MDS myelodysplastic syndrome
  • the WT-1 -expressing cancer is an MDS.
  • the WT-1-expressing cancer is a non-small cell lung cancer (NSCLC).
  • the WT-1 -expressing cancer is a Wilms'
  • the WT-1 -expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT-1 -expressing cancer is a mesothelioma. In another embodiment, the WT-1 -expressing cancer is a malignant mesothelioma. In another embodiment, the WT-1 -expressing cancer is a gastric cancer. In another embodiment, the WT-1 -expressing cancer is a colon cancer. In another embodiment, the WT-1 -expressing cancer is a lung cancer. In another embodiment, the WT-1 -expressing cancer is abreast cancer. In another embodiment, the WT-1 -expressing cancer is a germ cell tumor. In another embodiment, the WT-1 -expressing cancer is an ovarian cancer.
  • the WT 1 -expressing cancer is a uterine cancer. In another embodiment, the WT 1 - expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a liver cancer. In another embodiment, the WT-1- expressing cancer is a renal cancer. In another embodiment, the WT-1 -expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT-1- expressing cancer is a sarcoma. In another embodiment, the WT-1 -expressing cancer is any other carcinoma or sarcoma.
  • the WT-1 -expressing cancer is a solid tumor.
  • the solid tumor is associated with a WT-1 -expressing cancer.
  • the solid tumor is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • NSCLC non-small cell lung cancer
  • the solid tumor is associated with a lung cancer.
  • the solid tumor is associated with a breast cancer.
  • the solid tumor is associated with a colorectal cancer.
  • the solid tumor is associated with a prostate cancer.
  • the solid tumor is associated with an ovarian cancer.
  • the solid tumor is associated with a renal cancer.
  • the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
  • a cancer or tumor treated by a method of the present invention is suspected to express WT-1.
  • WT-1 expression has not been verified by testing of the actual tumor sample.
  • the cancer or tumor is of a type known to express WT-1 in many cases. In another embodiment, the type expresses WT-1 in the majority of cases.
  • Each type of WT-1 -expressing cancer or tumor, and cancer or tumor suspected to express WT- 1 represents a separate embodiment of the present invention.
  • Any embodiments enumerated herein, regarding peptides, vaccines and compositions of this invention can be employed in any of the methods of this invention, and each represents an embodiment thereof.
  • multiple peptides of this invention are used to stimulate an immune response in methods of the present invention.
  • vaccines of the present invention have the advantage of activating or eliciting WT-1 -specific CD4 + T cells containing a variety of different HLA class II alleles.
  • the vaccines have the advantage of activating or eliciting WT-1 -specific CD4 + T cells in a substantial proportion of the population (e.g. in different embodiments, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%, 90%, 95%, or greater than 95%).
  • the vaccines activate or elicit WT- 1 -specific CD4 + T cells in a substantial proportion of a particular population (e.g. American Caucasians). Each possibility represents a separate embodiment of the present invention.
  • methods of the present invention provide for an improvement in an immune response that has already been mounted by a subject.
  • methods of the present invention comprise administering the peptide, composition, or vaccine 2 or more times.
  • the peptides are varied in their composition, concentration, or a combination thereof.
  • the peptides provide for the initiation of an immune response against an antigen of interest in a subject who has not yet initiated an immune response against the antigen.
  • the CTL that are induced proliferate in response to presentation of the peptide on the APC or cancer cell.
  • reference to modulation of the immune response involves, either or both the humoral and cell-mediated arms of the immune system, which is accompanied by the presence of Th2 and Thl T helper cells, respectively, or in another embodiment, each arm individually.
  • each arm individually.
  • tumor inhibition is determined by measuring the actual tumor size over a period of time.
  • tumor inhibition can be determined by estimating the size of a tumor (over a period of time) utilizing methods well known to those of skill in the art. More specifically, a variety of radiologic imaging methods (e.g., single photon and positron emission computerized tomography; see generally, "Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.) Springer- Verlag, New York, 1986), can be utilized to estimate tumor size.
  • radiologic imaging methods e.g., single photon and positron emission computerized tomography; see generally, "Nuclear Medicine in Clinical Oncology," Winkler, C. (ed.) Springer- Verlag, New York, 1986
  • imaging agents can also utilize a variety of imaging agents, including for example, conventional imaging agents (e.g., Gallium-67 citrate), as well as specialized reagents for metabolite imaging, receptor imaging, or immunologic imaging (e.g., radiolabeled monoclonal antibody specific tumor markers).
  • conventional imaging agents e.g., Gallium-67 citrate
  • immunologic imaging e.g., radiolabeled monoclonal antibody specific tumor markers
  • non-radioactive methods such as ultrasound (see, "Ultrasonic Differential Diagnosis of Tumors", Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984), can also be utilized to estimate the size of a tumor.
  • lymphocyte mediated anti-tumor cytolytic activity determined for example, by a 51 Cr release assay (Examples), tumor dependent lymphocyte proliferation (Ioannides, et al., J. Immunol. 146(5): 1700-1707, 1991), in vitro generation of tumor specific antibodies (Herlyn, et al., J. Immunol. Meth.
  • cell e.g., CTL, helper T-cell
  • humoral e.g., antibody
  • methods of suppressing tumor growth indicate a growth state that is curtailed compared to growth without contact with, or exposure to a peptide of this invention.
  • Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a 3 H-thymidine incorporation assay, or counting tumor cells.
  • "Suppressing" tumor cell growth refers, in other embodiments, to slowing, delaying, or stopping tumor growth, or to tumor shrinkage. Each possibility represents a separate embodiment of the present invention.
  • WT-1 expression is measured.
  • WT-1 transcript expression is measured.
  • WT-1 protein levels in the tumor are measured.
  • lymphocyte proliferation assays wherein T cell uptake of a radioactive substance, e.g. 3 H-thymidine is measured as a function of cell proliferation.
  • detection of T cell proliferation is accomplished by measuring increases in interleukin-2 (IL-2) production, Ca 2+ flux, or dye uptake, such as 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium.
  • IL-2 interleukin-2
  • dye uptake such as 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium.
  • CTL stimulation is determined by means known to those skilled in the art, including, detection of cell proliferation, cytokine production and others. Analysis of the types and quantities of cytokines secreted by T cells upon contacting ligand-pulsed targets can be a measure of functional activity. Cytokines can be measured by ELISA or ELISPOT assays to determine the rate and total amount of cytokine production. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160: 181 ; Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259).
  • CTL activity is determined by 51 Cr-release lysis assay. Lysis of peptide- pulsed 51 Cr-labeled targets by antigen-specific T cells can be compared for target cells pulsed with control peptide.
  • T cells are stimulated with a peptide of this invention, and lysis of target cells expressing the native peptide in the context of MHC can be determined. The kinetics of lysis as well as overall target lysis at a fixed timepoint (e.g., 4 hours) are used, in another embodiment, to evaluate ligand performance. (Ware C. F. et al. (1983) J Immunol 131 : 1312).
  • affinity is determined by TAP stabilization assays.
  • affinity is determined by competition radioimmunoassay.
  • the following protocol is utilized: Target cells are washed two times in PBS with 1 % bovine serum albumin (BSA; Fisher Chemicals, Fairlawn, NJ). Cells are resuspended at 10 7 /ml on ice, and the native cell surface bound peptides are stripped for 2 minutes at 0[deg.] C using citrate -phosphate buffer in the presence of 3 mg/ml beta2 microglobulin.
  • BSA bovine serum albumin
  • the pellet is resuspended at 5 x 10 6 cells/ml in PBS/1 % BSA in the presence of 3 mg/ml beta 2 microglobulin and 30 mg/ml deoxyribonuclease, and 200 ml aliquots are incubated in the presence or absence of HLA-specific peptides for 10 min at 20°C, then with 125 I-labeled peptide for 30 min at 20 °C. Total bound 125 I is determined after two washes with PBS/2% BSA and one wash with PBS. Relative affinities are determined by comparison of escalating concentrations of the test peptide versus a known binding peptide.
  • HLA on surface of live cells is conducted to confirm that the binding is to the appropriate HLA molecule and to characterize its restriction.
  • This assay is performed, in another embodiment, on live fresh or 0.25% paraformaldehyde-fixed human PBMC, leukemia cell lines and EBV-transformed T-cell lines of specific HLA types. The relative avidity of the peptides found to bind MHC molecules on live cells (e.g. SKLY-16 cells) is conducted to confirm that the binding is to the appropriate HLA molecule and to characterize its restriction.
  • This assay is performed, in another embodiment, on live fresh or 0.25% paraformal
  • an HLA class Il-binding peptide of methods and compositions of the present invention is longer than the minimum length for binding to an HLA class II molecule, which is, in another embodiment, about 12 AA.
  • increasing the length of the HLA class II- binding peptide enables binding to more than one HLA class II molecule.
  • increasing the length enables binding to an HLA class II molecule whose binding motif is not known.
  • increasing the length enables binding to an HLA class I molecule.
  • the binding motif of the HLA class I molecule is known. In another embodiment, the binding motif of the HLA class I molecule is not known. Each possibility represents a separate embodiment of the present invention.
  • the peptides utilized in methods and compositions of the present invention comprise a non-classical amino acid such as: 1,2,3,4- tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991) J. Am Chem. Soc.
  • histidine isoquinoline carboxylic acid Zachel et al. (1991) Int. J. Pep. Protein Res. 38(2): 131-138); and HIC (histidine cyclic urea), (Dharanipragada et al.(1993) Int. J. Pep. Protein Res.42(l):68-77) and ((1992) Acta. Crst, Crystal Struc. Comm. 48(IV): 1239-124).
  • a peptide of this invention comprises an AA analog or peptidomimetic, which, in other embodiments, induces or favors specific secondary structures.
  • Such peptides comprise, in other embodiments, the following: LL-Acp (LL-3- amino-2-propenidone-6-carboxylic acid), a [beta]-turn inducing dipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838); [beta]-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5081-5082); [beta]-turn inducing analogs (Kemp et al.
  • apeptide of this invention is conjugated to one of various other molecules, as described hereinbelow, which can be via covalent or non-covalent linkage (complexed), the nature of which varies, in another embodiment, depending on the particular purpose.
  • the peptide is covalently or non-covalently complexed to a macromolecular carrier, (e.g. an immunogenic carrier), including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids.
  • a peptide of this invention is linked to a substrate.
  • the peptide is conjugated to a fatty acid, for introduction into a liposome (U.S. Pat. No.5, 837,249).
  • a peptide of the invention is complexed covalently or non-covalently with a solid support, a variety of which are known in the art.
  • linkage of the peptide to the carrier, substrate, fatty acid, or solid support serves to increase an elicited an immune response.
  • the carrier is thyroglobulin, an albumin (e.g.
  • amino acid refers to a natural or, in another embodiment, an unnatural or synthetic AA, and can include, in other embodiments, glycine, D- or L optical isomers, AA analogs, peptidomimetics, or combinations thereof.
  • cancer in another embodiment, the terms "cancer,” “neoplasm,” “neoplastic” or
  • tumor are used interchangeably and refer to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a tumor is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation, and in another embodiment, is identified by biochemical or immunologic findings, the latter which is used to identify cancerous cells, as well, in other embodiments.
  • the peptides of this invention are synthesized using an appropriate solid-state synthetic procedure (see for example,Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco, Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23: 451). The activity of these peptides is tested, in other embodiments, using assays as described herein.
  • the peptides of this invention are purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • immuno- affinity chromatography is used, whereby an epitope is isolated by binding it to an affinity column comprising antibodies that were raised against that peptide, or a related peptide of the invention, and were affixed to a stationary support.
  • affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Meth. Enzymol. 194:508-509), glutathione-S-transferase, or others, are attached to the peptides of this invention to allow easy purification by passage over an appropriate affinity column.
  • Isolated peptides can also be physically characterized, in other embodiments, using such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallography.
  • the peptides of this invention are produced by in vitro translation, through known techniques, as will be evident to one skilled in the art.
  • the peptides are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).
  • the peptides of this invention further comprise a detectable label, which in another embodiment, is fluorescent, or in another embodiment, luminescent, or in another embodiment, radioactive, or in another embodiment, electron dense.
  • the detectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-
  • galactosidase luciferase
  • P, I, H and C fluorescein and its derivatives
  • rhodamine and its derivatives fluorescein and its derivatives
  • dansyl and umbelliferone fluorescein or its derivatives
  • luciferin any number of other such labels known to one skilled in the art.
  • the particular label used will depend upon the type of immunoassay used.
  • a peptide of this invention is linked to a substrate, which, in another embodiment, serves as a carrier.
  • linkage of the peptide to a substrate serves to increase an elicited an immune response.
  • peptides of this invention are linked to other molecules, as described herein, using conventional cross-linking agents such as carbodiimide.
  • carbodiimide are 1- cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), l-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and l-ethyl-3-(4-azonia-44- dimethylpentyl) carbodiimide.
  • the cross-linking agents comprise cyanogen bromide, glutaraldehyde and succinic anhydride.
  • any of a number of homo-bifunctional agents including a homo- bifunctional aldehyde, a homo-bifunctional epoxide, a homo- bifunctional imido-ester, a homo- bifunctional N-hydroxysuccinimide ester, a homo- bifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyl disulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo- bifunctional diazonium derivative and a homo-bifunctional photoreactive compound can be used.
  • hetero-bifunctional compounds for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine- reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.
  • the homo-bifunctional cross-linking agents include the bifunctional N- hydroxy succinimide esters dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers l,4-di-[3'-(2'- pyridyldithio)propionamido]butane, bismaleimidohexane, and bis- N-maleimido-l,8-octane; the bifunctional aryl halides 1 ,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'-dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b
  • hetero-bifunctional cross-linking agents used to link the peptides to other molecules include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate), MB S (m- maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4- iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p- maleimidophenyl)butyrate), GMBS (N-(.gamma.-maleimidobutyryloxy)succmimide ester), MPBH (4-(4- N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl) cyclohexane-1- carboxyl-
  • the peptides of the invention are formulated as non- covalent attachment of monomers through ionic, adsorptive, or biospecific interactions.
  • Complexes of peptides with highly positively or negatively charged molecules can be accomplished, in another embodiment, through salt bridge formation under low ionic strength environments, such as in deionized water.
  • Large complexes can be created, in another embodiment, using charged polymers such as poly- (L- glutamic acid) or poly- (L-lysine), which contain numerous negative and positive charges, respectively.
  • peptides are adsorbed to surfaces such as microparticle latex beads or to other hydrophobic polymers, forming non-covalently associated peptide-superantigen complexes effectively mimicking cross-linked or chemically polymerized protein, in other embodiments.
  • peptides are non- covalently linked through the use of biospecific interactions between other molecules. For instance, utilization of the strong affinity of biotin for proteins such as avidin or streptavidin or their derivatives could be used to form peptide complexes.
  • peptides can be modified to possess biotin groups using common biotinylation reagents such as the N- hydroxysuccinimidyl ester of D-biotin (NHS-biotin), which reacts with available amine groups.
  • a peptide of the present invention is linked to a carrier.
  • the carrier is KLH.
  • the carrier is any other carrier known in the art, including, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like.
  • thyroglobulin albumins such as human serum albumin, tetanus toxoid
  • polyamino acids such as poly (lysine: glutamic acid)
  • influenza hepatitis B virus core protein
  • hepatitis B virus recombinant vaccine recombinant vaccine
  • the peptides of this invention are conjugated to a lipid, such as P3 CSS. In another embodiment, the peptides of this invention are conjugated to a bead. [0208] In another embodiment, the compositions of this invention further comprise immunomodulating compounds. In other embodiments, the immunomodulating compound is a cytokine, chemokine, or complement component that enhances expression of immune system accessory or adhesion molecules, their receptors, or combinations thereof.
  • the immunomodulating compound include interleukins, for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte- macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M- CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia and MIP-Ib, complement components, or combinations thereof.
  • interleukins for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte- macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M- CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and
  • the immunomodulating compound stimulate expression, or enhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or combination thereof.
  • the immunomodulatory compound induces or enhances expression of co- stimulatory molecules that participate in the immune response, which include, in some embodiments, CD40 or its ligand, CD28, CTLA-4 or a B7 molecule.
  • the immunomodulatory compound induces or enhances expression of a heat stable antigen (HSA) (Liu Y. et al. (1992) J. Exp.Med. 175:437-445), chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas M. F. et al (1993) Cell 74:257-268), or an intracellular adhesion molecule 1 (ICAM-I) (Van R. H.
  • HSA heat stable antigen
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • ICM-I intracellular adhesion molecule 1
  • the composition comprises a solvent, including water, dispersion media, cell culture media, isotonic agents and the like.
  • the solvent is an aqueous isotonic buffered solution with a pH of around 7.0.
  • the composition comprises a diluent such as water, phosphate buffered saline, or saline.
  • the composition comprises a solvent, which is nonaqueous, such as propyl ethylene glycol, polyethylene glycol and vegetable oils.
  • the composition is formulated for administration by any of the many techniques known to those of skill in the art.
  • this invention provides for administration of the pharmaceutical composition parenterally, intravenously, subcutaneously, intradermally, intramucosally, topically, orally, or by inhalation.
  • the vaccine comprising a peptide of this invention further comprises a cell population, which, in another embodiment, comprises lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells or combinations thereof, which, in another embodiment are autologous, syngeneic or allogeneic, with respect to each other.
  • the cell population comprises a peptide of the present invention.
  • the cell population takes up the peptide.
  • the cell populations of this invention are obtained from in vivo sources, such as, for example, peripheral blood, leukopheresis blood product, apheresis blood product, peripheral lymph nodes, gut associated lymphoid tissue, spleen, thymus, cord blood, mesenteric lymph nodes, liver, sites of immunologic lesions, e.g. synovial fluid, pancreas, cerebrospinal fluid, tumor samples, granulomatous tissue, or any other source where such cells can be obtained.
  • the cell populations are obtained from human sources, which are, in other embodiments, from human fetal, neonatal, child, or adult sources.
  • the cell populations of this invention are obtained from animal sources, such as, for example, porcine or simian, or any other animal of interest.
  • the cell populations of this invention are obtained from subjects that are normal, or in another embodiment, diseased, or in another embodiment, susceptible to a disease of interest.
  • the cell populations of this invention are separated via affinity-based separation methods. Techniques for affinity separation include, in other embodiments, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or use in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and "panning" with an antibody attached to a solid matrix, such as a plate, or any other convenient technique.
  • separation techniques include the use of fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • any technique that enables separation of the cell populations of this invention can be employed, and is to be considered as part of this invention.
  • the dendritic cells are from the diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, qualified as such (Steinman (1991) Ann. Rev. Immunol.9:271-296).
  • the dendritic cells used in this invention are isolated from bone marrow, or in another embodiment, derived from bone marrow progenitor cells, or, in another embodiment, from isolated from/derived from peripheral blood, or in another embodiment, derived from, or are a cell line.
  • the cell populations described herein are isolated from the white blood cell fraction of a mammal, such as a murine, simian or a human (See, e.g., WO 96/23060).
  • the white blood cell fraction can be, in another embodiment, isolated from the peripheral blood of the mammal.
  • the DC are isolated via a method which includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation; (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF and IL-4, (d) identifying the dendritic cell-enriched fraction from step (c); and (e) collecting the enriched fraction of step (d), preferably at about 4[deg.] C.
  • the dendritic cell-enriched fraction is identified by fluorescence-activated cell sorting, which identifies at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absence of the following markers: CD3, CD14, CD16, 56, 57, and CD 19, 20.
  • the cell population comprises lymphocytes, which are, in another embodiment, T cells, or in another embodiment, B cells.
  • the T cells are, in other embodiments, characterized as NK cells, helper T cells, cytotoxic T lymphocytes (CTL), TBLs, native T cells, or combinations thereof. It is to be understood that T cells which are primary, or cell lines, clones, etc. are to be considered as part of this invention.
  • the T cells are CTL, or CTL lines, CTL clones, or CTLs isolated from tumor, inflammatory, or other infiltrates.
  • hematopoietic stem or early progenitor cells comprise the cell populations used in this invention.
  • such populations are isolated or derived, by leukaphoresis.
  • the leukapheresis follows cytokine administration, from bone marrow, peripheral blood (PB) or neonatal umbilical cord blood.
  • the stem or progenitor cells are characterized by their surface expression of the surface antigen marker known as CD34 + , and exclusion of expression of the surface lineage antigen markers, Lin-.
  • the subject is administered a peptide, composition or vaccine of this invention, in conjunction with bone marrow cells.
  • the administration together with bone marrow cells embodiment follows previous irradiation of the subject, as part of the course of therapy, in order to suppress, inhibit or treat cancer in the subject.
  • the phrase "contacting a cell” or “contacting a population” refers to a method of exposure, which can be, in other embodiments, direct or indirect.
  • such contact comprises direct injection of the cell through any means well known in the art, such as microinjection.
  • supply to the cell is indirect, such as via provision in a culture medium that surrounds the cell, or administration to a subject, via any route well known in the art, and as described herein.
  • CTL generation of methods of the present invention is accomplished in vivo, and is effected by introducing into a subject an antigen presenting cell contacted in vitro with a peptide of this invention (See for example Paglia et al. (1996) J. Exp. Med. 183:317-322).
  • the peptides of methods and compositions of the present invention are delivered to APC.
  • the peptide-pulsed APC are administered to a subject to elicit and immune response or treat or inhibit growth or recurrence of a tumor.
  • Each possibility represents a separate embodiment of the present invention.
  • the peptides are delivered to APC in the form of cDNA encoding the peptides.
  • the term "antigen-presenting cells” refers to dendritic cells (DC), monocytes/macrophages, B lymphocytes or other cell type(s) expressing the necessary MHC/co- stimulatory molecules, which effectively allow for T cell recognition of the presented peptide.
  • the APC is a cancer cell.
  • the CTL are contacted with 2 or more APC populations.
  • the 2 or more APC populations present different peptides. Each possibility represents a separate embodiment of the present invention.
  • techniques that lead to the expression of antigen in the cytosol of APC are used to deliver the peptides to the APC.
  • Methods for expressing antigens on APC are well known in the art.
  • the techniques include (1) the introduction into the APC of naked DNA encoding a peptide of this invention, (2) infection of APC with recombinant vectors expressing a peptide of this invention, and (3) introduction of a peptide of this invention into the cytosol of an APC using liposomes.
  • foster APC such as those derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150: 1763-1771), are used, as exemplified herein.
  • the subject is exposed to a peptide, or a composition/cell population comprising a peptide of this invention, which differs from the native protein expressed, wherein subsequently a host immune cross-reactive with the native protein/antigen develops.
  • the subject as referred to in any of the methods or embodiments of this invention is a human.
  • the subject is a mammal, which can be a mouse, rat, rabbit, hamster, guinea pig, horse, cow, sheep, goat, pig, cat, dog, monkey, or ape. Each possibility represents a separate embodiment of the present invention.
  • peptides, vaccines, and compositions of this invention stimulate an immune response that results in tumor cell lysis.
  • any of the methods described herein is used to elicit any of the methods described herein.
  • CTL which are elicited in vitro.
  • the CTL are elicited ex-vivo.
  • the CTL are elicited in vitro.
  • the resulting CTL are, in another embodiment, administered to the subject, thereby treating the condition associated with the peptide, an expression product comprising the peptide, or a homologue thereof.
  • the method entails introduction of the genetic sequence that encodes the peptides of this invention using, e.g., one or more nucleic acid delivery techniques.
  • Nucleic acids of the invention include, in another embodiment, DNA, RNA and mixtures of DNA and RNA, alone or in conjunction with non-nucleic acid components.
  • the method comprises administering to the subject a vector comprising a nucleotide sequence, which encodes a peptide of the present invention (Tindle, R. W. et al. Virology (1994) 200:54).
  • the method comprises administering to the subject naked DNA which encodes a peptide, or in another embodiment, two or more peptides of this invention (Nabel, et al. PNAS-USA (1990) 90: 11307).
  • label, et al. PNAS-USA (1990) 90: 11307 In another embodiment, multi-epitope, analogue -based cancer vaccines are utilized (Fikes et al, Design of multi- epitope, analogue-based cancer vaccines. Expert Opin Biol Ther.2003 Sep;3(6):985-93). Each possibility represents a separate embodiment of the present invention.
  • Nucleic acids can be administered to a subject via any means as is known in the art, including parenteral or intravenous administration, or in another embodiment, by means of a gene gun. In another embodiment, the nucleic acids are administered in a composition, which correspond, in other embodiments, to any embodiment listed herein.
  • Vectors for use according to methods of this invention can comprise any vector that facilitates or allows for the expression of a peptide of this invention.
  • Vectors comprise, in some embodiments, attenuated viruses, such as vaccinia or fowlpox, such as described in, e.g., U.S. Pat. No. 4,722,848, incorporated herein by reference.
  • the vector is BCG (Bacille Calmette Guerin), such as described in Stover et al. (Nature 351 :456-460 (1991)).
  • BCG Bacille Calmette Guerin
  • Salmonella typhi vectors and the like will be apparent to those skilled in the art from the description herein.
  • the vector further encodes for an immunomodulatory compound, as described herein.
  • the subject is administered an additional vector encoding same, concurrent, prior to or following administration of the vector encoding a peptide of this invention to the subject.
  • the peptides, compositions and vaccines of this invention are administered to a subject, or utilized in the methods of this invention, in combination with other anticancer compounds and chemotherapeutics, including monoclonal antibodies directed against alternate cancer antigens, or, in another embodiment, epitopes that consist of an AA sequence which corresponds to, or in part to, that from which the peptides of this invention are derived.
  • the dosage is 20 ⁇ g per peptide per day. In another embodiment, the dosage is 10 ⁇ g/peptide/day. In another embodiment, the dosage is 30 ⁇ g/peptide/day. In another embodiment, the dosage is 40 ⁇ g/peptide/day. In another embodiment, the dosage is 60 ⁇ g/peptide/day. In another embodiment, the dosage is 80 ⁇ g/peptide/day. In another embodiment, the dosage is 100 ⁇ g/peptide/day. In another embodiment, the dosage is 150 ⁇ g/peptide/day. In another embodiment, the dosage is 200 ⁇ g/peptide/day. In another embodiment, the dosage is 300 ⁇ g/peptide/day.
  • the dosage is 400 ⁇ g/peptide/day. In another embodiment, the dosage is 600 ⁇ g/peptide/day. In another embodiment, the dosage is 800 ⁇ g/peptide/day. In another embodiment, the dosage is 1000 ⁇ g/ e tide/day. In another embodiment, the dosage is 1500 ⁇ g/peptide/day. In another embodiment, the dosage is 2000 ⁇ g/peptide/day.
  • the dosage is 10 ⁇ g/peptide/dose. In another embodiment, the dosage is 30 ⁇ g/peptide/dose. In another embodiment, the dosage is 40 ⁇ g/peptide/dose. In another embodiment, the dosage is 60 ⁇ g/peptide/dose. In another embodiment, the dosage is 80 ⁇ g/peptide/dose. In another embodiment, the dosage is 100 ⁇ g/peptide/dose. In another embodiment, the dosage is 150 ⁇ g/peptide/dose. In another embodiment, the dosage is 200 ⁇ g/peptide/dose. In another embodiment, the dosage is 300 ⁇ g/peptide/dose. In another embodiment, the dosage is 400 ⁇ g/peptide/dose. In another embodiment, the dosage is 600 ⁇ g/peptide/dose.
  • the dosage is 800 ⁇ g/peptide/dose. In another embodiment, the dosage is 1000 ⁇ g/peptide/dose. In another embodiment, the dosage is 1500 ⁇ g/peptide/dose. In another embodiment, the dosage is 2000 ⁇ g/peptide/dose.
  • the dosage is 10-20 ⁇ g/peptide/dose. In another embodiment, the dosage is 20-30 ⁇ g/peptide/dose. In another embodiment, the dosage is 20- 40 ⁇ g/peptide/dose. In another embodiment, the dosage is 30-60 ⁇ g/peptide/dose. In another embodiment, the dosage is 40-80 ⁇ g/peptide/dose. In another embodiment, the dosage is 50- 100 ⁇ g/peptide/dose. In another embodiment, the dosage is 50-150 ⁇ g/peptide/dose. In another embodiment, the dosage is 100-200 ⁇ g/peptide/dose. In another embodiment, the dosage is 200-300 ⁇ g/peptide/dose. In another embodiment, the dosage is 300- 400 ⁇ g/peptide/dose.
  • the dosage is 400-600 ⁇ g/peptide/dose. In another embodiment, the dosage is 500-800 ⁇ g/peptide/dose. In another embodiment, the dosage is 800-1000 ⁇ g/peptide/dose. In another embodiment, the dosage is 1000-1500 ⁇ g/peptide/dose. In another embodiment, the dosage is 1500-2000 ⁇ g/peptide/dose. [0241] In another embodiment, the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts.
  • the dosage is 20 mg per peptide per day. In another embodiment, the dosage is 10 mg/peptide/day. In another embodiment, the dosage is 30 mg/peptide/day. In another embodiment, the dosage is 40 mg/peptide/day. In another embodiment, the dosage is 60 mg/peptide/day. In another embodiment, the dosage is 80 mg/peptide/day.In another embodiment, the dosage is 100 mg/peptide/day. In another embodiment, the dosage is 150 mg/peptide/day. In another embodiment, the dosage is 200 mg/peptide/day. In another embodiment, the dosage is 300 mg/peptide/day. In another embodiment, the dosage is 400 mg/peptide/day.
  • the dosage is 600 mg/peptide/day. In another embodiment, the dosage is 800 mg/peptide/day. In another embodiment, the dosage is 1000 mg/peptide/day. [0244] In another embodiment, the dosage is 10 mg/peptide/dose. In another embodiment, the dosage is 30 mg/peptide/dose. In another embodiment, the dosage is 40 mg/peptide/dose. In another embodiment, the dosage is 60 mg/peptide/dose. In another embodiment, the dosage is 80 mg/peptide/dose. In another embodiment, the dosage is 100 mg/peptide/dose. In another embodiment, the dosage is 150 mg/peptide/dose. In another embodiment, the dosage is 200 mg/peptide/dose. In another embodiment, the dosage is 300 mg/peptide/dose. In another embodiment, the dosage is 400 mg/peptide/dose. In another embodiment, the dosage is 600 mg/peptide/dose. In another embodiment, the dosage is 800 mg/peptide/dose. In another embodiment, the dosage is 1000 mg/peptide/dose.
  • the dosage is 10-20 mg/peptide/dose. In another embodiment, the dosage is 20-30 mg/peptide/dose. In another embodiment, the dosage is 20- 40 mg/peptide/dose. In another embodiment, the dosage is 30-60 mg/peptide/dose. In another embodiment, the dosage is 40-80 mg/peptide/dose. In another embodiment, the dosage is 50- 100 mg/peptide/dose. In another embodiment, the dosage is 50-150 mg/peptide/dose.In another embodiment, the dosage is 100-200 mg/peptide/dose. In another embodiment, the dosage is 200-300 mg/peptide/dose. In another embodiment, the dosage is 300-400 mg/peptide/dose. In another embodiment, the dosage is 400-600 mg/peptide/dose. In another embodiment, the dosage is 500-800 mg/peptide/dose. In another embodiment, the dosage is 800-1000 mg/peptide/dose.
  • the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts. [0247] Each of the above doses represents a separate embodiment of the present invention.
  • the present invention provides a kit comprising a peptide, composition or vaccine of the present invention.
  • the kit further comprises a label or packaging insert.
  • the kit is used for detecting a WT-1 -specific CD4 response through the use of a delayed- type hypersensitivity test.
  • the kit is used for any other method enumerated herein.
  • the kit is used for any other method known in the art. Each possibility represents a separate embodiment of the present invention.
  • WT-1 is considered one of the most promising (47).
  • the number of immunogenic WT-1 peptide antigens previously identified and reported is very limited, and largely confined to a set of peptides presented by the HLA alleles A0201, A2402 and DRB 10401.
  • WT-1 peptide-specific IFNy-f- CD4+ and CD8 T-cell responses were generated from the blood of 41/56 (78%) normal donors, and thereafter the epitopes eliciting these responses and their presenting HLA alleles were identified.
  • the new immunogenic peptides identified include 36 peptides presented by class I HLA alleles and 5 presented by class II HLA alleles. Of the peptides presented by class I HLA alleles, 10 nonamer epitopes were identified which could be presented by from 2-4 different HLA alleles. Also identified, within 4 pentadecapeptides, were overlapping 11-mer and nonamer sequences that co-induced distinguishable CD4+ IFNy-f- and CD8+ IFNy+T-cells.
  • T-cell lines specific for 29 epitopes including 2/4 epitopes presented by class II and 27/32 presented by class I alleles, were also able to lyse WT-1+ leukemic blasts sharing the T-cells' restricting HLA allele.
  • FIG. 4 maps are shown of the WT-1 protein.
  • Fig. 4C defines the localization of all previously reported antigenic epitopes presented by HLA class I and II alleles;
  • Fig 4D depicts the location of immunogenic peptides identified in this report.
  • the 11 epitopes previously reported to be presented by class I and 10 presented by class II HLA alleles are principally clustered in sequences encoded by exons 1, 7 and 10
  • the epitopes recognized by normal T-cells sensitized with the WT-1 peptide pool are principally clustered in sequences encoded by the first 5 exons.
  • 26 of the new epitopes are included in each of the four major isoforms of WT-1 resulting from splice variants that do or do not include the 17 amino acid sequence (aas 250-266) in exon 5 or the three amino acid sequence (400-41 0 KTS) between zinc fingers 3 and 4. While the epitopes are broadly distributed, clusters of epitopes were detected in the RNA recognition domain in exon 1 and the activation domain (aa 181-250) (Fig. 4F) proximal to the spliced 17aa segment in exon 5. The latter area also contained those epitopes most frequently recognized by multiple donors (Fig. 4E).
  • epitopes map to a 126 amino acid sequence at the N terminus encoded by a segment of the WT-1 gene initially described by Gessler et al (37) that is centromeric to exon 1 of the (Exon 5+, KTS+) isoform of WT-1 and includes the long isoform of WT-1 initiated at a CUG codon upstream of the AUG initiator for exon 1.50 Strikingly, each of the epitopes identified in this sequence elicits IFNy+T-cells that are cytolytic against leukemic blasts coexpressing WT-1 and the T-cells' restricting HLA allele.
  • EBVBLCL loaded with the WT-1 peptide pool preferentially induced the generation of CD8+ and CD4+ IFNy+ WT-1 peptide- specific T-cells from 41/56 normal donors (73%).
  • each donor recognized only 1-3 epitopes of WT-1
  • peptides from the WT-1 protein sequence that are upstream from exon 1, i.e., within the first 126 amino acids of SEQ ID NO: 194, are heretofore unrecognized sites of immunogenic epitopes and therefore peptides useful for the purposes herein.
  • Peptide sequence analysis was performed using 2 databases. The first was the software of the Bioinformatics & Molecular Analysis Section (National Institutes of Health, Washington, DC) (Parker KC et al, Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side- chains. J Immunol 152: 163-175, 1994), which ranks 9-mer or 10-mer peptides on a predicted half-time dissociation coefficient from HLA class I molecules. The second database, SYFPEITHI prediction software, is described in Rammensee HG et al (SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213-219, 1999).
  • Irrelevant control peptides used in in vitro experiments were: RAS (TEYKLVVVGAPGVGKSALTIQ; SEQ ID No: 49) or CML b2a2 (VHSIPLTINKEEALQRPVASDFE; SEQ ID No: 50) for Class II, and HIV pol (ILKEPVHGV; SEQ ID No: 51) or CML F (YLKALQRPY; SEQ ID No: 52) for Class I.
  • RAS TEYKLVVVGAPGVGKSALTIQ
  • CML b2a2 VHSIPLTINKEEALQRPVASDFE
  • HIV pol HIV pol
  • YLKALQRPY YLKALQRPY
  • Cell lines were cultured in RPMI 1640 medium supplemented with
  • T2 is a human cell line lacking TAPl and TAP2 and therefore unable to present peptides derived from cytosolic proteins.
  • Raji cells are a human Burkitt lymphoma cells that exhibit a high level of TAP expression.
  • H-MesolA NCI, Bethesda, MD
  • JMN and VAMT Dr. Sirotnak, Memorial Sloan Kettering Cancer Center (MSKCC)
  • H-2452 and H2373 Dr. Pass, Karmanos Cancer Institute, Wayne State University, Detroit, MI
  • H28 and MSTO American Type Culture Collection (ATCC, Manassas, VA).
  • ATCC Manassas, VA
  • Mesothelioma cell lines Meso 11, Meso 34, Meso 37, Meso 47 and Meso 56 were obtained from Dr. M Gregoire (Institute of Biology, France) and cultured in RPMI 1640 (Life Technologies) + 10% fetal calf serum (FCS), 1% penicillin-streptomycin, and 1 % L-glutamine. All cells were HLA typed by the Department of Cellular Immunology at MSKCC. Melanoma cell line Mewo (WT-1- A201+) was obtained from the ATCC. SKRC-52 renal cell carcinoma was obtained from L. Old of the Ludwig Institute.
  • Leukemia cell lines were cultured in RPMI 1640 + 10% FCS, 1 % penicillin-streptomycin, 2mM glutamine and 2-mercaptoethanol at 37oC/5% C02.
  • SKLY-16 is a human B cell lymphoma (WT-1-, A0201+); K562, RwLeu4 and HL60, all WT-1+ leukemias, were obtained from the ATCC.
  • T2 assay for peptide binding and stabilization of HLA A0201 molecules T2 cells (TAP-, HLA-A0201 +) were incubated overnight at 27° C at a concentration of 1 x 10 6 cells/ml in FCS-free RPMI medium supplemented with 5 ⁇ g/ml human (Sigma, St Louis, MO) in the absence (negative control) or presence of either a positive reference tyrosinase peptide or test peptides at various final concentrations (50, 10, 1, and 0.1 micrograms ⁇ g)/ml).
  • T2 cells were labeled for 30 minutes at 4 °C with a saturating concentration of anti-HLA- A2.1 (BB7.2) mAb, then washed twice.
  • Cells were then incubated for 30 minutes, 4° C with a saturating concentration of FITC-conjugated goat IgG F(ab')2 anti-mouse Ig (Caltag, San Francisco, CA), washed twice, fixed in PBS/1% paraformaldehyde and analyzed using a FACS Calibur® cytofluorometer (Becton Dickinson, Immunocytometry Systems, San Jose, CA).
  • MIF mean intensity of fluorescence
  • Stabilization assays were performed similarly. Following initial evaluation of peptide binding at time 0, cells were washed in RPMI complete medium to remove free peptides and incubated in the continuous presence of 0.5 ⁇ g/ml brefeldin-A for 2, 4, 6 or 8 hours.
  • the number of stable peptide-HLA- A2.1 complexes was estimated as described above by immunofluorescence.
  • WT-1 peptides The sequence of the WT-1 protein published by Gessler et al.
  • CAMs Cytokine-activated monocytes
  • peripheral blood monocytes were separated by adherence on plastic and cultured in RPMI1640 containing 1 % autologous serum.
  • GM-CSF Bolex, Montville, NJ
  • IL-4 interleukin-4
  • IL-4 interleukin-4
  • TNFa 10ng/ml
  • IL-6 interleukin-6
  • ⁇ 400IU/ml
  • PGE2 25mM-3) (R&D Systems, Minneapolis, MN) together with GM-CSF and IL-4 at the same doses.
  • EBV-BLCL were also used as WT-1 peptide loaded and control APCs or as targets as specified in the experiments. They were generated by infection of peripheral blood mononuclear cells (PBMC) with EBV strain B95.8 (38,39) as previously described.
  • PBMC peripheral blood mononuclear cells
  • EBV transformed BLCL were cultured in RPMI1640(Gemini) with 10% fetal calf serum (Gemini) in the presence of Acyclovir.
  • PBMC peripheral blood mononuclear cells
  • Monocytes were depleted by adherence on plastic and NK cells by absorption to immunomagnetic CD56 pre-coated microbeads (Miltenyi Biotech Inc, MA) as previously described (32).
  • Enriched T-cell fractions were stimulated at a 20: 1 responder: stimulator ratio with autologous CAMs or EBV-BLCL that had been pre-loaded for 3 hours with the total pool of the WT-1 pentadecapeptides in serum- free medium and irradiated to 3000cGy.
  • T- cells were cultured in Yssel's medium supplemented with 5% AB human serum (YH5, Gemini), re-stimulated weekly with the autologous WT-1 total pool-loaded CAMs or EBV- BLCL and fed with interleukin-2(IL-2) (Collaborative Biomedical Products, Bedford, MA) every 2-3 days at 10-50U/ml.
  • IL-2 Interleukin-2
  • Cell Targets - leukemic cells Twenty-four primary leukemic cells and 1 leukemic cell line were characterized for their expression of WT-1 by intracellular FACS staining using murine anti-human WT-1 monoclonal antibodies (Neomarkers, Fremont, CA) as previously described (32,38)
  • the WT-1+ leukemias included blast cells from 11 primary AMLs, 3 primary ALLs and 1 B-cell precursor ALL cell line. Ten WT-1- leukemias, were used as controls, and inclOuded 3 B-cell precursor ALLs and 7 AMLs.
  • EBV BLCL and leukemia cells were typed for HLA A, B, C, DR and DQ alleles at high resolution by standard techniques.
  • T-cell response Assessment of T-cell response.
  • IFNy production by WT-1 specific T-cells The proportion and phenotype (CD4and CD8) of T-cells generating IFNy in response to secondary stimulation with the WT- 1 total pool, WT-1 subpools or single WT-1 15-mer or 9-mer WT-1 peptides loaded on autologous PBMC were measured by FACS analysis of T-cells containing intracellular IFNy as previously described (38,40).
  • Mapping of immunogenic epitopes Mapping of immunogenic epitopes.
  • Cytotoxic activity The W-l -specific and HLA-restricted cytotoxic activity of sensitized T-cells was measured in standard Cr51 release assays against a panel of HLA- matched and mismatched CAM targets either unmodified or loaded with the total pool, the identified 15-mer, or the 9-mer or 11-mer epitope of WT-1 eliciting T-cell responses, as previously described (32).
  • the restricting HLA allele presenting each immunogenic epitope was identified by measuring the cytotoxicity of the sensitized T-cells against a panel of allogeneic CAMs pre-loaded with the peptide, each sharing a single HLA allele expressed on the responding WT-l-specific T-cells as previously described (41).
  • the cytotoxic activity of the WT-1 epitope- specific CTLs against WT-1- and WT-1+ leukemia cell lines or primary leukemic cells expressing the restricting HLA alleles was also assessed in this cytotoxicity assay Cr51 assay as previously described (32).
  • Immunogenicity of the identified immunodominant WT-1 derived epitopes To estimate the immunogenicity of identified WT-1 peptide epitopes in different individuals, enriched T-cells separated from PBMC of groups of normal donors expressing one of a series of prevalent HLA alleles (i.e. HLA- A0201, A0301 , A2402, B0702) which were previously identified as a presenter of a newly identified WT-1 epitope were sensitized in vitro with artificial antigen-presenting cells (AAPC) (42) expressing that HLA allele and loaded with the pre-identified WT-1 epitope or an irrelevant peptide.
  • AAPC artificial antigen-presenting cells
  • the panel of AAPCs includes AAPCs expressing one of the following single HLA alleles: HLA A0201, A0101, A0301 , A2402, B0702 or B0801, which were generated as previously described (42). After 35 days of co-culture of T-cells with the peptide-loaded AAPCs in the presence of IL2, CTLs were secondarily stimulated overnight with autologous PBMC loaded with the sensitizing peptide or an unrelated peptide and tested for their IFNy response.
  • the responses were registered as positive if the proportion of T-cells producing IFNy in response to the secondary stimulation with autologous PBMC loaded with the stimulating WT-1 derived peptide exceeded the background proportion of IFNy T-cells incubated with PBMC alone by two fold or more.
  • T-cells generated from 38/56 donors also exhibited cytotoxic activity against autologous PHA blasts loaded with the WT-1 total pool (Figure IB), including T-cells from 38 of the 41 donors that produced IFNy in response to secondary stimulation with the WT-1 peptide pool.
  • IFNy response measured by FACS staining in different responder cell populations peripheral blood derived PBMC, pre-generated CTLs sensitized in vitro with the RMF peptide loaded on autologous CAM and pre-generated CTLs sensitized with the total pool of WT-1 15-mers
  • secondary overnight stimulation with autologous PBMC either unmodifiedor loaded with one of the following: RMF peptide, dominant epitopes of WT-1 identified by the epitope mapping approach in the WT-1 -total pool sensitized CTL, WT-1 total pool of the 141 pentadecapeptides; D.
  • WT-1 CTLs generated by sensitization with the pooled peptides are epitope specific and HLA restricted.
  • the epitopes recognized by T-cells sensitized in vitro with the total pool of overlapping WT-1 pentadecapeptides were identified by quantitatinglFNy-i- T-cells responding to a mapping grid of subpools of WT-1 15-mers formed sothat any single 15-mer is shared by only 2 intersecting subpools ( Figure 2B).As shown for a representative example in Figure 2C, significantly increased numbers of IFNy-f- T-cells are selectively generated in response to subpools # 3 and #19 which share the pentadecapeptide #75.
  • T-cells were then stimulated with neighboring 15-mers, each overlapping peptide #75 by l laa.
  • IFNy+ T-cells are selectively generated in response to peptide #75 ( Figure 2D).
  • the newly identified immunogenic WT-1 epitope is 174- 182 HSFKHEDPM.
  • the cytotoxic activity of these T-cells was assessed against a panel of allogeneic CAMs either unmodified or loaded with this peptide, each sharing one HLA allele expressed by the tested CTLs.
  • the T-cells selectively lysed peptide loaded autologous targets and targets expressing the HLA-B3501 allele, and did not lyse peptide-loaded targets sharing other HLA alleles inherited by the T-cell donor. These T-cells also lysed WT-1+ BALL cells coexpressing the HLA-B3501 allele.
  • WT-1 isoform D In order to match the sequentialnumbers of aminoacids within the WT-1 sequence used with the longest, most frequently described WT-1 isoform D we numbered the first 126aa with negative values and used the positive values to number the subsequent 449 aminoacids described in the longest isoform D;
  • C IFNy production by WT-1 sensitized CTLs in response to secondary overnight stimulation with the subpools of WT-1 pentadecapeptides loaded on autologous PBMC.
  • the CAMs or PHA blasts used in the assay are unmodified (grey bars) orloaded with the WT-1 dominant epitope (black bars).
  • the WT-1 specific cytotoxic activity of the WT- 1 CTLs is restricted by the B3501 HLA allele.
  • HLA alleles are listed in Table 1. Of the 42 WT-1 peptides eliciting T-cell responses, 41 are newly identified; only one of these WT-1 peptides, the 126 1 3 4RMFPNAPYL nonamer presented by HLA-A0201 , has been previously described and shown to be immunogenic when presented by this allele (43) Peptide 91, 235-249CMTWNQMNLGATLKG contains an epitope which, in the study, elicited CD4+ T-cell responses restricted by HLA DRB 1 0402, but also contains the 235-24 3 CMT nonamer known to be presented by HLA A0201 and HLA A2402 (29).
  • WT-1 derived immunogenic epitopes identified by IFNy production assay for T cells responses using pool of overlapping pentadecapeptides spanning the whole sequence of WT-1 protein. Shaded rows represent peptides that can be presented by more than one HLA allele. Bolded peptide sequences represent those tested in Example 5 and results shown in Table 3.
  • epitope mapping of T-cells sensitized with the complete pool of WT-1 15-mers identified specific 15-mers eliciting both CD4+ and CD8+ T-cell responses (15-mer peptides # 20, 41 , 91, 112). Fine mapping of the sequences eliciting these responses identified four 11 -mers that stimulated HLA class Il-restricted CD4+ T-cell responses which also contained, within their sequences, 9-mers that elicited HLA class I- restricted CD8+ T-cell responses.
  • a representative example of one of these dual stimulating peptides is presented in Figure 3.
  • peptide 41 was found to elicit both CD4+ and CD8+ IFNy-f- T-cell responses ( Figure 3A). Fine mapping of the 11 -mers within peptide 41 eliciting the CD4+ IFNy+ T-cell response ( Figure 3A) suggested the 38 ⁇ 8 LDFAPPGASAY peptide as the most immunogenic sequence inducing both CD4+ and CD8+ IFNy+ T-cell responses.
  • the peptide 41 sensitized T-cells lysed PHA blasts sensitized with either the 9aa sequence ( 38 _4 6 LDFAPPGAS) or the 1 laa sequence ( 38 _ 48 LDFAPPGASAY), but did not lyse PHA blasts loaded with the or 37- 4? VLDFAPPGASA 11- mers.
  • HLA class I and II restricted WT-1 specific T cell respond to the same immunodominant peptide 15-mer derived from WT-1 protein in the WT-1 CTL sensitized with the WT-1 total pool of overlapping 15-mers loaded on autologous CAMs .
  • T-cells generated against newly identified WT-1 epitopes exhibit cytotoxic activity against WT-1+ leukemias.
  • T-cell cultures exhibiting epitope-specific cytotoxic activity against peptide loaded targets 36 could be tested for cytotoxic activity against WT-1+ leukemic cells coexpressing the T-cell' s restricting HLA allele. Of these 36, 27 exhibited HLA-restricted cytotoxic activity against the WT-1+ leukemic cells (Table 2). T-cells specific for five peptides, 6-15RDL, 46-54SAY, 58 _6 6 PAP, 225-2 33 NLY, and 436-445NMH, presented by HLA A0201 , could not lyse HLA-A0201 + WT-1+ leukemic cells.
  • HLA B4001 restricted T-cells specific for the 46-54SAY peptide, could lyse WT-1+ leukemic coexpressing this HLA allele.
  • NMH peptide-specific HLA-restricted T-cell lines that lysed targets loaded with the NMH peptide coexpressing HLA A0201, B4001 or A2402 were only able to lyse WT-1+ leukemic cells expressing the HLA B4001 allele.
  • Table 2 WT-1 derived immunogenic epitopes identified by IFNy production assay for T cells responses using pool of overlapping pentadecapeptides spanning the whole sequence of WT-1 protein.Bold sequences indicate peptides tested as described in Example 5 and results provided in Table 3.
  • Table 3a Cytotoxic activity of the T cells specific for WT-1 derivedimmunogenic epitopes identified by IFNy production assay for T cells responses using pool of overlapping pentadecapeptides spanning the whole sequence of WT-1 protein and tested against WT-1 positive primary leukemic cellsand PHA blasts of the same origin.
  • Table 3b Leukemocidal activity of defined epitope-specific and HLA restricted T cells from normal donors sensitized with either autologous EBV BLCL or EBV BLCL loaded with pooled WT-1 peptides against primary WT-1 + leukemia sharing the T cells restricting HLA alleles.
  • the T-cells from each donor were sensitized with the identified epitope loaded on a panel of artificial antigen presenting cells (AAPC) (42) each expressing a single HLA allele, specifically A0201 , A0301, A2402 or B0702.As shown in Table 4, of 9 peptides identified that are presented by HLA-A0201 , all were able to stimulate WT-1 -specific IFNy-f- T-cell responses in a proportion of HLA-A0201+ individuals.
  • the previously reported 126- 134RMFPN AP YL peptide presented by HLA-A0201 allele elicited responses in 5/12 (42%) HLA-A0201+ normal donors tested.
  • the mapped peptide specificity (i.e. (_99)_(_9i)THS, (- 22 M- i9)KLG, 22 -3 I GGC, 12 6- 1 3 4 RMF and (_i25)-(-i i7)RQR) was identical to the peptide with the highest affinity for the presenting HLA allele predicted by the binding algorithm within the stimulating 15-mer.
  • the proportion of donors responding to the mapped sequences and the sequences predicted to have the highest binding index differed, the proportion of donors responding to
  • Figure 5 presents maps of the WT-1 protein.
  • Fig. 5C defines the localization of all previously reported antigenic epitopes presented by HLA class I and II alleles;
  • Fig 5D depicts the location of immunogenic peptides identified in this report.
  • the 11 epitopes previously reported to be presented by class I and 10 presented by class II HLA alleles are principally clustered in sequences encoded by exons 1, 7 and 10, while the epitopes recognized by normal T-cells sensitized with the WT- 1 peptide pool are principally clustered in sequences encoded by the first 5 exons.
  • 26 of the new epitopes are included in each of the four major isoforms of WT-1 resulting from splice variants that do or do not include the 17 amino acid sequence (aas 250-266) in exon 5 or the three amino acid sequence (400-41 0 KTS) between zinc fingers 3 and 4.
  • epitopes map to a 126 amino acid sequence at the N terminus encoded by a segment of the WT-1 gene initially described by Gessler et al 37 that is centromeric to exon 1 of the (Exon 5 + , KTS + ) isoform of WT-1 and includes the long isoform of WT-1 initiated at a CUG codon upstream of the AUG initiator for exon l . 50 Strikingly, each of the epitopes identified in this sequence elicits IFNy + T-cells that are cytolytic against leukemic blasts coexpressing WT-1 and the T-cells' restricting HLA allele.
  • T cell cultures specific for the following immunodominant epitopes were prepared using methods described above: A0201 restricted WT- 1 peptide LKTHTTRTHT (SEQ ID NO: 182) specific T cells; A0301 restricted WT-1 peptide RQRPHPGAL (SEQ ID NO: 142) specific T cells, and A0201 restricted WT-1 peptide HFPPSLPPT (SEQ ID NO: 145) T cells.
  • T cells to tumor cell ratios tested were 50: 1, 10: 1, 5: 1 and control (no T cells). Following tumor injection, the tumor burden was monitored by bioluminescent imaging. For all three T cell lines at each dose, a significant reduction in tumor burden was observed over timevs. control.
  • mice survival was prolonged by pre-incubation of tumor cells with WT-1 peptide specific T cells.
  • all mice were dead by 70 days post tumor injection.
  • Increased survival was seen dose-responsively with the T cell: tumor cell dose, which for the 50: 1 dose for all T cell lines still had some animals alive at 96 days, and also at 10 : 1 for the LKTHTTRTHT specific line.
  • WT-1 peptide specific T cells were administered intravenously to NOD/SCID mice bearing pre-established ovarian carcinoma SKOV3-A2 xenografts.
  • T cell lines evaluated were: A0201 restricted WT-1 peptide LKTHTTRTHT (SEQ ID NO: 182) specific T cells and A0301 restricted WT-1 peptide RQRPHPGAL (SEQ ID NO: 142) specific T cells.
  • Tumor burden was monitored by bioluminescence, tumor infiltration by human CD3+ cells evaluated and survival recorded. In both cases, the WT-1 specific T cells afforded reduced tumor burden vs. control, increased tumor infiltration by human CD3+ cells and increased survival.
  • the HLA restricting alleles and corresponding immunodominant WT-1 epitopes are as follows: A0201 , SEQ ID NO: 147; A0203, SEQ ID NOs: 176 and 183; B3503 and C0401, SEQ ID NOs: 161 and 169; A6901, SEQ ID NO: 177; A0201 , SEQ ID NO: 182; B4701 and DRBj 0102, SEQ ID NOs: 148 and 149; A3101 , SEQ ID NO: 145; B4402, SEQ ID NOs: 158 and 166; B3503, SEQ ID NOs: 146 and 162; DRBj 1104, SEQ ID NO: 149.
  • Pentadecapeptides [0303] The following pentadecapeptides were synthesized. H2N refers to the N- terminal end of the peptide, and -COOH the C-terminus.
  • H2N-AGTAA QAPGP RRLLA-COOH SEQ ID NO: l l .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Hospice & Palliative Care (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

This invention provides peptides, immunogenic compositions and vaccines, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1 -expressing cancer, comprising peptides derived from the WT-1 protein.

Description

IMMUNOGENIC WT-1 PEPTIDES AND METHODS OF USE THEREOF
GOVERNMENT SUPPORT
[01] This work was supported by grants CA23766, CA59350 and CA08748 from the National Institutes of Health. The US government has certain rights in the invention.
FIELD OF INVENTION
[02] This invention provides peptides, compositions and vaccines comprising same, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1- expressing cancer, comprising administering same. BACKGROUND OF THE INVENTION
[03] Wilms tumor (WT), a pediatric nephroblastoma that occurs with a frequency of 1 in 10,000 births, has been the subject of intense clinical and basic research for several years. The tumor is embryonic in origin; it is detected in children usually during the first 5 years of life and can occur unilaterally or bilaterally. A WT arises when condensed metanephric mesenchymal cells of the developing kidney fail to properly differentiate. The implication of the Wilms tumor 1 (WT-1) tumor suppressor gene in the etiology of WT illustrated the impact that genetic alterations can have on both development and tumorigenesis.
[04] Wilms tumor protein I (WT-1) is a zinc finger transcription factor expressed during normal ontogenesis such as in fetal kidney, testis and ovary. In adults, WT-1 expression is limited to low levels on hematopoietic stem cells, myoepithelial progenitor cells, renal podocytes and some cells in testis and ovary. Recent demonstration that WT-1 is over expressed in several types of leukemia suggested that WT-1 would be an attractive target for immunotherapy for various cancers.
SUMMARY OF THE INVENTION [05] This invention provides peptides, compositions, and immunogenic compositions such as vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1 -expressing cancer, comprising administering immunogenic peptides.
[06] In one embodiment, the present invention provides an isolated WT-1 peptide having an amino acid (AA) sequence consisting ofany one of the sequences SEQ ID NO: 1-160, 162- 185, 190, 191 and 193. In one embodiment, the present invention provides an isolated HLA class I binding WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 190, 191 and 193. In one embodiment, the present invention provides an isolated HLA class II binding WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 149, 156, 173, 174 and 180.
[07] In one embodiment, the present invention provides an isolated WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 1-160, 162-185, 190, 191 and 193, or a fragment of any of the foregoing.In one embodiment, the present invention provides an isolated HLA class I binding WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 190, 191 and 193. In one embodiment, the present invention provides an isolated HLA class II binding WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 149, 156, 173, 174 and 180.
[08] In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) a peptide selected from SEQ ID NO: 1-160, 162-185, 190, 191 and 193. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class I binding peptide selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class II binding peptide selected from SEQ ID NO: 149, 156, 173, 174 and 180. [09] In another embodiment, the present invention provides a vaccine comprising one or more peptides of SEQ ID NO: 1-160, 162-185, 190, 191 and 193. In another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In another embodiment, the present invention provides a vaccine comprising one or more HLA class II binding peptides selected from SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, and one or more HLA class II binding peptides selected from SEQ ID NO: 149, 156, 173, 174 and 180.
[010] In another embodiment, the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a WT- lpeptide or vaccine of the present invention, thereby treating a subject with a WT-1- expressing cancer.
[011] In another embodiment, the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT-lpeptide or vaccine of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, orits relapse, in a subject.
[012] In another embodiment, the present invention provides a method of inducing formation and proliferation of a WT-1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT-1 protein-specific CTL. This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
[013] In another embodiment, the present invention provides a method of inducing formation and proliferation of (a) a WT-1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT- 1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT-1 protein- specific CD8 lymphocyte; or (b) a CD4+ lymphocyte specific for the WT-1 protein; or a combination thereof. This method can be conducted in vitro, ex vivo or in vivo.When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect. [014] In another embodiment, the present invention provides a method of inducing an anticancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby inducing an anti-mesothelioma immune response in a subjectln one embodiment, the fragment of a WT-1 protein consists of a peptide or comprises a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 and 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.
[015] In another embodiment, the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby treating a subject with a mesothelioma. In one embodiment, the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.
[016] In another embodiment, the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In one embodiment, the fragment of a WT- lprotein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.
[017] In another embodiment, the cancer is a WT-1 -expressing cancer. In one embodiment, the WT-1 -expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT-1 -expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT-1 -expressing cancer is an MDS. In another embodiment, the WT-1 -expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT-1 -expressing cancer is a Wilms' tumor. In another embodiment, the WT-1 -expressing cancer is a leukemia. In another embodiment, the WT-1 -expressing cancer is a hematological cancer. In another embodiment, the WT-1- expressing cancer is a lymphoma. In another embodiment, the WT-1 -expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT-1 -expressing cancer is a mesothelioma. In another embodiment, the WT-1 -expressing cancer is a malignant mesothelioma. In another embodiment, the WT-1 -expressing cancer is a gastric cancer. In another embodiment, the WT-1 -expressing cancer is a colon cancer. In another embodiment, the WT-1 -expressing cancer is a lung cancer. In another embodiment, the WT-1 -expressing cancer is abreast cancer. In another embodiment, the WT-1 -expressing cancer is a germ cell tumor. In another embodiment, the WT-1 -expressing cancer is an ovarian cancer. In another embodiment, the WT-1 -expressing cancer is a uterine cancer. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a liver cancer. In another embodiment, the WT-1- expressing cancer is a renal cancer. In another embodiment, the WT- 1 -expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT-1 -expressing cancer is a sarcoma. In another embodiment, the WT-1 -expressing cancer is any other carcinoma or sarcoma. [018] In another embodiment, the WT-1 -expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT-1 -expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with a non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with a lung cancer. In another embodiment, the solid tumor is associated with a breast cancer. In another embodiment, the solid tumor is associated with a colorectal cancer. In another embodiment, the solid tumor is associated with a prostate cancer. In another embodiment, the solid tumor is associated with an ovarian cancer. In another embodiment, the solid tumor is associated with a renal cancer. In another embodiment, the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
[019] In another embodiment, the present invention provides a composition comprising an isolated peptide of the invention in combination with at least ladditionalWT-1 peptide. In certain embodiments, a composition comprising at least 2 different isolated peptides of the present invention is provided. In certain embodiments, a composition comprising at least 3 or at least 4 different isolated peptides of the present invention is provided. Each possibility represents a separate embodiment of the present invention.In certain embodiments, the composition of the present invention is a vaccine.
[020] In another embodiment, the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a peptideor composition of the present invention, thereby treating a subject with a WT-1- expressing cancer.
[021] In another embodiment, the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a peptide or composition of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject.
[022] In another embodiment, the present invention provides a method of inducing formation and proliferation of a WT-1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptideor composition of the present invention, thereby inducing formation and proliferation of a WT-1 protein- specific CTL. [023] In another embodiment, the present invention provides a method of inducing formation and proliferation of (a) a WT-1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT-1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT-1 protein- specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT-1 protein; or a combination thereof.
[024] In another embodiment, the invention is directed to a peptide of the invention with at least one amino acid change that increases the affinity of the peptide for binding to a HLA molecule.
[025] This application claims priority to US provisional applications serial number 61/586, 177, filed January 13, 2012; and serial number 61/647,207, filed May 15, 2012; both of which are incorporated herein by reference in their entireties.
BRIEF DESCRIPTION OF THE FIGURES [026] So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention are briefly summarized. Details of the above may be had by reference to certain embodiments thereof, which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted; however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.
[027] Figure 1 A-D showsWT-1 specific responses of CTL generated from PBMC of normaldonors (n=56) by stimulation with autologous APCs loaded with total pool of WT-1 derivedpentadecapeptides ; Figure 2 A-E depicts the strategy for the generation of the total pool of overlapping pentadecapeptides spanning the whole sequence of the WT-1 protein and epitope mapping;
Figure 3 A-D shows that the combined HLA class I and II restricted WT-1 specific T cell response to the same immunodominant peptide sequence derived from WT-1 protein in the WT-1 CTL after 40 days of co-culture with the WT-1 total pool of overlapping 15-mers loaded on autologous CAMs;
Figure 4A-Fdepicts schema of WT-1 ; and
Figure 5 depicts results using mixed A0201 epitopes loaded on A0201-AAPC in 8 normal A0201+ donors. DETAILED DESCRIPTION OF THE INVENTION
[028] This invention provides immunogenic peptides, and compositions and vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1 -expressing cancer, comprising administering one or more immunogenic peptides. [029] This invention provides WT-1 peptides and methods of treating, reducing the incidence of, and inducing immune responses against a WT-1 -expressing cancer, comprising immunogenic peptides.
[030] The WT-1 molecule from which the peptides of the present invention are derived has, in another embodiment, the sequence: 1 SRQRPHPGAL RNPTACPLPH FPPSLPPTHS PTHPPRAGTA AQAPGPRRLL
51 AAILDFLLLQ DPASTCVPEP ASQHTLRSGP GCLQQPEQQG VRDPGGIWAK 101 LGAAEASAER LQGRRSRGAS GSEPQQMGSD VRDLNALLPA VPSLGGGGGC 151 ALPVSGAAQW APVLDFAPPG ASAYGSLGGP APPPAPPPPP PPPPHSFIKQ 201 EPSWGGAEPH EEQCLSAFTV HFSGQFTGTA GACRYGPFGP PPPSQASSGQ 251 ARMFPNAPYL PSCLESQPAI RNQGYSTVTF DGTPSYGHTP SHHAAQFPNH 301 SFKHEDPMGQ QGSLGEQQYS VPPPVYGCHT PTDSCTGSQA LLLRTPYSSD 351 NLYQMTSQLE CMTWNQMNLG ATLKGVAAGS SSSVKWTEGQ SNHSTGYESD 401 NHTTPILCGA QYRIHTHGVF RGIQDVRRVP GVAPTLVRSA SETSEKRPFM 451 CAYPGCNKRY FKLSHLQMHS RKHTGEKPYQ CDFKDCERRF SRSDQLKRHQ 501 RRHTGVKPFQ CKTCQRKFSR SDHLKTHTRT HTGKTSEKPF SCRWPSCQKK 551 FARSDELVRH HNMHQRNMTK LQLAL (SEQ ID NO: 194)
The foregoing sequence of the WT-1 protein is that published by Gessler et al. (37) which comprises 575 aminoacids and includes the first 126 aminoacids in the N-terminus missing in the (Exon 5+, KTS+) isoform of WT-116.
[031] In another embodiment, the WT-1 sequence is MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPA PPPAPP
PPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQA SSGQA RMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPM GQQGS
LGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNL GATLK
GVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPG VAPTL
VRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSR SDQLK
RHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARS DELVR HHNMHQRNMTKLQLAL (GenBank Accession number AY245105; SEQ ID NO: 195).
[032] In another embodiment, the WT-1 molecule has the sequence:
AAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAA QWAP
VLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVH FSGQF
TGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTP SYGHT
PSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTP YSSDN LYQMTSQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQ DVRRV
PGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKD CERRF
SRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQK KFARS DELVRHHNMHQRNMTKLQLAL (GenBank Accession number NM_000378; SEQ ID NO: 196).
[033] In another embodiment, the WT-1 molecule has the sequence: MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGR RSRGA
SGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAY GSLGGP APPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFG PPPPSQ
ASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFK HEDP
MGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMT WNQM
NLGATLKGVAAGS SS S VKWTEGQSNHSTGYESDNHTTPILCG AQYRIHTHGVFRGIQ DVRRV
PGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKD CERRF SRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQK KFARS DELVRHHNMHQRNMTKLQLAL (GenBank Accession number NP_077742; SEQ ID No: 197).
[034] In another embodiment, the WT-1 protein has the sequence set forth in GenBank Accession # NM_024426. In other embodiments, the WT-1 protein has or comprises one of the sequences set forth in one of the following sequence entries: NM_024425, NM_024424,
NM_000378, S95530, D13624, D12496, D 12497, or X77549. In another embodiment, the WT-1 protein has any other WT-1 sequence known in the art. This invention provides peptides, compositions, and immunogenic compositions such as vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1 -expressing cancer, comprising administering immunogenic peptides.
[035] In one embodiment, the present invention provides an isolated WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 1-160, 162- 185, 190, 191 and 193. In one embodiment, the present invention provides an isolated HLA class I binding WT- 1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In one embodiment, the present invention provides an isolated HLA class II binding WT-1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151 , and the HLA class II peptide consists of or comprises SEQ ID NO: 149.
[036] In one embodiment, the present invention provides an isolated WT-1 peptide having an amino acid (AA) sequence comprising any one of the sequences SEQ ID NO: 1-53 or 43- XXX, or a fragment thereof. In one embodiment, the present invention provides an isolated HLA class I binding WT-1 peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In one embodiment, the present invention provides an isolated HLA class II binding WT-1 peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO: 149.
[037] In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) a peptide selected from SEQ ID NO: 1-160, 162-185, 190,
191 and 193. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class I binding peptide selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class II binding peptide selected from SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO: 149. [038] In another embodiment, the present invention provides a vaccine comprising one or more peptides of SEQ ID NO: 1-160, 162-185, 190, 191 and 193. In another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptidesselected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, and 183. In another embodiment, the present invention provides a vaccine comprising one or more HLA class II binding peptides selected from SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected fromSEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176,
177, 178, 179, 180, 181, 182, and 183, and one or more HLA class II binding peptides selected from SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class
I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO: 149.
[039] In another embodiment, the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a WT- lpeptide or vaccine of the present invention, thereby treating a subject with a WT-1- expressing cancer.
[040] In another embodiment, the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT- lpeptide or vaccine of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, orits relapse, in a subject.
[041] In another embodiment, the present invention provides a method of inducing an anticancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby inducing an anti-mesothelioma immune response in a subjectln one embodiment, the fragment of a WT-1 protein consists of a peptide or comprises a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 and 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180. [042] In another embodiment, the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby treating a subject with a mesothelioma. In one embodiment, the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 and 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151 , and the HLA class II peptide consists of or comprises SEQ ID NO: 149.
[043] In another embodiment, the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In one embodiment, the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147,
148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO: 149.
[044] In another embodiment, the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a WT- lpeptide or vaccine of the present invention, thereby treating a subject with a WT-1- expressing cancer.
[045] In another embodiment, the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT-lpeptide or vaccine of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, orits relapse, in a subject.
[046] In another embodiment, the present invention provides a method of inducing an anticancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby inducing an anti-mesothelioma immune response in a subject.In one embodiment, the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193.1n another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO: 149, 156, 173, 174 and 180.1n another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151 , and the HLA class II peptide consists of or comprises SEQ ID NO: 149.
[047] In another embodiment, the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby treating a subject with a mesothelioma. In one embodiment, the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193.
[048] In another embodiment, the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) a nucleotide molecule encoding a fragment of a WT-1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In one embodiment, the fragment of a WT-1 protein is a peptide from among SEQ ID NO: 1-160, 162-185, 190, 191 or 193. [049] In another embodiment, the cancer is a WT-1 -expressing cancer. In one embodiment, the WT-1 -expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT-1 -expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT-1 -expressing cancer is an MDS. In another embodiment, the WT-1 -expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT-1 -expressing cancer is a Wilms' tumor. In another embodiment, the WT-1 -expressing cancer is a leukemia. In another embodiment, the WT-1 -expressing cancer is a hematological cancer. In another embodiment, the WT-1- expressing cancer is a lymphoma. In another embodiment, the WT-1 -expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT-1 -expressing cancer is a mesothelioma. In another embodiment, the WT-1 -expressing cancer is a malignant mesothelioma. In another embodiment, the WT-1 -expressing cancer is a gastric cancer. In another embodiment, the WT-1 -expressing cancer is a colon cancer. In another embodiment, the WT-1 -expressing cancer is a lung cancer. In another embodiment, the WT-1 -expressing cancer is abreast cancer. In another embodiment, the WT-1 -expressing cancer is a germ cell tumor. In another embodiment, the WT-1 -expressing cancer is an ovarian cancer. In another embodiment, the WT-1 -expressing cancer is a uterine cancer. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a liver cancer. In another embodiment, the WT-1- expressing cancer is a renal cancer. In another embodiment, the WT- 1 -expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT-1 -expressing cancer is a sarcoma. In another embodiment, the WT-1 -expressing cancer is any other carcinoma or sarcoma.
[050] In another embodiment, the WT-1 -expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT-1 -expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with a non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with a lung cancer. In another embodiment, the solid tumor is associated with a breast cancer. In another embodiment, the solid tumor is associated with a colorectal cancer. In another embodiment, the solid tumor is associated with a prostate cancer. In another embodiment, the solid tumor is associated with an ovarian cancer. In another embodiment, the solid tumor is associated with a renal cancer. In another embodiment, the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
[051] In another embodiment, the present invention provides a composition comprising an isolated peptide of the invention in combination with at least 1 additional WT-1 peptide. In certain embodiments, a composition comprising at least 2 different isolated peptides of the present invention is provided. In certain embodiments, a composition comprising at least 3 or at least 4 different isolated peptides of the present invention is provided. Each possibility represents a separate embodiment of the present invention.In certain embodiments, the composition of the present invention is a vaccine.
[052] In another embodiment, the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a peptide or composition of the present invention, thereby treating a subject with a WT-1 -expressing cancer. [053] In another embodiment, the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a peptide or composition of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer, or its relapse, in a subject.
[054] In another embodiment, the present invention provides a method of inducing formation and proliferation of a WT-1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT-1 protein- specific CTL.
[055] In another embodiment, the present invention provides a method of inducing formation and proliferation of (a) a WT-1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT- 1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT-1 protein- specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT-1 protein; or a combination thereof. [056] In another embodiment, the invention is directed to a peptide of the invention with at least one amino acid change that increases the affinity of the peptide for binding to a HLA molecule.
[057] "Peptide," in another embodiment of methods and compositions of the present invention, refers to a compound of subunit A A connected by peptide bonds. In another embodiment, the peptide comprises an AA analogue. In another embodiment, the peptide comprises a peptidomimetic. The different AA analogues and peptidomimetics that can be included in the peptides of methods and compositions of the present invention are enumerated hereinbelow. The subunits are, in another embodiment, linked by peptide bonds. In another embodiment, the subunit is linked by another type of bond, e.g. ester, ether, etc. Each possibility represents a separate embodiment of the present invention.
[058] The unaltered peptides of the present invention (as described both above and below) are referred to collectively herein as " WT- 1 peptides. " Each of the embodiments enumerated below for "WT-1 peptides" applies to unaltered WT-1 peptides and HLA class I and class II heteroclitic peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
[059] In another embodiment, a WT-1 peptide of the present invention binds to an HLA class I molecule or a class II molecule. In another embodiment the peptide binds to both a class I and a class II molecule. In another embodiment, the HLA class II molecule is an HLA- DRB molecule. In another embodiment, the HLA class Il-molecule is an HLA-DRA molecule. In another embodiment, the HLA molecule is an HLA-DQAl molecule. In another embodiment, the HLA molecule is an HLA-DQB 1 molecule. In another embodiment, the HLA molecule is an HLA-DPA1 molecule. In another embodiment, the HLA molecule is an HLA-DPB 1 molecule. In another embodiment, the HLA molecule is an HLA-DMA molecule. In another embodiment, the HLA molecule is an HLA-DMB molecule. In another embodiment, the HLA molecule is an HLA-DOA molecule. In another embodiment, the HLA molecule is an HLA-DOB molecule. In another embodiment, the HLA molecule is any other HLA class Il-molecule known in the art. Each possibility represents a separate embodiment of the present invention. [060] In another embodiment, the HLA class I molecule whose binding motif is contained in or comprising a peptide of the present invention is, in another embodiment, an HLA-A molecule. In another embodiment, the HLA class I molecule is an HLA-B molecule. In another embodiment, the HLA class I molecule is an HLA-C molecule. In another embodiment, the HLA class I molecule is an HLA-A0201 molecule. In another embodiment, the molecule is HLA Al. In another embodiment, the HLA class I molecule is HLA A2. In another embodiment, the HLA class I molecule is HLA A2.1. In another embodiment, the HLA class I molecule is HLA A3. In another embodiment, the HLA class I molecule is HLA A3.2. In another embodiment, the HLA class I molecule is HLA Al l. In another embodiment, the HLA class I molecule is HLA A24. In another embodiment, the HLA class I molecule is HLA B7. In another embodiment, the HLA class I molecule is HLA B27. In another embodiment, the HLA class I molecule is HLA B8.Each possibility represents a separate embodiment of the present invention.
[061] In another embodiment, the HLA class I molecule-binding WT-1 peptide of methods and compositions of the present invention binds to a superfamily of HLA class I molecules. In another embodiment, the superfamily is the A2 superfamily. In another embodiment, the superfamily is the A3 superfamily. In another embodiment, the superfamily is the A24 superfamily. In another embodiment, the superfamily is the B7 superfamily. In another embodiment, the superfamily is the B27 superfamily. In another embodiment, the superfamily is the B44 superfamily. In another embodiment, the superfamily is the CI superfamily. In another embodiment, the superfamily is the C4 superfamily. In another embodiment, the superfamily is any other superfamily known in the art. Each possibility represents a separate embodiment of the present invention.
[062] In another embodiment, the HLA molecule is a AO 101, A0201, A0203, A2402, A6901, B0702, A3101, B3501, B3503, B3508, B3802, B3801, B3901, B4001, B4402, B4701, B5701, C0401, C1701, DRBjOlOl, DRBJ0402, DRBJ0402, DRBi0401 or DRBJ1104 molecule.In another embodiment, the peptides of SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, and SEQ ID NO:149, 156, 173, 174 and 180, bind to the HLA class I or class II molecules described for each peptide in Tables 1 or 2. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO: 149, and bind to the corresponding HLA molecule or molecules indicated for each peptide in Table 1 or Table 2.1n one embodiment, certain peptides can bind to more than one HLA allele.
[063] In another embodiment, a modification of a peptide of the invention is provided.In one embodiment the modification comprises at least one heteroclitic amino acid change, also referred to as a mutation or mutated, or an anchor residue mutation (see below). An HLA class I molecule binding motif of a modified peptide of the present invention exhibits an increased affinity for the HLA class I molecule, relative to the unmutated counterpart of the peptide. In another embodiment, the point mutation increases the affinity of the isolated, mutated WT- 1 peptide for the HLA class I molecule. In another embodiment, the increase in affinity is relative to the affinity (for the same HLA class I molecule) of the isolated, unmutated WT-1 peptide wherefrom the isolated, mutated WT-1 peptide was derived. Each possibility represents a separate embodiment of the present invention.
[064] In another embodiment, a WT- 1 peptide of methods and compositions of the present invention is so designed as to exhibit affinity for an HLA molecule. In another embodiment, the affinity is a high affinity, as described herein.
[065] HLA molecules, known in another embodiment as major histocompatibility complex (MHC) molecules, bind peptides and present them to immune cells. Thus, in another embodiment, the immunogenicity of a peptide is partially determined by its affinity for HLA molecules. HLA class I molecules interact with CD8 molecules, which are generally present on cytotoxic T lymphocytes (CTL). HLA class II molecules interact with CD4 molecules, which are generally present on helper T lymphocytes.
[066] In another embodiment, a peptide of the present invention is immunogenic. In another embodiment, "immunogenic" refers to an ability to stimulate, elicit or participate in an immune response. In another embodiment, the immune response elicited is a cell-mediated immune response. In another embodiment, the immune response is a combination of cell- mediated and humoral responses.
[067] In another embodiment, T cells that bind to the MHC molecule-peptide complex become activated and induced to proliferate and lyse cells expressing a protein comprising the peptide. T cells are typically initially activated by "professional" antigen presenting cells ("APC"; e.g. dendritic cells, monocytes, and macrophages), which present costimulatory molecules that encourage T cell activation as opposed to anergy or apoptosis. In another embodiment, the response is heteroclitic, as described herein, such that the CTL lyses a neoplastic cell expressing a protein which has an AA sequence homologous to a peptide of this invention, or a different peptide than that used to first stimulate the T cell.
[068] In another embodiment, an encounter of a T cell with a peptide of this invention induces its differentiation into an effector and/or memory T cell. Subsequent encounters between the effector or memory T cell and the same peptide, or, in another embodiment, with a related peptide of this invention, leads to a faster and more intense immune response. Such responses are gauged, in another embodiment, by measuring the degree of proliferation of the T cell population exposed to the peptide. In another embodiment, such responses are gauged by any of the methods enumerated hereinbelow.
[069] In another embodiment, the peptides of methods and compositions of the present invention bind an HLA class II molecule with high affinity. In other embodiments, the HLA class II molecule is any HLA class II molecule enumerated herein. Each possibility represents a separate embodiment of the present invention. [070] In another embodiment, derivatives of peptides of methods and compositions of the present invention bind an HLA class I molecule with high affinity. In other embodiments, the MHC class I molecule is any MHC class I molecule enumerated herein. Each possibility represents a separate embodiment of the present invention.
[071] In another embodiment, a peptide of methods and compositions of the present invention binds an HLA class II molecule with significant affinity, while a peptide derived from the original peptide binds an HLA class I molecule with significant affinity.
[072] In another embodiment, "affinity" refers to the concentration of peptide necessary for inhibiting binding of a standard peptide to the indicated MHC molecule by 50%. In another embodiment, "high affinity" refers to an affinity is such that a concentration of about 500 nanomolar (nM) or less of the peptide is required for 50% inhibition of binding of a standard peptide. In another embodiment, a concentration of about 400 nM or less of the peptide is required. In another embodiment, the binding affinity is 300 nM. In another embodiment, the binding affinity is 200 nM. In another embodiment, the binding affinity is 150 nM. In another embodiment, the binding affinity is 100 nM. In another embodiment, the binding affinity is 80 nM. In another embodiment, the binding affinity is 60 nM. In another embodiment, the binding affinity is 40 nM. In another embodiment, the binding affinity is 30 nM. In another embodiment, the binding affinity is 20 nM. In another embodiment, the binding affinity is 15 nM. In another embodiment, the binding affinity is 10 nM. In another embodiment, the binding affinity is 8 nM. In another embodiment, the binding affinity is 6 nM. In another embodiment, the binding affinity is 4 nM. In another embodiment, the binding affinity is 3 nM. In another embodiment, the binding affinity is 2 nM. In another embodiment, the binding affinity is 1.5 nM. In another embodiment, the binding affinity is 1 nM. In another embodiment, the binding affinity is 0.8 nM. In another embodiment, the binding affinity is 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. In another embodiment, the binding affinity is 0.4 nM. In another embodiment, the binding affinity is 0.3 nM. In another embodiment, the binding affinity is less than 0.3 nM.
[073] In another embodiment, "affinity" refers to a measure of binding strength to the MHC molecule. In another embodiment, affinity is measured using a method known in the art to measure competitive binding affinities. In another embodiment, affinity is measured using a method known in the art to measure relative binding affinities. In another embodiment, the method is a competitive binding assay. In another embodiment, the method is radioimmunoassay or RIA. In another embodiment, the method is BiaCore analyses. In another embodiment, the method is any other method known in the art. In another embodiment, the method yields an IC50 in relation to an IC50 of a reference peptide of known affinity. [074] Each type of affinity and method of measuring affinity represents a separate embodiment of the present invention.
[075] In another embodiment, "high affinity" refers to an IC50 of 0.5-500 nM. In another embodiment, the IC50 is 1-300 nM. In another embodiment, the IC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. In another embodiment, the IC50 is 3-100 nM. In another embodiment, the IC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. In another embodiment, the IC50 is 10-100 nM. In another embodiment, the IC50 is 30- 100 nM. In another embodiment, the IC50 is 3-80 nM. In another embodiment, the IC50 is 4- 60 nM. In another embodiment, the IC50 is 5-50 nM. In another embodiment, the IC50 is 6- 50 nM. In another embodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is 10- 50 nM. In another embodiment, the IC50 is 20-50 nM. In another embodiment, the IC50 is 6- 40 nM. In another embodiment, the IC50 is 8-30 nM. In another embodiment, the IC50 is 10- 25 nM. In another embodiment, the IC50 is 15-25 nM. Each affinity and range of affinities represents a separate embodiment of the present invention.
[076] In another embodiment, a peptide of methods and compositions of the present invention binds to a superfamily of HLA molecules. Superfamilies of HLA molecules share very similar or identical binding motifs. In another embodiment, the superfamily is a HLA class I superfamily. In another embodiment, the superfamily is a HLA class II superfamily. Each possibility represents a separate embodiment of the present invention.
[077] The terms "HLA-binding peptide," "HLA class I molecule-binding peptide," and "HLA class II molecule-binding peptide" refer, in another embodiment, to a peptide that binds an HLA molecule with measurable affinity. In another embodiment, the terms refer to a peptide that binds an HLA molecule with high affinity. In another embodiment, the terms refer to a peptide that binds an HLA molecule with sufficient affinity to activate a T cell precursor. In another embodiment, the terms refer to a peptide that binds an HLA molecule with sufficient affinity to mediate recognition by a T cell. The HLA molecule is, in other embodiments, any of the HLA molecules enumerated herein. Each possibility represents a separate embodiment of the present invention.
[078] "Heteroclitic" refers, in another embodiment, to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived (e.g. the peptide not containing the anchor residue mutations). In another embodiment, "original peptide" refers to a peptide of the present invention. In another embodiment, "heteroclitic" refers to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide. In another embodiment, a "heteroclitic" immune response refers to an immune response that recognizes the original peptide from which the improved peptide was derived (e.g. the peptide not containing the anchor residue mutations). In another embodiment, a "heteroclitic" immune response refers to an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide. In another embodiment, the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response substantially equal to the response to vaccination with the original peptide. In another embodiment, the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response less than the response to vaccination with the original peptide. In another embodiment, a heteroclitic peptide of the present invention is an HLA class I heteroclitic peptide. Methods for identifying HLA class I and class II residues, and for improving HLA binding by mutating the residues, are well known in the art, as described below. Each possibility represents a separate embodiment of the present invention.
[079] In another embodiment, a heteroclitic peptide of the present invention induces an immune response that is increased at least 2-fold relative to the WT- 1 peptide from which the heteroclitic peptide was derived ("native peptide"). In another embodiment, the increase is 3- fold relative to the native peptide. In another embodiment, the increase is 5 -fold relative to the native peptide. In another embodiment, the increase is 7-fold relative to the native peptide. In another embodiment, the increase is 10-fold relative to the native peptide. In another embodiment, the increase is 15 -fold relative to the native peptide. In another embodiment, the increase is 20-fold relative to the native peptide. In another embodiment, the increase is 30-fold relative to the native peptide. In another embodiment, the increase is 50- fold relative to the native peptide. In another embodiment, the increase is 100-fold relative to the native peptide. In another embodiment, the increase is 150-fold relative to the native peptide. In another embodiment, the increase is 200-fold relative to the native peptide. In another embodiment, the increase is 300-fold relative to the native peptide. In another embodiment, the increase is 500-fold relative to the native peptide. In another embodiment, the increase is 1000-fold relative to the native peptide. In another embodiment, the increase is more than 1000-fold relative to the native peptide. Each possibility represents a separate embodiment of the present invention. [080] In another embodiment, the present invention provides a HLA class II heteroclitic peptide derived from an isolated WT-1 peptide of the present invention. In another embodiment, the process of deriving comprises introducing a mutation that enhances a binding of the peptide to an HLA class II molecule. In another embodiment, the process of deriving consists of introducing a mutation that enhances a binding of the peptide to an HLA class I molecule. In another embodiment, the mutation is in an HLA class II anchor residue.
In another embodiment, a heteroclitic class II peptide of the present invention is identified and tested in a manner analogous to identification and testing of HLA class I heteroclitic peptides, as exemplified herein. Each possibility represents a separate embodiment of the present invention.
[081] In another embodiment, the HLA class II binding site in a peptide of the present invention is created or improved by mutation of an HLA class II motif anchor residue. In another embodiment, theanchor residue that is modified is in the PI position. In another embodiment, the anchor residue is at the P2 position. In another embodiment, the anchor residue is at the P6 position. In another embodiment, the anchor residue is at the P9 position. In another embodiment, the anchor residue is selected from the PI, P2, P6, and P9 positions. In another embodiment, the anchor residue is at the P3 position. In another embodiment, the anchor residue is at the P4 position. In another embodiment, the anchor residue is at the P5 position. In another embodiment, the anchor residue is at the P6 position. In another embodiment, the anchor residue is at the P8 position. In another embodiment, the anchor residue is at the P10 position. In another embodiment, the anchor residue is at the PI 1 position. In another embodiment, the anchor residue is at the PI 2 position. In another embodiment, the anchor residue is at the PI 3 position. In another embodiment, the anchor residue is at any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than PI, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding. Each possibility represents a separate embodiment of the present invention. [082] In another embodiment, a heteroclitic peptide is generated by introduction of a mutation that creates an anchor motif. "Anchor motifs" or "anchor residues" refers, in another embodiment, to 1 or a set of preferred residues at particular positions in an HLA-binding sequence. In another embodiment, the
[083] HLA-binding sequence is an HLA class Il-binding sequence. In another embodiment, the HLA-binding sequence is an HLA class I-binding sequence. In another embodiment, the positions corresponding to the anchor motifs are those that play a significant role in binding the HLA molecule. In another embodiment, the anchor residue is a primary anchor motif. In another embodiment, the anchor residue is a secondary anchor motif. Each possibility represents a separate embodiment of the present invention. [084] Methods for predicting MHC class H epitopes are well known in the art. In another embodiment, the MHC class II epitope is predicted using TEPITOPE (Meister GE, Roberts CG et al, Vaccine 1995 13: 581-91). In another embodiment, the MHC class II epitope is predicted using EpiMatrix (De Groot AS, Jesdale BM et al, AIDS Res Hum Retroviruses 1997 13: 529-31). In another embodiment, the MHC class II epitope is predicted using the Predict Method (Yu K, Petrovsky N et al, Mol Med. 2002 8: 137- 48). In another embodiment, the MHC class II epitope is predicted using the SYFPEITHI epitope prediction algorithm (Examples). In another embodiment, the MHC class II epitope is predicted using Rankpep. In another embodiment, the MHC class II epitope is predicted using any other method known in the art. Each possibility represents a separate embodiment of the present invention. [085] In another embodiment, in the case of HLA class II-binding peptides (e.g. HLA-DR- binding peptides), the anchor residue that is modified is in the PI position. In another embodiment, the anchor residue is in the P2 position. In another embodiment, the anchor residue is in the P6 position. In another embodiment, the anchor residue is in the P9 position. In other embodiments, the anchor residue is the P3, P4, P5, P6, P8, P10, Pl l, P12, or P13 position. In another embodiment, the anchor residue is any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than PI, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding. In another embodiment, any combination of the above residues is mutated. Each possibility represents a separate embodiment of the present invention. [086] In another embodiment, a WT-1 peptide of the present invention binds to 2 distinct HLA class II molecules. In another embodiment, the peptide binds to three distinct HLA class II molecules. In another embodiment, the peptide binds to four distinct HLA class II molecules. In another embodiment, the peptide binds to five distinct HLA class II molecules. In another embodiment, the peptide binds to six distinct HLA class II molecules. In another embodiment, the peptide binds to more than six distinct HLA class II molecules.
[087] In another embodiment, the HLA class II molecules that are bound by a WT-1 peptide of the present invention are encoded by two or more distinct alleles at a given HLA class II locus. In another embodiment, the HLA class II molecules are encoded by 3 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 4 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 5 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 6 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by more than six distinct alleles at a locus.
[088] In another embodiment, the HLA class II molecules bound by the WT-1 peptide are encoded by HLA class II genes at 2 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 2 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at more than 4 distinct loci. In other embodiments, the loci are selected from HLA-DRB loci. In another embodiment, the HLA class Il-binding peptide is an HLA-DRA binding peptide. In another embodiment, the peptide is an HLA- DQA1 binding peptide. In another embodiment, the peptide is an HLA-DQB 1 binding peptide. In another embodiment, the peptide is an HLA-DPAl binding peptide. In another embodiment, the peptide is an HLA-DPB 1 binding peptide. In another embodiment, the peptide is an HLA-DMA binding peptide. In another embodiment, the peptide is an HLA- DMB binding peptide. In another embodiment, the peptide is an HLA-DOA binding peptide. In another embodiment, the peptide is an HLA-DOB binding peptide. In another embodiment, the peptide binds to any other HLA class II molecule known in the art. Each possibility represents a separate embodiment of the present invention.
[089] In another embodiment, a WT-1 peptide of the present invention binds to 2 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 3 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 4 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 5 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 6 distinct HLA- DRB molecules. In another embodiment, the peptide binds to more than 6 distinct HLA-DRB molecules.
[090] In another embodiment, a WT-1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 3 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 4 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 5 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 6 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by more than 6 distinct HLA- DRB alleles. Each possibility represents a separate embodiment of the present invention.
[091] In another embodiment, a WT-1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT-1 peptide binds to HLA-DRB molecules encoded by 3 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT-1 peptide binds to HLA-DRB molecules encoded by 4 distinct HLA- DRB alleles selected from DRB 101 , DRB 301, DRB 401 , DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT-1 peptide binds to HLA-DRB molecules encoded by 5 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101 , DRB 1104 and DRB 1501. In another embodiment, the WT-1 peptide binds to HLA- DRB molecules encoded by each of the following HLA-DRB alleles: DRB 101, DRB 301, DRB 401 , DRB 701, DRB 1101 , and DRB 1501. Each possibility represents a separate embodiment of the present invention.
[092] In another embodiment, the present invention provides a composition comprising 2 distinct WT-1 peptides of the present invention. In another embodiment, the 2 distinct WT-1 peptides are both unaltered. In another embodiment, 1 of the WT-1 peptides is unaltered, while the other is heteroclitic. In another embodiment, both of the WT-1 peptides are heteroclitic.
[093] In another embodiment, the composition comprises 3 distinct WT-1 peptides of the present invention. In another embodiment, the composition comprises 4 distinct WT-1 peptides of the present invention. In another embodiment, the composition comprises 5 distinct WT-1 peptides of the present invention. In another embodiment, the composition comprises more than 5 distinct isolated WT-1 peptides of the present invention.
[094] In another embodiment, 2 of the WT-1 peptides in the composition are unaltered. In another embodiment, 2 of the WT-1 peptides in the composition are heteroclitic. In another embodiment, 2 of the WT-1 peptides in the composition are unaltered, and 2 are heteroclitic. In another embodiment, more than 2 of the WT-1 peptides in the composition are unaltered.
In another embodiment, more than 2 of the WT-1 peptides in the composition are heteroclitic. In another embodiment, more than 2 of the WT-1 peptides in the composition are unaltered, and more than 2 are heteroclitic. Each possibility represents a separate embodiment of the present invention.
[095] In another embodiment, 1 of the additional WT-1 peptides in a composition of the present invention has a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. In another embodiment, 2 of the additional WT-1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. In another embodiment, 3 of the additional WT-1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. [096] In another embodiment, any other immunogenic WT-1 peptide known in the art is utilized as an additional WT-1 peptide. In another embodiment, any combination of immunogenic WT-1 peptides known in the art is utilized. Non-limiting sources of other WT peptides include WO2005053618, WO2007047764 and WO2007120673.
[097] Each additional WT-1 peptide, and each combination thereof, represents a separate embodiment of the present invention.
[098] In another embodiment, a composition of the present invention contains 2 HLA class II heteroclitic peptides that are derived from the same isolated WT-1 peptide of the present invention. In another embodiment, the 2 HLA class II heteroclitic peptides contain mutations in different HLA class II molecule anchor residues. In another embodiment, the 2 HLA class II heteroclitic peptides contain different mutations in the same anchor residues. In another embodiment, 2 of the HLA class II heteroclitic peptides are derived from different isolated WT-1 peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
[099] In another embodiment, 2 WT-1 peptides of the present invention, or the WT-1 peptides that correspond to two HLA class II heteroclitic peptides of the present invention, overlap with one another. In another embodiment, the overlap between the peptides is at least 7 amino acids (AA). In another embodiment, the overlap is at least 8 AA. In another embodiment, the overlap is at least 9 AA. In another embodiment, the overlap is 7 AA. In another embodiment, the overlap is 8 AA. In another embodiment, the overlap is 9 AA. In another embodiment, the overlap is 10 AA. In another embodiment, the overlap is 11 AA. In another embodiment, the overlap is 12 AA. In another embodiment, the overlap is 13 AA. In another embodiment, the overlap is 14 AA. In another embodiment, the overlap is 15 AA. In another embodiment, the overlap is 16 AA. In another embodiment, the overlap is more than 16 AA. Each possibility represents a separate embodiment of the present invention.
[0100] In another embodiment, the peptides in a composition of the present invention bind to 2 distinct HLA class II molecules. In another embodiment, the peptides bind to 3 distinct HLA class II molecules. In another embodiment, the peptides bind to 4 distinct HLA class II molecules. In another embodiment, the peptides bind to 5 distinct HLA class II molecules. In another embodiment, the peptides bind to more than 5 distinct HLA class II molecules. In another embodiment, the peptides in the composition bind to the same HLA class II molecules.
[0101] In another embodiment, each of the WT 1 peptides in a composition of the present invention binds to a set of HLA class II molecules. In another embodiment, each of the WT-1 peptides binds to a distinct set of HLA class II molecules. In another embodiment, the WT-1 peptides in the composition bind to the same set of HLA class II molecules. In another embodiment, 2 of the WT- 1 peptides bind to a distinct but overlapping set of HLA class II molecules. In another embodiment, 2 or more of the WT-1 peptides bind to the same set of HLA class II molecules, while another of the WT-1 peptides binds to a distinct set. In another embodiment, 2 or more of the WT-1 peptides bind to an overlapping set of HLA class II molecules, while another of the WT-1 peptides binds to a distinct set. [0102] In another embodiment, 2 or more of the WT-1 peptides in a composition of the present invention each binds to more than 1 HLA-DRB molecule. In another embodiment, the 4 or more HLA-DRB molecules bound by the peptides in the composition are distinct from one another. In another embodiment, the HLA-DRB molecules are encoded by different HLA-DRB alleles. Each possibility represents a separate embodiment of the present invention.
[0103] In another embodiment, 2 or more of the HLA class II molecules bound by
WT-1 peptides in a composition of the present invention are HLA-DRB molecules. In another embodiment, 3 or more of the HLA class II molecules that are bound are HLA-DRB molecules. In other embodiments, the HLA class II molecules that are bound can be any of the HLA class II molecules enumerated herein. In another embodiment, the HLA class II molecules that are bound are encoded by 2 or more distinct HLA class II alleles at a given locus. In another embodiment, the HLA class II molecules that are bound are encoded by HLA class II genes at 2 or more distinct loci.
[0104] Each of the above compositions represents a separate embodiment of the present invention. [0105] In another embodiment, a "set of HLA class II molecules" refers to the HLA class II molecules encoded by different alleles at a particular locus. In another embodiment, the term refers to HLA class II molecules with a particular binding specificity. In another embodiment, the term refers to HLA class II molecules with a particular peptide consensus sequence. In another embodiment, the term refers to a superfamily of HLA class II molecules. Each possibility represents a separate embodiment of the present invention.
[0106] In another embodiment, the present invention provides a composition comprising an unaltered HLA class II molecule-binding WT-1 peptide of the present invention and a second, HLA class I molecule-binding WT-1 peptide. In another embodiment, the composition comprises more than 1 HLA class II molecule-binding WT-1 peptide of the present invention, in addition to the HLA class I molecule- binding WT-1 peptide. In another embodiment, the composition comprises more than 1 HLA class I molecule-binding WT-1 peptide, in addition to the HLA class II molecule-binding WT-1 peptide. Each possibility represents a separate embodiment of the present invention.
[0107] In another embodiment, the AA sequence of the HLA class I molecule-binding WT-1 peptide comprises a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or
193. In another embodiment, the AA sequence of the HLA class I molecule-binding WT-1 peptide is selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. Each possibility represents a separate embodiment of the present invention.
[0108] In another embodiment, the HLA class I molecule-binding WT-1 peptide is an HLA class I heteroclitic peptide. In another embodiment, the HLA class I molecule-binding WT-1 peptide contains a mutation in an HLA class I molecule anchor residue thereof, as described further herein. As provided herein, WT- 1 -derived peptides were modified in HLA anchor residues to generate heteroclitic peptides with increased predicted binding to HLA- A0201 and HLA-A0301. Peptides with increased predicted binding also exhibited enhanced ability to bind HLA class I molecules and increased immunogenicity. [0109] In another embodiment, the mutation that enhances MHC binding is in the residue at position 1 of the HLA class I heteroclitic peptide. In another embodiment, the residue is changed to tyrosine. In another embodiment, the residue is changed to glycine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to phenylalanine. In another embodiment, the residue is changed to any other residue known in the art. In another embodiment, a substitution in position 1 (e.g. to tyrosine) stabilizes the binding of the position 2 anchor residue.
[0110] In another embodiment, the mutation is in position 2 of the HLA class I heteroclitic peptide. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to methionine. In another embodiment, the residue is changed to any other residue known in the art.
[0111] In another embodiment, the mutation is in position 6 of the HLA class I heteroclitic peptide. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to glutamine. In another embodiment, the residue is changed to histidine. In another embodiment, the residue is changed to any other residue known in the art.
[0112] In another embodiment, the mutation is in position 9 of the HLA class I heteroclitic peptide. In another embodiment, the mutation changes the residue at the C- terminal position thereof. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to alanine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to any other residue known in the art.
[0113] In another embodiment, the point mutation is in a primary anchor residue. In another embodiment, the HLA class I primary anchor residues are positions 2 and 9. In another embodiment, the point mutation is in a secondary anchor residue. In another embodiment, the HLA class I secondary anchor residues are positions 1 and 8. In another embodiment, the HLA class I secondary anchor residues are positions 1, 3, 6, 7, and 8. In another embodiment, the point mutation is in a position selected from positions 4, 5, and 8. Each possibility represents a separate embodiment of the present invention.
[0114] In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1 , 2, 8, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1 , 2, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 2 and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 6 and 9. Each possibility represents a separate embodiment of the present invention. [0115] Each of the above anchor residues and substitutions represents a separate embodiment of the present invention.
[0116] In another embodiment, the HLA class I molecule-binding WT peptide has length of 9 AA. In another embodiment, the peptide has length of 10 AA. As provided herein, native and heteroclitic peptides of 9- 10 AA exhibited substantial binding to HLA class I molecules and ability to elicit cytokine secretion and cytolysis by CTL.
[0117] In another embodiment, the HLA class I molecule that is bound by the HLA class I molecule- binding WT- 1 peptide is an HLA- A molecule. In another embodiment, the HLA class I-molecule is an HLA-A2 molecule. In another embodiment, the HLA class I- molecule is an HLA-A3 molecule. In another embodiment, the HLA class I-molecule is an HLA-A1 1 molecule. In another embodiment, the HLA class I-molecule is an HLA-B 8 molecule. In another embodiment, the HLA class I-molecule is an HLA-0201 molecule. In another embodiment, the HLA class I-molecule binds any other HLA class I molecule known in the art. Each possibility represents a separate embodiment of the present invention.
[0118] In another embodiment, a WT-1 peptide of methods and compositions of the present invention has a length of 8-30 amino acids. In another embodiment, the peptide has a length of 9-11 AA. In another embodiment, the peptide ranges in size from 7-25 AA, or in another embodiment, 8-11, or in another embodiment, 8-15, or in another embodiment, 9-20, or in another embodiment, 9-18, or in another embodiment, 9-15, or in another embodiment, 8-12, or in another embodiment, 9-11 AA in length. In another embodiment, the peptide is 8 AA in length, or in another embodiment, 9 AA or in another embodiment, 10 AA or in another embodiment, 12 AA or in another embodiment, 25 AA in length, or in another embodiment, any length therebetween. In another embodiment, the peptide is of greater length, for example 50, or 100, or more. In this embodiment, the cell processes the peptide to a length of 7 and 25 AA in length. In this embodiment, the cell processes the peptide to a length of 9-11 AA Each possibility represents a separate embodiment of the present invention.
[0119] In another embodiment, the peptide is 15-23 AA in length. In another embodiment, the length is 15-24 AA. In another embodiment, the length is 15-25 AA. In another embodiment, the length is 15-26 AA. In another embodiment, the length is 15-27 AA. In another embodiment, the length is 15-28 AA. In another embodiment, the length is 14-30 AA. In another embodiment, the length is 14-29 AA. In another embodiment, the length is 14-28 AA. In another embodiment, the length is 14-26 AA. In another embodiment, the length is 14-24 AA. In another embodiment, the length is 14-22 AA. In another embodiment, the length is 14-20 AA. In another embodiment, the length is 16-30 AA. In another embodiment, the length is 16-28 AA. In another embodiment, the length is 16-26 AA. In another embodiment, the length is 16-24 AA. In another embodiment, the length is 16-22 AA. In another embodiment, the length is 18-30 AA. In another embodiment, the length is 18-28 AA. In another embodiment, the length is 18-26 AA. In another embodiment, the length is 18-24 AA. In another embodiment, the length is 18-22 AA. In another embodiment, the length is 18-20 AA. In another embodiment, the length is 20-30 AA. In another embodiment, the length is 20-28 AA. In another embodiment, the length is 20-26 AA. In another embodiment, the length is 20-24 AA. In another embodiment, the length is 22-30 AA. In another embodiment, the length is 22-28 AA. In another embodiment, the length is 22-26 AA. In another embodiment, the length is 24-30 AA. In another embodiment, the length is 24-28 AA. In another embodiment, the length is 24-26 AA. [0120] Each of the above peptides, peptide lengths, and types of peptides represents a separate embodiment of the present invention. [0121] In another embodiment, minor modifications are made to peptides of the present invention without decreasing their affinity for HLA molecules or changing their TCR specificity, utilizing principles well known in the art. In the case of HLA class I-binding peptides, "minor modifications" refers, in another embodiment, to e.g. insertion, deletion, or substitution of one AA, inclusive, or deletion or addition of 1-3 AA outside of the residues between 2 and 9, inclusive. While the computer algorithms described herein are useful for predicting the MHC class I-binding potential of peptides, they have 60- 80% predictive accuracy; and thus, the peptides should be evaluated empirically before a final determination of MHC class I-binding affinity is made. Thus, peptides of the present invention are not limited to peptides predicated by the algorithms to exhibit strong MHC class I-binding affinity. The types are modifications that can be made are listed below. Each modification represents a separate embodiment of the present invention.
[0122] In another embodiment, a peptide enumerated in the Examples of the present invention is further modified by mutating an anchor residue to an MHC class I preferred anchor residue, which can be, in other embodiments, any of the anchor residues enumerated herein. In another embodiment, a peptide of the present invention containing an MHC class I preferred anchor residue is further modified by mutating the anchor residue to a different MHC class I preferred residue for that location. The different preferred residue can be, in other embodiments, any of the preferred residues enumerated herein. [0123] In another embodiment, the anchor residue that is further modified is in the 1 position. In another embodiment, the anchor residue is in the 2 position. In another embodiment, the anchor residue is in the 3 position. In another embodiment, the anchor residue is in the 4 position. In another embodiment, the anchor residue is in the 5 position. In another embodiment, the anchor residue is in the 6 position. In another embodiment, the anchor residue is in the 7 position. In another embodiment, the anchor residue is in the 8 position. In another embodiment, the anchor residue is in the 9 position. In the case of HLA class I-binding peptides, residues other than 2 and 9 can serve as secondary anchor residues; therefore, mutating them can improve MHC class I binding. Each possibility represents a separate embodiment of the present invention. [0124] In another embodiment, a peptide of methods and compositions of the present invention is a length variant of a peptide enumerated in the Examples. In another embodiment, the length variant is one amino acid (AA) shorter than the peptide from the Examples. In another embodiment, the length variant is two AA shorter than the peptide from the Examples. In another embodiment, the length variant is more than two A A shorter than the peptide from the Examples. In another embodiment, the shorter peptide is truncated on the N-terminal end. In another embodiment, the shorter peptide is truncated on the C- terminal end. In another embodiment, the truncated peptide is truncated on both the N- terminal and C- terminal ends. Peptides are, in another embodiment, amenable to truncation without changing affinity for HLA molecules, as is well known in the art.
[0125] Each of the above truncated peptides represents a separate embodiment of the present invention. [0126] In another embodiment, the length variant is longer than a peptide enumerated in the Examples of the present invention. In another embodiment, the longer peptide is extended on the N-terminal end in accordance with the surrounding WT- 1 sequence. Peptides are, in another embodiment, amenable to extension on the N-terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples. In another embodiment, the N-terminal extended peptide is extended by one residue. In another embodiment, the N- terminal extended peptide is extended by two residues. In another embodiment, the N-terminal extended peptide is extended by three residues. In another embodiment, the N-terminal extended peptide is extended by more than three residues. [0127] In another embodiment, the longer peptide is extended on the C terminal end in accordance with the surrounding WT-1 sequence. Peptides are, in another embodiment, amenable to extension on the C- terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention. In another embodiment, the C -terminal extended peptide is extended by one residue. In another embodiment, the C- terminal extended peptide is extended by two residues. In another embodiment, the C-terminal extended peptide is extended by three residues. In another embodiment, the C-terminal extended peptide is extended by more than three residues.
[0128] In another embodiment, the extended peptide is extended on both the N- terminal and C-terminal ends in accordance with the surrounding WT- 1 sequence. [0129] Each of the above extended peptides represents a separate embodiment of the present invention.
[0130] In another embodiment, a truncated peptide of the present invention retains the
HLA anchor residues (e.g. the HLA class I anchor residues) on the second residue and the C- terminal residue, with a smaller number of intervening residues (e.g.5) than a peptide enumerated in the Examples of the present invention. Peptides are, in another embodiment, amenable to such mutation without changing affinity for
[0131] HLA molecules. In another embodiment, such a truncated peptide is designed by removing one of the intervening residues of one of the above sequences. In another embodiment, the HLA anchor residues are retained on the second and eighth residues. In another embodiment, the HLA anchor residues are retained on the first and eighth residues. Each possibility represents a separate embodiment of the present invention.
[0132] In another embodiment, an extended peptide of the present invention retains the HLA anchor residues (e.g. the HLA class I anchor residues) on the second residue and the C-terminal residue, with a larger number of intervening residues (e.g. 7 or 8) than a peptide enumerated in the Examples of the present invention. In another embodiment, such an extended peptide is designed by adding one or more residues between two of the intervening residues of one of the above sequences. It is well known in the art that residues can be removed from or added between the intervening sequences of HLA-binding peptides without changing affinity for HLA. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention. In another embodiment, the HLA anchor residues are retained on the second and ninth residues. In another embodiment, the HLA anchor residues are retained on the first and eighth residues. In another embodiment, the HLA anchor residues are retained on the two residues separated by six intervening residues. Each possibility represents a separate embodiment of the present invention.
[0133] "Fragment," in another embodiment, refers to a peptide of 11 or more AA in length. In another embodiment, a peptide fragment of the present invention is 16 or more AA long. In another embodiment, the fragment is 12 or more AA long. In another embodiment, the fragment is 13 or more AA. In another embodiment, the fragment is 14 or more AA. In another embodiment, the fragment is 15 or more AA. In another embodiment, the fragment is
17 or more AA. In another embodiment, the fragment is 18 or more AA. In another embodiment, the fragment is 19 or more AA. In another embodiment, the fragment is 22 or more AA. In another embodiment, the fragment is 8-12 AA. In another embodiment, the fragment is about 8-12 AA. In another embodiment, the fragment is 16- 19 AA. In another embodiment, the fragment is about 16-19 AA. In another embodiment, the fragment 10-25 AA. In another embodiment, the fragment is about 10-25 AA. In another embodiment, the fragment has any other length. Each possibility represents a separate embodiment of the present invention.
[0134] "Fragment of a WT-1 protein," in another embodiment, refers to any of the definitions of "fragment" found herein. Each definition represents a separate embodiment of the present invention.
[0135] In another embodiment, a peptide of the present invention is homologous to a peptide enumerated in the Examples. The terms "homology," "homologous," etc., when in reference to any protein or peptide, refer, in another embodiment, to a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art.
[0136] In another embodiment, the term "homology," when in reference to any nucleic acid sequence similarly indicates a percentage of nucleotides in a candidate sequence that are identical with the nucleotides of a corresponding native nucleic acid sequence.
[0137] Homology is, in another embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art. In other embodiments, computer algorithm analysis of nucleic acid sequence homology includes the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.
[0138] In another embodiment, "homology" refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 70%. In another embodiment, "homology" refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 72%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 75%. In another embodiment, "homology" refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 78%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 80%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 82%. In another embodiment, "homology" refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 83%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 85%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 87%. In another embodiment, "homology" refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than[0128] 88%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 90%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 92%. In another embodiment, "homology" refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 93%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 95%. In another embodiment, "homology" refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 96%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 97%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 98%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 99%. In another embodiment, "homology" refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of 100%. Each possibility represents a separate embodiment of the present invention. [00114] In another embodiment, homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, "Nucleic AcidHybridization" Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y). In another embodiments, methods of hybridization are carried out under moderate to stringent conditions, to the complement of aDNA encoding a native caspase peptide. Hybridization conditions being, for example, overnight incubation at 42 °C in a solution comprising: 10-20 % formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate ( H 7.6), 5 X Denhardt's solution, 10 % dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.
[0139] Each of the above homologues and variants of peptides enumerated in the
Examples represents a separate embodiment of the present invention. [0140] In another embodiment, the present invention provides a composition comprising a peptide of this invention. In another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the composition further comprises an adjuvant. In another embodiment, the composition comprises 2 or more peptides of the present invention. In another embodiment, the composition further comprises any of the additives, compounds, or excipients set forth hereinbelow. In another embodiment, the adjuvantis KLH, QS21 , Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG or alum. In other embodiments, the carrier is any carrier enumerated herein. In other embodiments, the adjuvant is any adjuvant enumerated herein. Each possibility represents a separate embodiment of the present invention. [0141] In another embodiment, this invention provides a vaccine comprising a peptide of this invention. In another embodiment, this invention provides a vaccine comprising an antigen-presenting cell (APC) and a peptide of this invention. In another embodiment, the vaccine further comprises a carrier. In another embodiment, the vaccine further comprises an adjuvant. In another embodiment, the vaccine further comprises an APC. In another embodiment, the vaccine further comprises a combination of more than 1 of an antigen, carrier, and/or APC. In another embodiment, the vaccine is a cell-based composition. Each possibility represents a separate embodiment of the present invention.
[0142] In another embodiment, the term "vaccine" refers to a material or composition that, when introduced into a subject, provides a prophylactic or therapeutic response for a particular disease, condition, or symptom of same. In another embodiment, this invention comprises peptide-based vaccines, wherein the peptide comprises any embodiment listed herein, including immunomodulating compounds such as cytokines, adjuvants, etc.
[0143] In another embodiment, a vaccine of methods and compositions of the present invention further comprises an adjuvant. In another embodiment, the adjuvant is a Montanide. In another embodiment the adjuvant is Montanide ISA 51. Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier. In another embodiment, the adjuvant is GM-CSF. Recombinant GM-CSF is a human protein grown, in another embodiment, in a yeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, APC, and dendritic cells and T cells.
[0144] In another embodiment, the adjuvant is a cytokine. In another embodiment, the adjuvant is a growth factor. In another embodiment, the adjuvant is a cell population. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is Freund's incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. In another embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is BCG. In another embodiment, the adjuvant is alum. [0145] In another embodiment, the adjuvant is an interleukin. In another embodiment, the adjuvant is a chemokine. In another embodiment, the adjuvant is any other type of adjuvant known in the art. In another embodiment, the WT-1 vaccine comprises two the above adjuvants. In another embodiment, the WT-1 vaccine comprises more than two the above adjuvants. Each possibility represents a separate embodiment of the present invention. [0146] In other embodiments, a vaccine or composition of the present invention can comprise any of the embodiments of WT-1 peptides of the present invention and combinations thereof. Each possibility represents a separate embodiment of the present invention.
[0147] It is to be understood that any embodiments described herein, regarding peptides, vaccines and compositions of this invention can be employed in any of the methods of this invention. Each combination of peptide, vaccine, or composition with a method represents an embodiment thereof.
[0148] In another embodiment, the present invention provides a method of treating a subject with a WT-1 -expressing cancer, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby treating a subject with a WT-1 -expressing cancer.
[0149] In another embodiment, the present invention provides a method of treating a subject with an MDS, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby treating a subject with an MDS. [0150] In another embodiment, the present invention provides a method of suppressing or halting the progression of a WT-1 -expressing cancer in a subject, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby suppressing or halting the progression of a WT-1- expressing cancer. [0151] In another embodiment, the present invention provides a method of reducing the incidence of a WT-1 -expressing cancer in a subject, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby reducing the incidence of a WT-1 -expressing cancer in a subject.
[0152] In another embodiment, the present invention provides a method of reducing the incidence of an AML in a subject, the method comprising administering to the subject a WT- 1 vaccine of the present invention, thereby reducing the incidence of an AML.
[0153] In another embodiment, the present invention provides a method of reducing the incidence of relapse of a WT-1 -expressing cancer in a subject, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby reducing the incidence of relapse of a WT-1 -expressing cancer in a subject.
[0154] In another embodiment, the present invention provides a method of reducing the incidence ofrelapse of an AML in a subject, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby reducing the incidence of relapse of an AML in a subject. [0155] In another embodiment, the present invention provides a method of breaking a
T cell tolerance of a subject to a WT-1 -expressing cancer, the method comprising administering to the subject a WT-1 vaccine of the present invention, thereby breaking a T cell tolerance to a WT-1 -expressing cancer.
[0156] In another embodiment, the present invention provides a method of treating a subject having aWT-1 -expressing cancer, comprising (a) inducing in a donor formation and proliferation of human cytotoxic T lymphocytes (CTL) that recognize a malignant cell of the cancer by a method of the present invention; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer. [0157] In another embodiment, the present invention provides a method of treating a subject having a WT 1 -expressing cancer, comprising (a) inducing ex vivo formation and proliferation of human CTL that recognize a malignant cell of the cancer by a method of the present invention, wherein the human immune cells are obtained from a donor; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer.
[0158] In another embodiment, the present invention provides a method of inducing formation and proliferation of (a) a WT-1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT- 1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT-1 protein- specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT-1 protein; or a combination thereof. This method can be conducted in vitro, ex vivo or in vivo.When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
[0159] Methods for ex vivo immunotherapy are well known in the art and are described, for example, inUnited States Patent Application Serial Numbers 2006/0057130, 2005/0221481, 2005/0214268, 2003/0175272, 2002/0127718, and United States Patent Number 5,229,115, which are incorporated herein by reference. Additional methods are well known in the art and are described, for example, in Davis ID et al (Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients. J Immunother. 2006 Sep-Oct;29(5):499-511) and Mitchell MS et al (The cytotoxic T cell response to peptide analogs of the HLA-A*0201 -restricted MUC1 signal sequence epitope, Ml.2. Cancer Immunol Immunother. 2006 Jul 28). Each method represents a separate embodiment of the present invention.
[0160] In another embodiment, the present invention provides a method of inducing the formation and proliferation of CTL specific for cells of a WT- 1 -expressing cancer, the method comprising contacting a lymphocyte population with a vaccine of the present invention. In another embodiment, the vaccine is an APC associated with a peptide of the present invention. In another embodiment, the vaccine is an APC associated with a mixture of peptides of the present invention. Each possibility represents a separate embodiment of the present invention. [0161] In another embodiment, this invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against a WT-1 -expressing cancer, the method comprising administering to the subject a vaccine of the present invention, thereby generating a heteroclitic immune response. [0162] In another embodiment, the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT-1 protein; or (b) a fragment of a WT protein, thereby inducing an anti-mesothelioma immune response in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
[0163] In another embodiment, the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising a nucleotide molecule encoding (a) a WT-1 protein; or (b) a fragment of a WT-1 protein, thereby inducing an anti- mesothelioma immune response in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
[0164] In another embodiment, the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; or (b) a fragment of a WT protein, thereby treating a subject with a mesothelioma. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
[0165] In another embodiment, the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT-1 protein; or (b) a fragment of a WT-1 protein, thereby treating a subject with a mesothelioma. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention. [0166] In another embodiment, the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT-1 protein; or (b) a fragment of a WT protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention. [0167] In another embodiment, the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT-1 protein; or (b) a fragment of a WT-1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
[0168] In another embodiment, a target cell of an immune response elicited by a method of the present invention presents the WT-1 peptide of the present invention, or a corresponding WT-1 fragment, on an HLA molecule. In another embodiment, the HLA molecule is an HLA class I molecule. In other embodiments, the HLA molecule is any HLA class I subtype or HLA class I molecule known in the art. In another embodiment, the immune response against the WT-1 peptide or fragment is a heteroclitic immune response. Each possibility represents a separate embodiment of the present invention.
[0169] In another embodiment, the WT-1 -expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT-1 -expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT-1 -expressing cancer is an MDS. In another embodiment, the WT-1- expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT-1 -expressing cancer is a Wilms' tumor. In another embodiment, the WT-1 -expressing cancer is a leukemia. In another embodiment, the WT-1 -expressing cancer is a hematological cancer. In another embodiment, the WT-1- expressing cancer is a lymphoma. In another embodiment, the WT-1 -expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT-1 -expressing cancer is a mesothelioma. In another embodiment, the WT-1 -expressing cancer is a malignant mesothelioma. In another embodiment, the WT-1 -expressing cancer is a gastric cancer. In another embodiment, the WT-1 -expressing cancer is a colon cancer. In another embodiment, the WT-1 -expressing cancer is a lung cancer. In another embodiment, the WT-1 -expressing cancer is abreast cancer. In another embodiment, the WT-1 -expressing cancer is a germ cell tumor. In another embodiment, the WT-1 -expressing cancer is an ovarian cancer. In another embodiment, the WT 1 -expressing cancer is a uterine cancer. In another embodiment, the WT 1 - expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT-1 -expressing cancer is a thyroid cancer. In another embodiment, the WT-1 -expressing cancer is a liver cancer. In another embodiment, the WT-1- expressing cancer is a renal cancer. In another embodiment, the WT-1 -expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT-1- expressing cancer is a sarcoma. In another embodiment, the WT-1 -expressing cancer is any other carcinoma or sarcoma. [0170] In another embodiment, the WT-1 -expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT-1 -expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with a non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with a lung cancer. In another embodiment, the solid tumor is associated with a breast cancer. In another embodiment, the solid tumor is associated with a colorectal cancer. In another embodiment, the solid tumor is associated with a prostate cancer. In another embodiment, the solid tumor is associated with an ovarian cancer. In another embodiment, the solid tumor is associated with a renal cancer. In another embodiment, the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
[0171] In another embodiment, a cancer or tumor treated by a method of the present invention is suspected to express WT-1. In another embodiment, WT-1 expression has not been verified by testing of the actual tumor sample. In another embodiment, the cancer or tumor is of a type known to express WT-1 in many cases. In another embodiment, the type expresses WT-1 in the majority of cases.
[0172] Each type of WT-1 -expressing cancer or tumor, and cancer or tumor suspected to express WT- 1 , represents a separate embodiment of the present invention. [0173] Any embodiments enumerated herein, regarding peptides, vaccines and compositions of this invention can be employed in any of the methods of this invention, and each represents an embodiment thereof.
[0174] In another embodiment, multiple peptides of this invention are used to stimulate an immune response in methods of the present invention.
[0175] The methods disclosed herein will be understood by those in the art to enable design of other WT-1 -derived peptides. The methods further enable design of peptides binding to other HLA molecules. The methods further enable design of vaccines combining WT-1 -derived peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
[0176] [00152] In another embodiment, vaccines of the present invention have the advantage of activating or eliciting WT-1 -specific CD4+ T cells containing a variety of different HLA class II alleles. In another embodiment, the vaccines have the advantage of activating or eliciting WT-1 -specific CD4+ T cells in a substantial proportion of the population (e.g. in different embodiments, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%, 90%, 95%, or greater than 95%). In another embodiment, the vaccines activate or elicit WT- 1 -specific CD4+ T cells in a substantial proportion of a particular population (e.g. American Caucasians). Each possibility represents a separate embodiment of the present invention.
[0177] In another embodiment, methods of the present invention provide for an improvement in an immune response that has already been mounted by a subject. In another embodiment, methods of the present invention comprise administering the peptide, composition, or vaccine 2 or more times. In another embodiment, the peptides are varied in their composition, concentration, or a combination thereof. In another embodiment, the peptides provide for the initiation of an immune response against an antigen of interest in a subject who has not yet initiated an immune response against the antigen. In another embodiment, the CTL that are induced proliferate in response to presentation of the peptide on the APC or cancer cell. In other embodiments, reference to modulation of the immune response involves, either or both the humoral and cell-mediated arms of the immune system, which is accompanied by the presence of Th2 and Thl T helper cells, respectively, or in another embodiment, each arm individually. [0178] In other embodiments, the methods affecting the growth of a tumor result in
(1) the direct inhibition of tumor cell division, or (2) immune cell mediated tumor cell lysis, or both, which leads to a suppression in the net expansion of tumor cells.
[0179] Inhibition of tumor growth by either of these two mechanisms can be readily determined by one of ordinary skill in the art based upon a number of well-known methods. In another embodiment, tumor inhibition is determined by measuring the actual tumor size over a period of time. In another embodiment, tumor inhibition can be determined by estimating the size of a tumor (over a period of time) utilizing methods well known to those of skill in the art. More specifically, a variety of radiologic imaging methods (e.g., single photon and positron emission computerized tomography; see generally, "Nuclear Medicine in Clinical Oncology," Winkler, C. (ed.) Springer- Verlag, New York, 1986), can be utilized to estimate tumor size. Such methods can also utilize a variety of imaging agents, including for example, conventional imaging agents (e.g., Gallium-67 citrate), as well as specialized reagents for metabolite imaging, receptor imaging, or immunologic imaging (e.g., radiolabeled monoclonal antibody specific tumor markers). In addition, non-radioactive methods such as ultrasound (see, "Ultrasonic Differential Diagnosis of Tumors", Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984), can also be utilized to estimate the size of a tumor.
[0180] In addition to the in vivo methods for determining tumor inhibition discussed above, a variety of in vitro methods can be utilized in order to predict in vivo tumor inhibition. Representative examples include lymphocyte mediated anti-tumor cytolytic activity determined for example, by a 51Cr release assay (Examples), tumor dependent lymphocyte proliferation (Ioannides, et al., J. Immunol. 146(5): 1700-1707, 1991), in vitro generation of tumor specific antibodies (Herlyn, et al., J. Immunol. Meth. 73: 157-167, 1984), cell (e.g., CTL, helper T-cell) or humoral (e.g., antibody) mediated inhibition of cell growth in vitro (Gazit, et al., Cancer Immunol Immunother 35: 135-144, 1992), and, for any of these assays, determination of cell precursor frequency (Vose, Int. J. Cancer 30: 135-142 (1982), and others.
[0181] In another embodiment, methods of suppressing tumor growth indicate a growth state that is curtailed compared to growth without contact with, or exposure to a peptide of this invention. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a 3H-thymidine incorporation assay, or counting tumor cells. "Suppressing" tumor cell growth refers, in other embodiments, to slowing, delaying, or stopping tumor growth, or to tumor shrinkage. Each possibility represents a separate embodiment of the present invention. [0182] In another embodiment of methods and compositions of the present invention,
WT-1 expression is measured. In another embodiment, WT-1 transcript expression is measured. In another embodiment, WT-1 protein levels in the tumor are measured. Each possibility represents a separate embodiment of the present invention.
[0183] Methods of determining the presence and magnitude of an immune response are well known in the art. In another embodiment, lymphocyte proliferation assays, wherein T cell uptake of a radioactive substance, e.g. 3H-thymidine is measured as a function of cell proliferation. In other embodiments, detection of T cell proliferation is accomplished by measuring increases in interleukin-2 (IL-2) production, Ca2+ flux, or dye uptake, such as 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Each possibility represents a separate embodiment of the present invention.
[0184] In another embodiment, CTL stimulation is determined by means known to those skilled in the art, including, detection of cell proliferation, cytokine production and others. Analysis of the types and quantities of cytokines secreted by T cells upon contacting ligand-pulsed targets can be a measure of functional activity. Cytokines can be measured by ELISA or ELISPOT assays to determine the rate and total amount of cytokine production. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160: 181 ; Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259).
[0185] In another embodiment, CTL activity is determined by 51Cr-release lysis assay. Lysis of peptide- pulsed 51Cr-labeled targets by antigen-specific T cells can be compared for target cells pulsed with control peptide. In another embodiment, T cells are stimulated with a peptide of this invention, and lysis of target cells expressing the native peptide in the context of MHC can be determined. The kinetics of lysis as well as overall target lysis at a fixed timepoint (e.g., 4 hours) are used, in another embodiment, to evaluate ligand performance. (Ware C. F. et al. (1983) J Immunol 131 : 1312). [0186] Methods of determining affinity of a peptide for an HLA molecule are well known in the art. In another embodiment, affinity is determined by TAP stabilization assays. [0187] In another embodiment, affinity is determined by competition radioimmunoassay. In another embodiment, the following protocol is utilized: Target cells are washed two times in PBS with 1 % bovine serum albumin (BSA; Fisher Chemicals, Fairlawn, NJ). Cells are resuspended at 107/ml on ice, and the native cell surface bound peptides are stripped for 2 minutes at 0[deg.] C using citrate -phosphate buffer in the presence of 3 mg/ml beta2 microglobulin. The pellet is resuspended at 5 x 106 cells/ml in PBS/1 % BSA in the presence of 3 mg/ml beta2 microglobulin and 30 mg/ml deoxyribonuclease, and 200 ml aliquots are incubated in the presence or absence of HLA-specific peptides for 10 min at 20°C, then with 125I-labeled peptide for 30 min at 20 °C. Total bound 125I is determined after two washes with PBS/2% BSA and one wash with PBS. Relative affinities are determined by comparison of escalating concentrations of the test peptide versus a known binding peptide.
[0188] In another embodiment, a specificity analysis of the binding of peptide to
HLA on surface of live cells (e.g. SKLY-16 cells) is conducted to confirm that the binding is to the appropriate HLA molecule and to characterize its restriction. This includes, in another embodiment, competition with excess unlabeled peptides known to bind to the same or disparate HLA molecules and use of target cells which express the same or disparate HLA types. This assay is performed, in another embodiment, on live fresh or 0.25% paraformaldehyde-fixed human PBMC, leukemia cell lines and EBV-transformed T-cell lines of specific HLA types. The relative avidity of the peptides found to bind MHC molecules on
125 the specific cells are assayed by competition assays as described above against I-labeled peptides of known high affinity for the relevant HLA molecule, e.g., tyrosinase or HBV peptide sequence. In another embodiment, an HLA class Il-binding peptide of methods and compositions of the present invention is longer than the minimum length for binding to an HLA class II molecule, which is, in another embodiment, about 12 AA. In another embodiment, increasing the length of the HLA class II- binding peptide enables binding to more than one HLA class II molecule. In another embodiment, increasing the length enables binding to an HLA class II molecule whose binding motif is not known. In another embodiment, increasing the length enables binding to an HLA class I molecule. In another embodiment, the binding motif of the HLA class I molecule is known. In another embodiment, the binding motif of the HLA class I molecule is not known. Each possibility represents a separate embodiment of the present invention. [0189] In another embodiment, the peptides utilized in methods and compositions of the present invention comprise a non-classical amino acid such as: 1,2,3,4- tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991) J. Am Chem. Soc. 113:2275- 2283); (2S,3S)-methyl-phenylalanine, (2S,3R)- methyl-phenylalanine, (2R,3S)-methyl- phenylalanine and (2R,3R)-methyl-phenylalanine (Kazmierski and Hruby (1991) Tetrahedron Lett. 32(41): 5769-5772); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) Ph.D. Thesis, University of Arizona); hydroxy- 1 ,2,3, 4-tetrahydroisoquinoline-3- carboxylate (Miyake et al. (1984) J. TakedaRes. Labs.43:53-76) histidine isoquinoline carboxylic acid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2): 131-138); and HIC (histidine cyclic urea), (Dharanipragada et al.(1993) Int. J. Pep. Protein Res.42(l):68-77) and ((1992) Acta. Crst, Crystal Struc. Comm. 48(IV): 1239-124).
[0190] In another embodiment, a peptide of this invention comprises an AA analog or peptidomimetic, which, in other embodiments, induces or favors specific secondary structures. Such peptides comprise, in other embodiments, the following: LL-Acp (LL-3- amino-2-propenidone-6-carboxylic acid), a [beta]-turn inducing dipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838); [beta]-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5081-5082); [beta]-turn inducing analogs (Kemp et al. (1988) Tetrahedron Left. 29:5057-5060); alpha-helix inducing analogs (Kemp et al. (1988) Tetrahedron Left. 29:4935-4938); gamma-turn inducing analogs (Kemp et al. (1989) J. Org. Chem. 54: 109: 115); analogs provided by the following references: Nagai and Sato (1985) Tetrahedron Left.26: 647-650; and DiMaio et al. (1989) J. Chem. Soc. Perkin Trans, p. 1687; a Gly- Ala turn analog (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bond isostere (Jones et al. (1988) Tetrahedron Left. 29(31):3853-3856); tretrazol (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880); DTC (Samanen et al. (1990) Int. J. ProteinPep. Res. 35:501 :509); and analogs taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 and Garveyet al. (1990) J. Org. Chem. 55(3):936-940. Conformationally restricted mimetics of beta turns and beta bulges, and peptides containing them, are described in U.S. Pat. No.5,440,013, issued Aug. 8, 1995 to Kahn.
[0191] In other embodiments, apeptide of this invention is conjugated to one of various other molecules, as described hereinbelow, which can be via covalent or non-covalent linkage (complexed), the nature of which varies, in another embodiment, depending on the particular purpose. In another embodiment, the peptide is covalently or non-covalently complexed to a macromolecular carrier, (e.g. an immunogenic carrier), including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids. In another embodiment, a peptide of this invention is linked to a substrate. In another embodiment, the peptide is conjugated to a fatty acid, for introduction into a liposome (U.S. Pat. No.5, 837,249). In another embodiment, a peptide of the invention is complexed covalently or non-covalently with a solid support, a variety of which are known in the art. In another embodiment, linkage of the peptide to the carrier, substrate, fatty acid, or solid support serves to increase an elicited an immune response. [0192] In other embodiments, the carrier is thyroglobulin, an albumin (e.g. human serum albumin), tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), an influenza protein, hepatitis B virus core protein, keyhole limpet hemocyanin, an albumin, or another carrier protein or carrier peptide; hepatitis B virus recombinant vaccine, or an APC. Each possibility represents a separate embodiment of the present invention. [0193] In another embodiment, the term "amino acid" (AA) refers to a natural or, in another embodiment, an unnatural or synthetic AA, and can include, in other embodiments, glycine, D- or L optical isomers, AA analogs, peptidomimetics, or combinations thereof.
[0194] In another embodiment, the terms "cancer," "neoplasm," "neoplastic" or
"tumor," are used interchangeably and refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. In another embodiment, a tumor is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation, and in another embodiment, is identified by biochemical or immunologic findings, the latter which is used to identify cancerous cells, as well, in other embodiments.
[0195] Methods for synthesizing peptides are well known in the art. In another embodiment, the peptides of this invention are synthesized using an appropriate solid-state synthetic procedure (see for example,Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco, Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23: 451). The activity of these peptides is tested, in other embodiments, using assays as described herein. [0196] In another embodiment, the peptides of this invention are purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. In another embodiment, immuno- affinity chromatography is used, whereby an epitope is isolated by binding it to an affinity column comprising antibodies that were raised against that peptide, or a related peptide of the invention, and were affixed to a stationary support.
[0197] In another embodiment, affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Meth. Enzymol. 194:508-509), glutathione-S-transferase, or others, are attached to the peptides of this invention to allow easy purification by passage over an appropriate affinity column. Isolated peptides can also be physically characterized, in other embodiments, using such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallography.
[0198] In another embodiment, the peptides of this invention are produced by in vitro translation, through known techniques, as will be evident to one skilled in the art. In another embodiment, the peptides are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).
[0199] In another embodiment, the peptides of this invention further comprise a detectable label, which in another embodiment, is fluorescent, or in another embodiment, luminescent, or in another embodiment, radioactive, or in another embodiment, electron dense. In other embodiments, the detectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-
32 125 3 14
galactosidase, luciferase, P, I, H and C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin or any number of other such labels known to one skilled in the art. The particular label used will depend upon the type of immunoassay used.
[0200] In another embodiment, a peptide of this invention is linked to a substrate, which, in another embodiment, serves as a carrier. In another embodiment, linkage of the peptide to a substrate serves to increase an elicited an immune response.
[0201] In another embodiment, peptides of this invention are linked to other molecules, as described herein, using conventional cross-linking agents such as carbodiimide. Examples of carbodiimide are 1- cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), l-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and l-ethyl-3-(4-azonia-44- dimethylpentyl) carbodiimide.
[0202] In other embodiments, the cross-linking agents comprise cyanogen bromide, glutaraldehyde and succinic anhydride. In general, any of a number of homo-bifunctional agents including a homo- bifunctional aldehyde, a homo-bifunctional epoxide, a homo- bifunctional imido-ester, a homo- bifunctional N-hydroxysuccinimide ester, a homo- bifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyl disulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo- bifunctional diazonium derivative and a homo-bifunctional photoreactive compound can be used. Also envisioned, in other embodiments, are hetero-bifunctional compounds, for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine- reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.
[0203] In other embodiments, the homo-bifunctional cross-linking agents include the bifunctional N- hydroxy succinimide esters dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers l,4-di-[3'-(2'- pyridyldithio)propionamido]butane, bismaleimidohexane, and bis- N-maleimido-l,8-octane; the bifunctional aryl halides 1 ,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'-dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4- azidosalicylamido)ethyl]disulfide; the bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as 1 ,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipic acid dihydrazide, carbohydrazide, and succinic acid dihydrazide; the bifunctional diazoniums o-tolidine, diazotized and bis-diazotized benzidine; the bifunctional alkylhalides N,N'-ethylene- bis (iodoacetamide) , NIN'-hexamethylene-bis (iodoacetamide) , N, N'-undecamethylene- bis(iodoacetamide), as well as benzylhalides and halomustards, such as ala'-diiodo-p-xylene sulfonic acid and tri(2-chloroethyl)amine, respectively,
[0204] In other embodiments, hetero-bifunctional cross-linking agents used to link the peptides to other molecules, as described herein, include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate), MB S (m- maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4- iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p- maleimidophenyl)butyrate), GMBS (N-(.gamma.-maleimidobutyryloxy)succmimide ester), MPBH (4-(4- N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl) cyclohexane-1- carboxyl- hydrazide), SMPT (succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate). [0205] In another embodiment, the peptides of the invention are formulated as non- covalent attachment of monomers through ionic, adsorptive, or biospecific interactions. Complexes of peptides with highly positively or negatively charged molecules can be accomplished, in another embodiment, through salt bridge formation under low ionic strength environments, such as in deionized water. Large complexes can be created, in another embodiment, using charged polymers such as poly- (L- glutamic acid) or poly- (L-lysine), which contain numerous negative and positive charges, respectively. In another embodiment, peptides are adsorbed to surfaces such as microparticle latex beads or to other hydrophobic polymers, forming non-covalently associated peptide-superantigen complexes effectively mimicking cross-linked or chemically polymerized protein, in other embodiments. In another embodiment, peptides are non- covalently linked through the use of biospecific interactions between other molecules. For instance, utilization of the strong affinity of biotin for proteins such as avidin or streptavidin or their derivatives could be used to form peptide complexes. The peptides, according to this aspect, and in another embodiment, can be modified to possess biotin groups using common biotinylation reagents such as the N- hydroxysuccinimidyl ester of D-biotin (NHS-biotin), which reacts with available amine groups. [0206] In another embodiment, a peptide of the present invention is linked to a carrier. In another embodiment, the carrier is KLH. In other embodiments, the carrier is any other carrier known in the art, including, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. Each possibility represents a separate embodiment of the present invention.
[0207] In another embodiment, the peptides of this invention are conjugated to a lipid, such as P3 CSS. In another embodiment, the peptides of this invention are conjugated to a bead. [0208] In another embodiment, the compositions of this invention further comprise immunomodulating compounds. In other embodiments, the immunomodulating compound is a cytokine, chemokine, or complement component that enhances expression of immune system accessory or adhesion molecules, their receptors, or combinations thereof. In some embodiments, the immunomodulating compound include interleukins, for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte- macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M- CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia and MIP-Ib, complement components, or combinations thereof. In other embodiments, the immunomodulating compound stimulate expression, or enhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or combination thereof.
[0209] In another embodiment, the immunomodulatory compound induces or enhances expression of co- stimulatory molecules that participate in the immune response, which include, in some embodiments, CD40 or its ligand, CD28, CTLA-4 or a B7 molecule. In another embodiment, the immunomodulatory compound induces or enhances expression of a heat stable antigen (HSA) (Liu Y. et al. (1992) J. Exp.Med. 175:437-445), chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas M. F. et al (1993) Cell 74:257-268), or an intracellular adhesion molecule 1 (ICAM-I) (Van R. H. (1992) Cell 71 : 1065-1068), which assists, in another embodiment, co- stimulation by interacting with their cognate ligands on the T cells. [0210] In another embodiment, the composition comprises a solvent, including water, dispersion media, cell culture media, isotonic agents and the like. In another embodiment, the solvent is an aqueous isotonic buffered solution with a pH of around 7.0. In another embodiment, the composition comprises a diluent such as water, phosphate buffered saline, or saline. In another embodiment, the composition comprises a solvent, which is nonaqueous, such as propyl ethylene glycol, polyethylene glycol and vegetable oils.
[0211] In another embodiment, the composition is formulated for administration by any of the many techniques known to those of skill in the art. For example, this invention provides for administration of the pharmaceutical composition parenterally, intravenously, subcutaneously, intradermally, intramucosally, topically, orally, or by inhalation.
[0212] In another embodiment, the vaccine comprising a peptide of this invention further comprises a cell population, which, in another embodiment, comprises lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells or combinations thereof, which, in another embodiment are autologous, syngeneic or allogeneic, with respect to each other. In another embodiment, the cell population comprises a peptide of the present invention. In another embodiment, the cell population takes up the peptide. Each possibility represents a separate embodiment of the present invention.
[0213] In another embodiment, the cell populations of this invention are obtained from in vivo sources, such as, for example, peripheral blood, leukopheresis blood product, apheresis blood product, peripheral lymph nodes, gut associated lymphoid tissue, spleen, thymus, cord blood, mesenteric lymph nodes, liver, sites of immunologic lesions, e.g. synovial fluid, pancreas, cerebrospinal fluid, tumor samples, granulomatous tissue, or any other source where such cells can be obtained. In another embodiment, the cell populations are obtained from human sources, which are, in other embodiments, from human fetal, neonatal, child, or adult sources. In another embodiment, the cell populations of this invention are obtained from animal sources, such as, for example, porcine or simian, or any other animal of interest. In another embodiment, the cell populations of this invention are obtained from subjects that are normal, or in another embodiment, diseased, or in another embodiment, susceptible to a disease of interest. [0214] In another embodiment, the cell populations of this invention are separated via affinity-based separation methods. Techniques for affinity separation include, in other embodiments, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or use in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and "panning" with an antibody attached to a solid matrix, such as a plate, or any other convenient technique. In other embodiment, separation techniques include the use of fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. In other embodiments, any technique that enables separation of the cell populations of this invention can be employed, and is to be considered as part of this invention. [0215] In another embodiment, the dendritic cells are from the diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, qualified as such (Steinman (1991) Ann. Rev. Immunol.9:271-296). In another embodiment, the dendritic cells used in this invention are isolated from bone marrow, or in another embodiment, derived from bone marrow progenitor cells, or, in another embodiment, from isolated from/derived from peripheral blood, or in another embodiment, derived from, or are a cell line.
[0216] In another embodiment, the cell populations described herein are isolated from the white blood cell fraction of a mammal, such as a murine, simian or a human (See, e.g., WO 96/23060). The white blood cell fraction can be, in another embodiment, isolated from the peripheral blood of the mammal.
[0217] Methods of isolating dendritic cells are well known in the art. In another embodiment, the DC are isolated via a method which includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation; (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF and IL-4, (d) identifying the dendritic cell-enriched fraction from step (c); and (e) collecting the enriched fraction of step (d), preferably at about 4[deg.] C.
[0218] In another embodiment, the dendritic cell-enriched fraction is identified by fluorescence-activated cell sorting, which identifies at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absence of the following markers: CD3, CD14, CD16, 56, 57, and CD 19, 20.
[0219] In another embodiment, the cell population comprises lymphocytes, which are, in another embodiment, T cells, or in another embodiment, B cells. The T cells are, in other embodiments, characterized as NK cells, helper T cells, cytotoxic T lymphocytes (CTL), TBLs, native T cells, or combinations thereof. It is to be understood that T cells which are primary, or cell lines, clones, etc. are to be considered as part of this invention. In another embodiment, the T cells are CTL, or CTL lines, CTL clones, or CTLs isolated from tumor, inflammatory, or other infiltrates. [0220] In another embodiment, hematopoietic stem or early progenitor cells comprise the cell populations used in this invention. In another embodiment, such populations are isolated or derived, by leukaphoresis. In another embodiment, the leukapheresis follows cytokine administration, from bone marrow, peripheral blood (PB) or neonatal umbilical cord blood. In another embodiment, the stem or progenitor cells are characterized by their surface expression of the surface antigen marker known as CD34+, and exclusion of expression of the surface lineage antigen markers, Lin-.
[0221] In another embodiment, the subject is administered a peptide, composition or vaccine of this invention, in conjunction with bone marrow cells. In another embodiment, the administration together with bone marrow cells embodiment follows previous irradiation of the subject, as part of the course of therapy, in order to suppress, inhibit or treat cancer in the subject.
[0222] In another embodiment, the phrase "contacting a cell" or "contacting a population" refers to a method of exposure, which can be, in other embodiments, direct or indirect. In another embodiment, such contact comprises direct injection of the cell through any means well known in the art, such as microinjection. It is also envisaged, in another embodiment, that supply to the cell is indirect, such as via provision in a culture medium that surrounds the cell, or administration to a subject, via any route well known in the art, and as described herein.
[0223] In another embodiment, CTL generation of methods of the present invention is accomplished in vivo, and is effected by introducing into a subject an antigen presenting cell contacted in vitro with a peptide of this invention (See for example Paglia et al. (1996) J. Exp. Med. 183:317-322).
[0224] In another embodiment, the peptides of methods and compositions of the present invention are delivered to APC. In another embodiment, the peptide-pulsed APC are administered to a subject to elicit and immune response or treat or inhibit growth or recurrence of a tumor. Each possibility represents a separate embodiment of the present invention.
[0225] In another embodiment, the peptides are delivered to APC in the form of cDNA encoding the peptides. In another embodiment, the term "antigen-presenting cells" (APC) refers to dendritic cells (DC), monocytes/macrophages, B lymphocytes or other cell type(s) expressing the necessary MHC/co- stimulatory molecules, which effectively allow for T cell recognition of the presented peptide. In another embodiment, the APC is a cancer cell. Each possibility represents a separate embodiment of the present invention.
[0226] In another embodiment, the CTL are contacted with 2 or more APC populations. In another embodiment, the 2 or more APC populations present different peptides. Each possibility represents a separate embodiment of the present invention.
[0227] In another embodiment, techniques that lead to the expression of antigen in the cytosol of APC (e.g. DC) are used to deliver the peptides to the APC. Methods for expressing antigens on APC are well known in the art. In another embodiment, the techniques include (1) the introduction into the APC of naked DNA encoding a peptide of this invention, (2) infection of APC with recombinant vectors expressing a peptide of this invention, and (3) introduction of a peptide of this invention into the cytosol of an APC using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med. 184:465-472; Rouse et al. (1994) J. Virol. 68:5685- 5689; and Nair et al. (1992) J. Exp. Med. 175:609-612). [0228] In another embodiment, foster APC such as those derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150: 1763-1771), are used, as exemplified herein. [0229] In another embodiment, as described herein, the subject is exposed to a peptide, or a composition/cell population comprising a peptide of this invention, which differs from the native protein expressed, wherein subsequently a host immune cross-reactive with the native protein/antigen develops. [0230] In another embodiment, the subject, as referred to in any of the methods or embodiments of this invention is a human. In other embodiments, the subject is a mammal, which can be a mouse, rat, rabbit, hamster, guinea pig, horse, cow, sheep, goat, pig, cat, dog, monkey, or ape. Each possibility represents a separate embodiment of the present invention.
[0231] In another embodiment, peptides, vaccines, and compositions of this invention stimulate an immune response that results in tumor cell lysis.
[0232] In another embodiment, any of the methods described herein is used to elicit
CTL, which are elicited in vitro. In another embodiment, the CTL are elicited ex-vivo. In another embodiment, the CTL are elicited in vitro. The resulting CTL, are, in another embodiment, administered to the subject, thereby treating the condition associated with the peptide, an expression product comprising the peptide, or a homologue thereof. Each possibility represents a separate embodiment of the present invention.
[0233] In another embodiment, the method entails introduction of the genetic sequence that encodes the peptides of this invention using, e.g., one or more nucleic acid delivery techniques. Nucleic acids of the invention include, in another embodiment, DNA, RNA and mixtures of DNA and RNA, alone or in conjunction with non-nucleic acid components. In another embodiment, the method comprises administering to the subject a vector comprising a nucleotide sequence, which encodes a peptide of the present invention (Tindle, R. W. et al. Virology (1994) 200:54). In another embodiment, the method comprises administering to the subject naked DNA which encodes a peptide, or in another embodiment, two or more peptides of this invention (Nabel, et al. PNAS-USA (1990) 90: 11307). In another embodiment, multi-epitope, analogue -based cancer vaccines are utilized (Fikes et al, Design of multi- epitope, analogue-based cancer vaccines. Expert Opin Biol Ther.2003 Sep;3(6):985-93). Each possibility represents a separate embodiment of the present invention.
[0234] Nucleic acids can be administered to a subject via any means as is known in the art, including parenteral or intravenous administration, or in another embodiment, by means of a gene gun. In another embodiment, the nucleic acids are administered in a composition, which correspond, in other embodiments, to any embodiment listed herein.
[0235] Vectors for use according to methods of this invention can comprise any vector that facilitates or allows for the expression of a peptide of this invention. Vectors comprise, in some embodiments, attenuated viruses, such as vaccinia or fowlpox, such as described in, e.g., U.S. Pat. No. 4,722,848, incorporated herein by reference. In another embodiment, the vector is BCG (Bacille Calmette Guerin), such as described in Stover et al. (Nature 351 :456-460 (1991)). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g., Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.
[0236] In another embodiment, the vector further encodes for an immunomodulatory compound, as described herein. In another embodiment, the subject is administered an additional vector encoding same, concurrent, prior to or following administration of the vector encoding a peptide of this invention to the subject. [0237] In another embodiment, the peptides, compositions and vaccines of this invention are administered to a subject, or utilized in the methods of this invention, in combination with other anticancer compounds and chemotherapeutics, including monoclonal antibodies directed against alternate cancer antigens, or, in another embodiment, epitopes that consist of an AA sequence which corresponds to, or in part to, that from which the peptides of this invention are derived.
[0238] Various embodiments of dosage ranges are contemplated by this invention. In another embodiment, the dosage is 20 μg per peptide per day. In another embodiment, the dosage is 10 μg/peptide/day. In another embodiment, the dosage is 30 μg/peptide/day. In another embodiment, the dosage is 40 μg/peptide/day. In another embodiment, the dosage is 60 μg/peptide/day. In another embodiment, the dosage is 80 μg/peptide/day. In another embodiment, the dosage is 100 μg/peptide/day. In another embodiment, the dosage is 150 μg/peptide/day. In another embodiment, the dosage is 200 μg/peptide/day. In another embodiment, the dosage is 300 μg/peptide/day. In another embodiment, the dosage is 400 μg/peptide/day. In another embodiment, the dosage is 600 μg/peptide/day. In another embodiment, the dosage is 800 μg/peptide/day. In another embodiment, the dosage is 1000 μg/ e tide/day. In another embodiment, the dosage is 1500 μg/peptide/day. In another embodiment, the dosage is 2000 μg/peptide/day.
[0239] In another embodiment, the dosage is 10 μg/peptide/dose. In another embodiment, the dosage is 30 μg/peptide/dose. In another embodiment, the dosage is 40 μg/peptide/dose. In another embodiment, the dosage is 60 μg/peptide/dose. In another embodiment, the dosage is 80 μg/peptide/dose. In another embodiment, the dosage is 100 μg/peptide/dose. In another embodiment, the dosage is 150 μg/peptide/dose. In another embodiment, the dosage is 200 μg/peptide/dose. In another embodiment, the dosage is 300 μg/peptide/dose. In another embodiment, the dosage is 400 μg/peptide/dose. In another embodiment, the dosage is 600 μg/peptide/dose. In another embodiment, the dosage is 800 μg/peptide/dose. In another embodiment, the dosage is 1000 μg/peptide/dose. In another embodiment, the dosage is 1500 μg/peptide/dose. In another embodiment, the dosage is 2000 μg/peptide/dose.
[0240] In another embodiment, the dosage is 10-20 μg/peptide/dose. In another embodiment, the dosage is 20-30 μg/peptide/dose. In another embodiment, the dosage is 20- 40 μg/peptide/dose. In another embodiment, the dosage is 30-60 μg/peptide/dose. In another embodiment, the dosage is 40-80 μg/peptide/dose. In another embodiment, the dosage is 50- 100 μg/peptide/dose. In another embodiment, the dosage is 50-150 μg/peptide/dose. In another embodiment, the dosage is 100-200 μg/peptide/dose. In another embodiment, the dosage is 200-300 μg/peptide/dose. In another embodiment, the dosage is 300- 400 μg/peptide/dose. In another embodiment, the dosage is 400-600 μg/peptide/dose. In another embodiment, the dosage is 500-800 μg/peptide/dose. In another embodiment, the dosage is 800-1000 μg/peptide/dose. In another embodiment, the dosage is 1000-1500 μg/peptide/dose. In another embodiment, the dosage is 1500-2000 μg/peptide/dose. [0241] In another embodiment, the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts.
[0242] Each of the above doses represents a separate embodiment of the present invention.
[0243] Various embodiments of dosage ranges are contemplated by this invention. In another embodiment, the dosage is 20 mg per peptide per day.In another embodiment, the dosage is 10 mg/peptide/day. In another embodiment, the dosage is 30 mg/peptide/day. In another embodiment, the dosage is 40 mg/peptide/day. In another embodiment, the dosage is 60 mg/peptide/day. In another embodiment, the dosage is 80 mg/peptide/day.In another embodiment, the dosage is 100 mg/peptide/day. In another embodiment, the dosage is 150 mg/peptide/day. In another embodiment, the dosage is 200 mg/peptide/day. In another embodiment, the dosage is 300 mg/peptide/day. In another embodiment, the dosage is 400 mg/peptide/day. In another embodiment, the dosage is 600 mg/peptide/day. In another embodiment, the dosage is 800 mg/peptide/day. In another embodiment, the dosage is 1000 mg/peptide/day. [0244] In another embodiment, the dosage is 10 mg/peptide/dose. In another embodiment, the dosage is 30 mg/peptide/dose. In another embodiment, the dosage is 40 mg/peptide/dose. In another embodiment, the dosage is 60 mg/peptide/dose. In another embodiment, the dosage is 80 mg/peptide/dose. In another embodiment, the dosage is 100 mg/peptide/dose. In another embodiment, the dosage is 150 mg/peptide/dose. In another embodiment, the dosage is 200 mg/peptide/dose. In another embodiment, the dosage is 300 mg/peptide/dose. In another embodiment, the dosage is 400 mg/peptide/dose. In another embodiment, the dosage is 600 mg/peptide/dose. In another embodiment, the dosage is 800 mg/peptide/dose. In another embodiment, the dosage is 1000 mg/peptide/dose.
[0245] In another embodiment, the dosage is 10-20 mg/peptide/dose. In another embodiment, the dosage is 20-30 mg/peptide/dose. In another embodiment, the dosage is 20- 40 mg/peptide/dose. In another embodiment, the dosage is 30-60 mg/peptide/dose. In another embodiment, the dosage is 40-80 mg/peptide/dose. In another embodiment, the dosage is 50- 100 mg/peptide/dose. In another embodiment, the dosage is 50-150 mg/peptide/dose.In another embodiment, the dosage is 100-200 mg/peptide/dose. In another embodiment, the dosage is 200-300 mg/peptide/dose. In another embodiment, the dosage is 300-400 mg/peptide/dose. In another embodiment, the dosage is 400-600 mg/peptide/dose. In another embodiment, the dosage is 500-800 mg/peptide/dose. In another embodiment, the dosage is 800-1000 mg/peptide/dose.
[0246] In another embodiment, the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts. [0247] Each of the above doses represents a separate embodiment of the present invention.
[0248] In another embodiment, the present invention provides a kit comprising a peptide, composition or vaccine of the present invention. In another embodiment, the kit further comprises a label or packaging insert. In another embodiment, the kit is used for detecting a WT-1 -specific CD4 response through the use of a delayed- type hypersensitivity test. In another embodiment, the kit is used for any other method enumerated herein. In another embodiment, the kit is used for any other method known in the art. Each possibility represents a separate embodiment of the present invention. [0249] Among those antigens uniquely or differentially expressed by malignant cells,
WT-1 is considered one of the most promising (47). However, the number of immunogenic WT-1 peptide antigens previously identified and reported is very limited, and largely confined to a set of peptides presented by the HLA alleles A0201, A2402 and DRB 10401. As will be seen from the examples presented below, using a pool of overlapping 15-mer peptides spanning the amino acid sequence of WT-1 loaded on autologous APCs for sensitization, WT-1 peptide-specific IFNy-f- CD4+ and CD8 T-cell responses were generated from the blood of 41/56 (78%) normal donors, and thereafter the epitopes eliciting these responses and their presenting HLA alleles were identified. Of the 42 WT-1 peptide antigens described, all but one have not been heretofore identified. The new immunogenic peptides identified include 36 peptides presented by class I HLA alleles and 5 presented by class II HLA alleles. Of the peptides presented by class I HLA alleles, 10 nonamer epitopes were identified which could be presented by from 2-4 different HLA alleles. Also identified, within 4 pentadecapeptides, were overlapping 11-mer and nonamer sequences that co-induced distinguishable CD4+ IFNy-f- and CD8+ IFNy+T-cells. Whether and to what degree epitopes that can be presented by more than one allele can elicit enhanced WT-1 specific responses in individuals inheriting both presenting HLA alleles or both the class I and class II presenting HLA alleles in those instances in which overlapping sequences are contained in the same 15- mer is readily determinable ;ho we ver, inclusion of such peptides in WT-1 vaccines could significantly broaden their applicability particularly among patients not inheriting HLA- A0201 or A2402.
[0250] As shown in the examples, those peptides presented by class I HLA alleles elicited IFNy+ CD8+ T-cells that were able to lyse peptide loaded autologous APCs as well as allogeneic APCs sharing the T-cells' restricting HLA allele in 50/51 (99%) and 48/51 (94%)cultures tested respectively (Table 1, 2). More importantly, of 36 HLA-restricted WT-1 peptide specific T-cell lines that could be tested, T-cell lines specific for 29 epitopes including 2/4 epitopes presented by class II and 27/32 presented by class I alleles, were also able to lyse WT-1+ leukemic blasts sharing the T-cells' restricting HLA allele. The failure of the HLA-restricted WT-1 epitope-specific T-cells to lyse allogeneic PHA blasts from the same leukemic patients (Table 3A), coupled with the differential leukemocidal activity of T- cells sensitized with WT-1 peptide-loaded autologous EBVBLCL when compared to aliquots of the same T-cells sensitized with autologous EBVBLCL alone (Table 3B) indicates that the leukemocidal activity is WT- 1 peptide-specific and not a result of contaminating alloreactive T-cells. Thus, these data show that 29/36 immunogenic peptides of WT-1 identified (80%) can be processed and presented by WT-1+ leukemic cells at concentrations adequate for WT- 1 epitope-specific T-cell recognition and cytolysis.
[0251] In Figure 4, maps are shown of the WT-1 protein. Fig. 4C defines the localization of all previously reported antigenic epitopes presented by HLA class I and II alleles; Fig 4D depicts the location of immunogenic peptides identified in this report. As can be seen, the 11 epitopes previously reported to be presented by class I and 10 presented by class II HLA alleles are principally clustered in sequences encoded by exons 1, 7 and 10, while the epitopes recognized by normal T-cells sensitized with the WT-1 peptide pool are principally clustered in sequences encoded by the first 5 exons. Thus, 26 of the new epitopes are included in each of the four major isoforms of WT-1 resulting from splice variants that do or do not include the 17 amino acid sequence (aas 250-266) in exon 5 or the three amino acid sequence (400-410KTS) between zinc fingers 3 and 4. While the epitopes are broadly distributed, clusters of epitopes were detected in the RNA recognition domain in exon 1 and the activation domain (aa 181-250) (Fig. 4F) proximal to the spliced 17aa segment in exon 5. The latter area also contained those epitopes most frequently recognized by multiple donors (Fig. 4E). Interestingly, 9 newly identified epitopes map to a 126 amino acid sequence at the N terminus encoded by a segment of the WT-1 gene initially described by Gessler et al (37) that is centromeric to exon 1 of the (Exon 5+, KTS+) isoform of WT-1 and includes the long isoform of WT-1 initiated at a CUG codon upstream of the AUG initiator for exon 1.50 Strikingly, each of the epitopes identified in this sequence elicits IFNy+T-cells that are cytolytic against leukemic blasts coexpressing WT-1 and the T-cells' restricting HLA allele. [0252] Of the several "self proteins such as WT-1, NY-ESO-1, HER2/neu, MAGE, and others, differentially expressed by specific tumors, only WT-1 and MART-1 have been shown to elicit responses in normal donors (31 ,32,51-54). In contrast, T-cells specific for each of these proteins have been recorded in a proportion of patients with tumors overexpressing them (55). In particular, T-cells specific for the RMF and CMT peptides of WT-1 have been detected in patients with leukemias, myeloma, carcinoma of the breast and prostate and other solid tumors (31, 32, 56-61). Responses to several of the WT-1 epitopes identified in the present study in 50-60% of patients with ovarian cancer have been documented. Given the high number of potentially immunogenic epitopes in proteins such as NY-ESO-1 and HER2/neu that have elicited responses in tumor-bearing hosts (62), the number of immunogenic WT-1 peptides we have identified is not sufficiently different to account for the differential presence of WT-1 responses in normal donors. Furthermore, Pospori et al (63) have shown that HSCs expressing a transduced TCR specific for a WT-1 peptide presented by HLA-A0201 are not deleted in the thymus of HLA-A0201 transgenic mice and generate functional memory T-cells. However, while the basis for this lack of "self tolerance is unclear, the studies of Rezvani et al (31) and data herein (Fig. 1A) indicate that the frequencies of WT- 1 specific T-cells in the blood of healthy donors is low. In part, this may reflect the low levels and limited tissue distribution of WT-1 expression in normal individuals (18-20). Recently, Rezvani et al (64) also demonstrated declining T-cell responses to WT-1 in patients repeatedly vaccinated with WT-1 peptides, suggesting that these responses are highly regulated. Lehe et al (65) have also recently shown that sensitization of T-cells with a WT-1 peptide presented by DRB 10402 in the presence of high concentrations of IL-2 preferentially stimulates the generation of CD25+ FOX P3+ GITR+ CD127- regulatory T-cells capable of inhibiting CD8+ WT-1 specific T-cell responses. [0253] Under the culture conditions employed herein, autologous DCs and
EBVBLCL loaded with the WT-1 peptide pool preferentially induced the generation of CD8+ and CD4+ IFNy+ WT-1 peptide- specific T-cells from 41/56 normal donors (73%). Although each donor recognized only 1-3 epitopes of WT-1 , the fact that T-cells specific for 80% of these epitopes could recognize WT-1+ leukemic cells sharing the T-cells' presenting HLA allele suggests that the turnover and processing of the aberrantly expressed WT-1 is high, permitting the simultaneous presentation of several different WT-1 epitopes by the restricting HLA allele expressed by these leukemic cells. Identification of these epitopes is useful both for in vitro generation of potent tumoricidal WT- 1 specific T-cells for adoptive cell therapies and for the generation of more broadly applicable vaccines for stimulating T-cell responses for eradication of clonogenic tumor cells expressing WT-1 in vivo.
[0254] In one embodiment, peptides from the WT-1 protein sequence that are upstream from exon 1, i.e., within the first 126 amino acids of SEQ ID NO: 194, are heretofore unrecognized sites of immunogenic epitopes and therefore peptides useful for the purposes herein.
EXAMPLE 1
BINDING OF HLA-A0201 AND -A0301 BY SYNTHETIC
PEPTIDE ANALOGUES DERIVED FROM WT-1
[0255] Materials and Experimental Methods.Peptides were synthesized by
Genemed Synthesis Inc, CA using fluorenylmethoxycarbonyl chemistry and solid phase synthesis, and were purified by high pressure liquid chromatography (HPLC). The quality of the peptides was assessed by HPLC analysis, and the expected molecular weight was measured using matrix-assisted laser desorption mass spectrometry. Peptides were sterile and > 90% pure. The peptides were dissolved in DMSO and diluted in PBS at pH 7.4 or saline solution to yield a concentration of 5 milligrams per milliliter (mg/ml) and were stored at - 80° C. For in vitro experiments, an irrelevant control peptide, HLA A24 consensus, was used.
[0256] Peptide sequence analysis. Peptide sequence analysis was performed using 2 databases. The first was the software of the Bioinformatics & Molecular Analysis Section (National Institutes of Health, Washington, DC) (Parker KC et al, Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side- chains. J Immunol 152: 163-175, 1994), which ranks 9-mer or 10-mer peptides on a predicted half-time dissociation coefficient from HLA class I molecules. The second database, SYFPEITHI prediction software, is described in Rammensee HG et al (SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213-219, 1999). Irrelevant control peptides used in in vitro experiments were: RAS (TEYKLVVVGAPGVGKSALTIQ; SEQ ID No: 49) or CML b2a2 (VHSIPLTINKEEALQRPVASDFE; SEQ ID No: 50) for Class II, and HIV pol (ILKEPVHGV; SEQ ID No: 51) or CML F (YLKALQRPY; SEQ ID No: 52) for Class I. [0257] Cell lines. Cell lines were cultured in RPMI 1640 medium supplemented with
5% FCS, penicillin, streptomycin, 2mM glutamine and 2-mercaptoethanol at 37 °C in humidified air containing 5% C02. T2 is a human cell line lacking TAPl and TAP2 and therefore unable to present peptides derived from cytosolic proteins. Raji cells are a human Burkitt lymphoma cells that exhibit a high level of TAP expression.
[0258] Human mesothelioma cell lines studied included: sarcomatoid (VAMT, H2373, H28), epithelioid (H2452) and biphasic (JMN, MSTO and H-MesolA). Cell lines were obtained from the following sources: H-MesolA: NCI, Bethesda, MD; JMN and VAMT: Dr. Sirotnak, Memorial Sloan Kettering Cancer Center (MSKCC); H-2452 and H2373: Dr. Pass, Karmanos Cancer Institute, Wayne State University, Detroit, MI; H28 and MSTO: American Type Culture Collection (ATCC, Manassas, VA). Cell lines were maintained in media recommended by the suppliers and incubated in a humidified incubator with 5% C02.
[0259] Mesothelioma cell lines Meso 11, Meso 34, Meso 37, Meso 47 and Meso 56 were obtained from Dr. M Gregoire (Institute of Biology, Nantes, France) and cultured in RPMI 1640 (Life Technologies) + 10% fetal calf serum (FCS), 1% penicillin-streptomycin, and 1 % L-glutamine. All cells were HLA typed by the Department of Cellular Immunology at MSKCC. Melanoma cell line Mewo (WT-1- A201+) was obtained from the ATCC. SKRC-52 renal cell carcinoma was obtained from L. Old of the Ludwig Institute. Leukemia cell lines were cultured in RPMI 1640 + 10% FCS, 1 % penicillin-streptomycin, 2mM glutamine and 2-mercaptoethanol at 37oC/5% C02. LAMA81, BV173 and 697, Ph+ leukemias that are all WT-1+ and A0201+, were provided by Dr. HJ Stauss (University
College London).SKLY-16 is a human B cell lymphoma (WT-1-, A0201+); K562, RwLeu4 and HL60, all WT-1+ leukemias, were obtained from the ATCC.
[0260] T2 assay for peptide binding and stabilization of HLA A0201 molecules. T2 cells (TAP-, HLA-A0201+) were incubated overnight at 27° C at a concentration of 1 x 106 cells/ml in FCS-free RPMI medium supplemented with 5 μg/ml human (Sigma, St Louis, MO) in the absence (negative control) or presence of either a positive reference tyrosinase peptide or test peptides at various final concentrations (50, 10, 1, and 0.1 micrograms ^g)/ml). Following a 4-hour incubation with 5 μg/ml brefeldin A (Sigma), T2 cells were labeled for 30 minutes at 4 °C with a saturating concentration of anti-HLA- A2.1 (BB7.2) mAb, then washed twice. Cells were then incubated for 30 minutes, 4° C with a saturating concentration of FITC-conjugated goat IgG F(ab')2 anti-mouse Ig (Caltag, San Francisco, CA), washed twice, fixed in PBS/1% paraformaldehyde and analyzed using a FACS Calibur® cytofluorometer (Becton Dickinson, Immunocytometry Systems, San Jose, CA).
[0261] The mean intensity of fluorescence (MIF) observed for each peptide concentration (after dividing by the MIF in the absence of peptide) was used as an indication of peptide binding and expressed as a "fluorescence index." Stabilization assays were performed similarly. Following initial evaluation of peptide binding at time 0, cells were washed in RPMI complete medium to remove free peptides and incubated in the continuous presence of 0.5 μg/ml brefeldin-A for 2, 4, 6 or 8 hours.
[0262] The number of stable peptide-HLA- A2.1 complexes was estimated as described above by immunofluorescence. The half time of complexes is an estimate of the time required for a 50% reduction of the MIF value at time = 0.
[0263] WT-1 peptides.The sequence of the WT-1 protein published by Gessler et al.
(37) which comprises 575 aminoacids and includes the first 126 aminoacids in the N- terminus missing in the (Exon 5+, KTS+) isoform of WT-116, was used to design the peptide sequences (SEQ ID NO: l ; Figure 2A). 141 pentadecapeptides spanning this sequence, each overlapping the next by l laa, were synthesized by Invitrogen (Baltimore, MD) to specifications of validated sequence, 95% purity, sterility and absence of endotoxin. These 141 15-mers were mixed in equal amounts to form a total pool of peptides, in which each peptide is at a concentration of 0.35mcg/ml. This pool was used for the T-cell sensitization. To identify peptides eliciting responses, subpools containing 12 pentadecapeptides (4.17mcg/ml/peptide) were established to form a mapping matrix in which each peptide is included in only two overlapping subpools (Figure 2B).
[0264] Generation of WT-1 Specific T-cells:Peripheral blood was obtained from 56 consenting normal donors according to protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (New York, NY). All donors were typed for HLA-A, B, C, DR and DQ at high resolution by standard techniques.
[0265] Cytokine-activated monocytes (CAMs) were used as antigen presenting cells
(APCs), and generated as previously described (32). Briefly, peripheral blood monocytes were separated by adherence on plastic and cultured in RPMI1640 containing 1 % autologous serum. GM-CSF (Berlex, Montville, NJ) and interleukin-4 (IL-4) (R&D Systems, Minneapolis, MN) were added to final concentrations of 2000U/ml and lOOOU/ml respectively on days 0, 2, 4. On day 5, these cells were additionally treated with TNFa(10ng/ml), interleukin-6 (IL-6) (lOOOIU/ml), Ιίΐβ (400IU/ml), PGE2 (25mM-3) (R&D Systems, Minneapolis, MN) together with GM-CSF and IL-4 at the same doses. CAMs harvested on day 7 of culture expressed CD83, CD80, CD86, and HLA class I and II alleles as determined by FACS analysis. [0266] EBV-BLCL were also used as WT-1 peptide loaded and control APCs or as targets as specified in the experiments. They were generated by infection of peripheral blood mononuclear cells (PBMC) with EBV strain B95.8 (38,39) as previously described. The EBV transformed BLCL (EBV-BLCL) were cultured in RPMI1640(Gemini) with 10% fetal calf serum (Gemini) in the presence of Acyclovir. [0267] Sensitization and propagation of WT-1 specific T-cells. To generate WT-1- specific CTLs, PBMC were isolated by Ficoll-Hypaque density gradient centrifugation. Monocytes were depleted by adherence on plastic and NK cells by absorption to immunomagnetic CD56 pre-coated microbeads (Miltenyi Biotech Inc, MA) as previously described (32). Enriched T-cell fractions were stimulated at a 20: 1 responder: stimulator ratio with autologous CAMs or EBV-BLCL that had been pre-loaded for 3 hours with the total pool of the WT-1 pentadecapeptides in serum- free medium and irradiated to 3000cGy. T- cells were cultured in Yssel's medium supplemented with 5% AB human serum (YH5, Gemini), re-stimulated weekly with the autologous WT-1 total pool-loaded CAMs or EBV- BLCL and fed with interleukin-2(IL-2) (Collaborative Biomedical Products, Bedford, MA) every 2-3 days at 10-50U/ml.
[0268] Cell Targets - leukemic cells:Twenty-four primary leukemic cells and 1 leukemic cell line were characterized for their expression of WT-1 by intracellular FACS staining using murine anti-human WT-1 monoclonal antibodies (Neomarkers, Fremont, CA) as previously described (32,38) The WT-1+ leukemias included blast cells from 11 primary AMLs, 3 primary ALLs and 1 B-cell precursor ALL cell line. Ten WT-1- leukemias, were used as controls, and inclOuded 3 B-cell precursor ALLs and 7 AMLs.
[0269] All EBV BLCL and leukemia cells were typed for HLA A, B, C, DR and DQ alleles at high resolution by standard techniques.
Assessment of T-cell response. [0270] IFNy production by WT-1 specific T-cells.The proportion and phenotype (CD4and CD8) of T-cells generating IFNy in response to secondary stimulation with the WT- 1 total pool, WT-1 subpools or single WT-1 15-mer or 9-mer WT-1 peptides loaded on autologous PBMC were measured by FACS analysis of T-cells containing intracellular IFNy as previously described (38,40). [0271] Mapping of immunogenic epitopes. Aliquots of the T-cells stimulated with the WT-1 total pool for 35-42 days were washed and re-stimulated overnight with autologous PBMC loaded with one of each of the subpools of WT-1 pentadecapeptides. T-cell responses to each subpool were quantitated by FACS analysis of T-cells bearing intracellular IFNy as previously described (41). The mapping grid (Figure 2B) was then used to identify specific WT-1 15-mers uniquely shared by 2 subpools eliciting T-cell responses. These 15-mers and 9-mer or 11-mer sequences within the 15-mers were then analyzed as secondary single peptide stimulators to confirm their immunogenicity and define the immunogenic epitope(s) within the 15-mer eliciting responses.
[0272] Cytotoxic activity. The W-l -specific and HLA-restricted cytotoxic activity of sensitized T-cells was measured in standard Cr51 release assays against a panel of HLA- matched and mismatched CAM targets either unmodified or loaded with the total pool, the identified 15-mer, or the 9-mer or 11-mer epitope of WT-1 eliciting T-cell responses, as previously described (32). In addition, the restricting HLA allele presenting each immunogenic epitope was identified by measuring the cytotoxicity of the sensitized T-cells against a panel of allogeneic CAMs pre-loaded with the peptide, each sharing a single HLA allele expressed on the responding WT-l-specific T-cells as previously described (41). The cytotoxic activity of the WT-1 epitope- specific CTLs against WT-1- and WT-1+ leukemia cell lines or primary leukemic cells expressing the restricting HLA alleles was also assessed in this cytotoxicity assay Cr51 assay as previously described (32). [0273] Immunogenicity of the identified immunodominant WT-1 derived epitopes.To estimate the immunogenicity of identified WT-1 peptide epitopes in different individuals, enriched T-cells separated from PBMC of groups of normal donors expressing one of a series of prevalent HLA alleles (i.e. HLA- A0201, A0301 , A2402, B0702) which were previously identified as a presenter of a newly identified WT-1 epitope were sensitized in vitro with artificial antigen-presenting cells (AAPC) (42) expressing that HLA allele and loaded with the pre-identified WT-1 epitope or an irrelevant peptide. The panel of AAPCs includes AAPCs expressing one of the following single HLA alleles: HLA A0201, A0101, A0301 , A2402, B0702 or B0801, which were generated as previously described (42). After 35 days of co-culture of T-cells with the peptide-loaded AAPCs in the presence of IL2, CTLs were secondarily stimulated overnight with autologous PBMC loaded with the sensitizing peptide or an unrelated peptide and tested for their IFNy response. The responses were registered as positive if the proportion of T-cells producing IFNy in response to the secondary stimulation with autologous PBMC loaded with the stimulating WT-1 derived peptide exceeded the background proportion of IFNy T-cells incubated with PBMC alone by two fold or more.
EXAMPLE 2.
Responses of normal donors to the WT-1 total pool of pentadecapeptides.
[0274] Frequencies of WT-1 -specific IFNy+ T-cells in the PBMC of 41 normal donors were measured initially. These frequencies ranged between 0.01% to 1.82%, and exceeded the background of IFNy+ T-cells detected in T-cells stimulated with autologous PBMC alone in only 10/41 individuals (Figure 1A). In vitro sensitization of T-cells from 56 normal donors with autologous CAMs loaded with the total pool of WT-1 pentadecapeptides for periods of 35-42 days resulted in significant expansion of IFNy+ T-cells in 41/56 cases (73%) (Figure 1A). T-cells generated from 38/56 donors also exhibited cytotoxic activity against autologous PHA blasts loaded with the WT-1 total pool (Figure IB), including T-cells from 38 of the 41 donors that produced IFNy in response to secondary stimulation with the WT-1 peptide pool.
[0275] The capacity of one of the previously reported WT-1 epitopes predicted to bind the HLA-A0201 allele, 126-i34RMFPNAPYL (SEQ ID NO:21 ; RMF) (43) were compared with the total pool of WT-1 pentadecapeptides to stimulate WT-1 reactive T-cells in HLA-A0201+ normal donors (n=14) when loaded on autologous CAMs. Increased frequencies of IFNy-f- T-cells initially sensitized with the RMF peptide were detected in 9/14 donors, 7 of whom also responded to secondary simulation with the pooled peptides (Figure 1C). In contrast, 12/14 CTL lines initially sensitized with the WT-1 peptide pool, generated high frequencies ofIFNy+ T-cells after secondary stimulation with the WT-1 total pool, including 6 CTL lines that also responded to RMF. The epitopes of WT- 1 recognized by the T-cells sensitized with the total pool (vide infra) were mapped and epitopes other than RMF in 12/14 donors were identified. The magnitude of the responses to those epitopes was much higher than to the RMF peptide (Figure 1C). Only 4/14 CTL lines initially sensitized with RMF exhibited cytotoxic activity against RMF- loaded autologous PHA blasts; of which 3 could also lyse autologous PHA blasts loaded with the WT-lpool (Figure ID). In contrast, 10/14 CTL sensitized with the pool of WT-1 peptides were cytotoxic against PHA blasts loaded with the WT-1 total pool including 3/14 that lysed RMF peptide loaded blasts (Figure ID). Thus, in a high proportion of HLA A0201+ donors, stimulation of T-cells with the WT- 1 total pool more consistently elicited WT-1 -specific T-cell responses than stimulation with the single HLA A0201 binding RMF peptide.
[0276] Detailed description of Figure l.WT-1 specific responses of CTL generated from PBMC of normal donors (n=56) by stimulation with autologous APCs loaded with total pool of WT-1 derivedpentadecapeptides: A. production of IFNyin PBMC alone (as a background), PBMC co-incubated overnight with the total pool of pentadecapeptides spanning the whole sequence of WT-1 protein (PBMC+WT-1 pool) and pre-generated WT-1 specific T cells co-incubated overnight with WT-1 peptide loaded PBMC; B. cytotoxic activity of the WT-1 specific CTLs generated in vitro by stimulation with WT-1 total pool against WT-1- (autologous PHA stimulated blasts) and WT-l+(autologous PHA stimulated blasts loaded with the total pool of WT-1 pentadecapeptides) targets at 50: 1 effector : stimulator ratio; C. IFNy response measured by FACS staining in different responder cell populations (peripheral blood derived PBMC, pre-generated CTLs sensitized in vitro with the RMF peptide loaded on autologous CAM and pre-generated CTLs sensitized with the total pool of WT-1 15-mers)after secondary overnight stimulation with autologous PBMC either unmodifiedor loaded with one of the following: RMF peptide, dominant epitopes of WT-1 identified by the epitope mapping approach in the WT-1 -total pool sensitized CTL, WT-1 total pool of the 141 pentadecapeptides; D. Cytotoxic activity of the WT specific T cells generated in vitro by sensitization with autologous CAMs loaded with the RMF 9-mer or with the total pool of the WT-1 15-mers. The cytotoxicity of the T cells was assessed against autologous WT-1 negative targets(PHA activated blasts) and the same targets loaded with RMF peptide, the total pool of WT-1 15-mers or the dominant WT-1 epitope identified for the same T cell line. EXAMPLE 3
Identification of immunogenic epitopes of WT-1 protein recognized by the WT-1 -reactive T-cells.
[0277] WT-1 CTLs generated by sensitization with the pooled peptides are epitope specific and HLA restricted. The epitopes recognized by T-cells sensitized in vitro with the total pool of overlapping WT-1 pentadecapeptides (Figure 2A) were identified by quantitatinglFNy-i- T-cells responding to a mapping grid of subpools of WT-1 15-mers formed sothat any single 15-mer is shared by only 2 intersecting subpools (Figure 2B).As shown for a representative example in Figure 2C, significantly increased numbers of IFNy-f- T-cells are selectively generated in response to subpools # 3 and #19 which share the pentadecapeptide #75. The T-cells were then stimulated with neighboring 15-mers, each overlapping peptide #75 by l laa. As can be seen, IFNy+ T-cells are selectively generated in response to peptide #75 (Figure 2D). The newly identified immunogenic WT-1 epitope is 174- 182HSFKHEDPM. Subsequently, the cytotoxic activity of these T-cells was assessed against a panel of allogeneic CAMs either unmodified or loaded with this peptide, each sharing one HLA allele expressed by the tested CTLs. As shown in Figure 2E, the T-cells selectively lysed peptide loaded autologous targets and targets expressing the HLA-B3501 allele, and did not lyse peptide-loaded targets sharing other HLA alleles inherited by the T-cell donor. These T-cells also lysed WT-1+ BALL cells coexpressing the HLA-B3501 allele.
[0278] Detailed description of Figure 2. Strategy for the generation of the total pool of overlapping pentadecapeptides spanning the whole sequence of the WT-1 protein and epitope mapping: A. The sequence of the WT-1 protein consisting of 575 amino acids and the principle of 11 amino acid overlapping pentadecapeptides are illustrated. A total of 141 pentadecapeptides are required to span the entire protein. The sequence of 575 aminoacids published by Gessler et al.(37), was employed. This sequence includes an additional 126 aminoacids in the N-terminus. In order to match the sequentialnumbers of aminoacids within the WT-1 sequence used with the longest, most frequently described WT-1 isoform D we numbered the first 126aa with negative values and used the positive values to number the subsequent 449 aminoacids described in the longest isoform D; B. The mapping grid consisting of 24 subpools each containing up to 12 WT-1 -derived pentadecapeptides. Each peptide is uniquely contained within two intersecting subpools: for example peptide 75 is uniquely shared by subpools 3 and 19; C. IFNy production by WT-1 sensitized CTLs in response to secondary overnight stimulation with the subpools of WT-1 pentadecapeptides loaded on autologous PBMC. Dominant responses are observed for the subpools #3 and #19 both containing one common pentadecapeptide #75; D. IFNy production by the WT-1 CTLs in response to secondary overnight stimulation with the single pentadecapeptide contained within the subpools eliciting the highest responses as per the analysis determined in 2C of this figure confirms that the dominant immunogenic sequence is contained within pentadecapeptide #75 ; E. HLA restriction of the WT- 1 specific T cells responding to peptide #75 identified by Cr51 release assay against a panel of allogeneic CAMs or PHA blasts matching single HLA alleles expressed by the WT-1CTL donors. These are presented along the X axis of the graph. The CAMs or PHA blasts used in the assay are unmodified (grey bars) orloaded with the WT-1 dominant epitope (black bars). The WT-1 specific cytotoxic activity of the WT- 1 CTLs is restricted by the B3501 HLA allele.
[0279] Mapping of WT-1 Peptides Eliciting T-Cell Responses Identifies a
Diversity of Immunogenic Epitopes Presented by Different Class I and II HLA Alleles.
The same approach was used to map and ultimately identify WT-1 epitopes eliciting responses by T-cells from the other 40 responding normal donors. Of these donors, 8 (19%) responded exclusively to one WT-1 peptide, while 18 (43%) responded to two and 16 (39%) to 3 peptides. In cultures eliciting responses to more than one WT- 1 peptide, the patterns of IFNy-f- T-cell responses to the subpools were sufficiently distinctive to permit initial segregation of potentially immunogenic peptides. Each candidate peptide was then evaluated individually to ascertain the specific peptide inducing a T-cell response. [0280] The immunogenic peptides of WT- 1 that were identified and their presenting
HLA alleles are listed in Table 1. Of the 42 WT-1 peptides eliciting T-cell responses, 41 are newly identified; only one of these WT-1 peptides, the 126 134RMFPNAPYL nonamer presented by HLA-A0201 , has been previously described and shown to be immunogenic when presented by this allele (43) Peptide 91, 235-249CMTWNQMNLGATLKG contains an epitope which, in the study, elicited CD4+ T-cell responses restricted by HLA DRB 1 0402, but also contains the 235-243CMT nonamer known to be presented by HLA A0201 and HLA A2402 (29). For 26 of the peptides presented by class I HLA alleles, a single presenting HLA allele was identified in the initially studied donor. However, when the HLA -restrictions of T- cells responding to these peptides in different donors was examined, 10 of these peptides were found that could elicit T-cell responses when presented by 2 or 3 different class I HLA alleles. One sequence, the 238-246WNQMNLGAT peptide, elicited strong IFNy+ CD8+ T-cell responses when presented in different donors by any one of 4 distinct HLA class I alleles. [0281] Table 1. WT-1 derived immunogenic epitopes identified by IFNy production assay for T cells responses using pool of overlapping pentadecapeptides spanning the whole sequence of WT-1 protein. Shaded rows represent peptides that can be presented by more than one HLA allele. Bolded peptide sequences represent those tested in Example 5 and results shown in Table 3.
Figure imgf000077_0002
Figure imgf000077_0003
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
*- the epitope previously predicted by the computer algorithm or described in the literature
**- T-cells cytotoxic against autologous WT-1 peptide loaded APCs but not leukemia cells
***- assignment of HLA restriction to one or other allele cannot be made due to lack of targets inheriting one allele without the other
[0282] Using this epitope mapping strategy, 5 new 11-mer peptides were identified that stimulated CD4+ T-cell responses restricted by HLA class II alleles. The CD4+ T-cells generated in response to each of these epitopes expressed high levels of IFNy-f- T-cells. The CD4+ T-cells responding to 3 of these 5 peptide epitopes also exhibited specific cytotoxic activity against peptide loaded PHA blasts as well as unmodified WT-1+ leukemic blasts selectively sharing the restricting class II HLA allele.
[0283] In 4 of the 56 donors tested, epitope mapping of T-cells sensitized with the complete pool of WT-1 15-mers identified specific 15-mers eliciting both CD4+ and CD8+ T-cell responses (15-mer peptides # 20, 41 , 91, 112). Fine mapping of the sequences eliciting these responses identified four 11 -mers that stimulated HLA class Il-restricted CD4+ T-cell responses which also contained, within their sequences, 9-mers that elicited HLA class I- restricted CD8+ T-cell responses. A representative example of one of these dual stimulating peptides is presented in Figure 3. In this case, peptide 41 was found to elicit both CD4+ and CD8+ IFNy-f- T-cell responses (Figure 3A). Fine mapping of the 11 -mers within peptide 41 eliciting the CD4+ IFNy+ T-cell response (Figure 3A) suggested the 38^8LDFAPPGASAY peptide as the most immunogenic sequence inducing both CD4+ and CD8+ IFNy+ T-cell responses. Strikingly, the peptide 41 sensitized T-cells lysed PHA blasts sensitized with either the 9aa sequence (38_46LDFAPPGAS) or the 1 laa sequence (38_48LDFAPPGASAY), but did not lyse PHA blasts loaded with the
Figure imgf000080_0001
or 37-4?VLDFAPPGASA 11- mers. Subsequent examination of the HLA restriction of the T-cells in the culture (Figure 3D) revealed that the class II HLA -restricted T-cells were selectively cytotoxic against targets sharing the alleles DRB1 0402 and DQB 1 0302 only when loaded with the LDF 11-mer, while the T-cells restricted by HLA A0201 were able to lyse targets loaded with either the 11-mer or the 9-mer LDF peptide. In this case, it was not possible to ascertain whether DRB 1 0402 or DQB 1 0302 was the restricting class II HLA allele because cells were not available in the panel expressing one without the other.
[0284] Detailed description of Figure 3. HLA class I and II restricted WT-1 specific T cell respond to the same immunodominant peptide 15-mer derived from WT-1 protein in the WT-1 CTL sensitized with the WT-1 total pool of overlapping 15-mers loaded on autologous CAMs . A. Production of IFNg by the CD8+ and CD4+WT-1 specific T cells in response to secondary overnight stimulation with the same dominant WT-1 derived 15-mer #41 ; B. Identification of the immunogenic sequence of aminoacids within pentadecapeptide #41 by IFNg production after secondary overnight stimulation with autologous PBMC loaded with a panel of 9-mers either unique for the peptide #41 (LDF- LDFAAPGAS) or contained within the neighboring overlapping 15-mer #40 (PVL - PVLDFAPPG, VLD - VLDFAPPGA) and #42 (DFA - DFAPPGASA). Only the 9-mer uniquely presented within the 15-mer #41, LDF, elicits an IFNg response; C. Peptide-specific cytotoxic activity of WT-1 CTL against the panel of 9-mers and 11-mers contained within peptide #41 and loaded on autologous PHA stimulated blasts is observed against both the 11-mer LDF and 9-mer LDF contained within the 11-mer LDF as determined in a standard Cr51 release assay at 25: 1 E:T ratio; D. HLA restriction of the cytotoxic activity of the WT-1 CTL: T-cells restricted by HLA-A0201 lyse targets loaded with either the 11-mer or the 9-mer, while those restricted by HLA DRB 10402 only lysed targets loaded with the 11-mer.
EXAMPLE 4
T-cells generated against newly identified WT-1 epitopes exhibit cytotoxic activity against WT-1+ leukemias.
[0285] Once the WT-1 peptide specificity was established and HLA restrictions of the IFNy+ T-cells responding to the pool of WT-1 peptides, their cytotoxic activity was examined against unmodified and peptide loaded autologous PHA blasts and against a series of allogeneic PHA blasts loaded with the identified peptides as well as primary acute leukemic cell blasts expressing WT-1 protein that coexpressed the WT-1 specific T-cells' restricting HLA allele. For the latter tests, WT-1+ leukemic cells not expressing the restricting allele and WT-1- cells sharing the restricting allele served as controls. Results are summarized in Tables 1 and 2.
[0286] As can be seen in Table 1 , of 51 cultures generating IFNy-f- CD8+ T-cells after secondary stimulation with an identified peptide loaded autologous APC, 50 also exhibited significant specific cytotoxic activity against autologous PHA blasts loaded with the targeted peptide. Of these, 48 also lysed allogeneic peptide loaded PHA blasts or DCs sharing the restricting HLA allele of the responding T-cells. CD4+ IFNy-f- T-cells responding to 3/5 identified 11-mer peptides presented by class II HLA alleles also lysed peptide loaded autologous and HLA-sharing allogeneic class 11+ targets.
[0287] Of the T-cell cultures exhibiting epitope-specific cytotoxic activity against peptide loaded targets, 36 could be tested for cytotoxic activity against WT-1+ leukemic cells coexpressing the T-cell' s restricting HLA allele. Of these 36, 27 exhibited HLA-restricted cytotoxic activity against the WT-1+ leukemic cells (Table 2). T-cells specific for five peptides, 6-15RDL, 46-54SAY, 58_66PAP, 225-233NLY, and 436-445NMH, presented by HLA A0201 , could not lyse HLA-A0201+WT-1+ leukemic cells. However, HLA B4001 restricted T-cells specific for the 46-54SAY peptide, could lyse WT-1+ leukemic coexpressing this HLA allele. Similarly, NMH peptide-specific HLA-restricted T-cell lines that lysed targets loaded with the NMH peptide coexpressing HLA A0201, B4001 or A2402 were only able to lyse WT-1+ leukemic cells expressing the HLA B4001 allele.
[0288] Table 2.WT-1 derived immunogenic epitopes identified by IFNy production assay for T cells responses using pool of overlapping pentadecapeptides spanning the whole sequence of WT-1 protein.Bold sequences indicate peptides tested as described in Example 5 and results provided in Table 3.
Figure imgf000082_0001
Presenting Sequence identified Prediction algorithm Cytotoxic CTL response, % (at 50: 1)E:T ratio vs
HLA allele
Binding Dis- WT-1- WT-l+allo WT-1- WT-1+ index associa- allo APC APC with leukemia leukemia tion with restricting
time restrictHLA allele
ing HLA loaded with
allele WT-1 peptide
PLPHFPPSL
SEQIDNO:144
(-99M-91) 3 0 1 21 0 38
THSPTHPP
SEQIDNO:146
(-75)-(-67) 19 0.272 3 17 3 19
AILDFLLLQ
SEQIDNO:147
(-47)-(-39) 0 0 7 27 5 19
PGCLQQPEQ
SEQIDNO:148
(-27M-19) 19 17 2 22 10 37
KLGAAEASA
SEQIDNO:150
6-15 18 0.2 4 31 0 9
RDLNALLPAV
SEQIDNO:152
22-31 13 0.003 3 25 3 47
GGCALPVSGA
SEQIDNO:153
38-46 11 0 1 62 0 40
LDFAPPGAS
SEQIDNO:155
46-54 14 0 5 31 0 0
SAYGSLGGP**
SEQIDNO:157
58-66 5 0 1 18 0 0
PAPPPPPPP**
SEQIDNO:158
126-134 22 313 1 52 2 25
RMFPNAPYL*
SEQIDNO:161
225-233 23 68 3 28 0 0
NLYQMTSQLE**
SEQIDNO:170
238-246 19 0.3 0 21 1 19
WNQMNLGAT
SEQIDNO:171
242-250 24 160 1 14 2 19
NLGATLKGV
SEQIDNO:175
390-398 11 0.054 1 27 ND ND
PvKFSPvSDHL
SEQIDNO:181
398-406 5 0.18 1 22 ND ND
LKTHTTRTHT
SEQIDNO:182
436-445 20 15 4 32 0 0
NMHQRNHTKL* * Presenting Sequence identified Prediction algorithm Cytotoxic CTL response, % (at 50: 1)E:T ratio vs HLA allele
Binding Dis- WT-1- WT-l+allo WT-1- WT-1+ index associa- allo APC APC with leukemia leukemia tion with restricting
time restrictHLA allele
ing HLA loaded with
allele WT-1 peptide
SEQIDNO:183
A0203 243-252 19 NA 0 21 ND ND
LGATLKGVAA SEQIDNO:176
A2402 239-248 10 7.2 0 2 1 17
NQMNLGATL
SEQIDNO:172
436-445 13 0.6 13 27 0 0
NMHQRNHTKL* *
SEQIDNO:183
A6901 246-253 NA NA 0 57 ND ND
TLGVAAGS
SEQIDNO:177
B0702 (-125H-117) 15 40 1 53 1 67
Q PHPGAL SEQIDNO:142
(-119M-111) 2 0.3 5 22 1 60 GALRNPTAC
SEQIDNO:143
A3101 (-107)-(-99) NA 0.01 0 27 ND ND
HFPPSLPPT
SEQIDNO:145
B3501 (-8)-(-l) NA 15 3 51 5 39
ASGSEPQQM
SEQIDNO:151
135-143 NA 0.075 0 21 ND ND
PSCLESQPA
SEQIDNO:162
174-182 NA 10 3 63 5 45
HSFKHEDPM
SEQIDNO:165
269-278 NA 0.004 0 23 ND ND
GYESDNHTT
SEQIDNO:178
323-332 NA 0.01 0 61 5 45
FMCAYPGCNK SEQIDNO:179
B3503 122-130 NA NA 3 41 ND ND
SGQARMFPN
SEQIDNO:160
218-226 NA NA 3 31 4 48
RTPYSSDNL
SEQIDNO:169
B3508 238-246 NA NA 2 21 4 19
WNQMNLGAT SEQIDNO:171
B3802 206-214 NA NA 1 53 ND ND
TDSCTGSQA
SEQIDNO:168 Presenting Sequence identified Prediction algorithm Cytotoxic CTL response, % (at 50: 1)E:T ratio vs HLA allele
Binding Dis- WT-1- WT-l+allo WT-1- WT-1+ index associa- allo APC APC with leukemia leukemia tion with restricting
time restrictHLA allele
ing HLA loaded with
allele WT-1 peptide
B3801 166-174 11 0.3 1 17 ND ND
HHAAQFPNH
SEQIDNO:164
B3901 30-38 12 3 0 19 ND ND
GAAQWAPVL SEQIDNO:154
B4001 (-99)-(-91) 3 0.02 0 31 3 65
THSPTHPPR
SEQIDNO:146
46-54 1 0.002 8 24 3 68
SAYGSLGGP SEQIDNO:157
436-445 1 0.002 1 26 3 72
NMHQRNHTKL SEQIDNO:183
B4402 202-210 3 NA 7 19 ND ND
CHTPTDSCT
SEQIDNO:166
206-214 2 NA 0 88 1 56
TDSCTGSQA
SEQIDNO:168
106-114 4 NA 7 23 ND ND
ACRYGPFGP
SEQIDNO:159
B4701 (-47M-37) 1 NA 1 25 ND ND
PGCLQQPEQ
SEQIDNO:148
B5701 6-15 NA NA 1 22 ND ND
RDLNALLPAV SEQIDNO:152
C0401 122-130 NA NA 3 41 ND ND
SGQARMFPN
SEQIDNO:160
C1701 238-246 NA NA 0 7 1 16
WNQMNLGAT SEQIDNO:171
D BiOlOl (-47)-(-37) 8 NA 1 25 ND ND
PGCLQQPEQQG
SEQIDNO:149
D B!0402 38-48 NA NA 1 71 0 40
LDFAPPGASAY SEQIDNO:156
D B!0402 235-249 NA NA 2 15 1 17
CMTWNQMNLGA TLKG
SEQIDNO:174
DRBitMOl 320-334 22 NA 3 0 5 5
KRPFMCAYPGC SEQIDNO:180
Figure imgf000086_0001
*- previously reported epitopes;
** - T cells cytotoxic against the autologous WT-1 peptide loaded APC but not the leukemic cells. [0289] To ascertain that the cytotoxic activity of the WT-1 peptide-specific T-cells observed against allogeneic WT-1+ leukemic cells sharing the T-cells restricting allele does not reflect the presence of alloresponsive T-cells in the T-cell lines, we tested the cytotoxic activity of 13 of these HLA-restricted WT-1 peptide specific T-cell lines against WT-1+ leukemic cells and WT-1- PHA blasts cultured from the same leukemic patient. As shown in Table 3a, the WT-1 specific T-cells lysed the WT-1+ leukemic cells but not PHA blasts from the same patient.
[0290] Table 3a. Cytotoxic activity of the T cells specific for WT-1 derivedimmunogenic epitopes identified by IFNy production assay for T cells responses using pool of overlapping pentadecapeptides spanning the whole sequence of WT-1 protein and tested against WT-1 positive primary leukemic cellsand PHA blasts of the same origin.
Figure imgf000086_0002
#24-25 (-27M-19) KLGAAEASA A0201 37 8
SEQ ID NO:150
#29-30 (.8)-(i) ASGSEPQQM B3501 39 9
SEQ ID NO:151
#37 22-31GGCALPVSGA A0201 47 6
SEQ ID NO:153
#43 46-54SAYGSLGGP* B4001 68 3
SEQ ID NO:157
#62-63 126.134RMFPNAPYL* A0201 25 3
SEQ ID NO:161
#86 218-226RTPYSSDNL B3503 48 1
SEQ ID NO: 169
C0401 48 1
#141 436-445NMHQRNHTKL* B4001 72 1
SEQ ID NO: 183
P<0.001
*- the epitope previously predicted by the computer algorithm or described in the literature ** - leukemia samples were presented either by immortalized leukemia cell lines or by
primary leukemia cells obtained from patients with WT-1+ leukemia
*** - PHA blasts were generated from PBMC derived from the same patients as the WT-1+ primary leukemia
[0291] PHA blasts were not available from every patient that provided leukemia blasts for this study. Nevertheless, these results provide evidence that the cytotoxicity of the WT-1 specific T-cells is not ascribable to contaminating alloreactivity. A second, more inclusive, but less direct line of evidence is provided by a paired comparison of the responses of T-cells derived from 35 of the donors that had been contemporaneously sensitized in vitro against either WT-1 peptide pool loaded or unmodified autologous EBVBLCL, against these primary leukemias. As shown in Table 3b, T-cells sensitized with the WT-1 peptide pool- loaded EBVBLCL lysed WT-1+ leukemic cells sharing the T-cells' restricting HLA allele in 25 of 35 cases. In contrast, T-cells sensitized with autologous EBVBLCL alone consistently failed to lyse the same WT-1+ leukemia targets.
[0292] Table 3b.Leukemocidal activity of defined epitope-specific and HLA restricted T cells from normal donors sensitized with either autologous EBV BLCL or EBV BLCL loaded with pooled WT-1 peptides against primary WT-1+ leukemia sharing the T cells restricting HLA alleles.
Figure imgf000087_0001
Figure imgf000088_0001
KG
SEQ ID NO: 174
#92 242-25oNLGATLKGV A0201 19 2
SEQ ID NO: 175
#99-100 269-27sGYESDNHTT A0101 33 0
SEQ ID NO: 178
#112-113 323.332EMCAYPGCNK B3501 45 5
SEQ ID NO: 179
320-334KRPFMCAYPGC DRB^ 5 5
SEQ ID NO: 180 01
#141 436-445NMHQRNHTKL* A0201 0 0
SEQ ID NO: 183
B4001 72 3
A2402 0 0
p<0.001
* - the epitope previously predicted by the computer algorithm or described in the literature
EXAMPLE 5
Immunogenicity of the newly identified WT-1 epitopes.
[0293] In order to ascertain that that the peptides identified by mapping responses in single donors were also immunogenic in a high proportion of individuals bearing the same presenting HLA allele, it was determined whether these epitopes could elicit appropriately restricted T-cell responses in groups of 6-12 individuals expressing that HLA allele. For this purpose, the T-cells from each donor were sensitized with the identified epitope loaded on a panel of artificial antigen presenting cells (AAPC) (42) each expressing a single HLA allele, specifically A0201 , A0301, A2402 or B0702.As shown in Table 4, of 9 peptides identified that are presented by HLA-A0201 , all were able to stimulate WT-1 -specific IFNy-f- T-cell responses in a proportion of HLA-A0201+ individuals. The previously reported 126- 134RMFPN AP YL peptide presented by HLA-A0201 allele elicited responses in 5/12 (42%) HLA-A0201+ normal donors tested. In comparison, 5 of the other 8 peptides tested elicited WT-1 peptide- specific responses in 50-75% of the same HLA-A0201+ donors. Two WT-1 epitopes presented by the HLA-B0702 allele also elicited WT-1 specific T-cell responses in 50% and 63% of the tested individuals respectively (Table 4). All of the peptides tested elicited specific responses in at least 2 additional donors bearing their presenting HLA allele.
[0294] Table 4. Proportion of normal donors responding to identified WT-1 peptides loaded on AAPCs expressing a single HLA allele. Identifie Predicted Predicted
Proportion Proportion d in # of
of normal WT-1 sequence of donors
HLA Sequence previously donors predicted to be responses after
allele identified to be respondin
total Bindi Dissoc immunogenic when Bindin Dissoc in normal expres presented by the g to the
pool ng iation presented by the g iation donors to sed by HLA allele expressed peptide es
stimulat index time HLA allel index time the AAPC by the AAPC loaded on expressed by the peptide ion
AAPCs AAPC stimulatio on
(%) n
CAMs
A0201 (-99M-91) 1 6/12 3 0 (-99M-91) 3 0 6/12 THSPTHPP (50%) THSPTHPPR (50%) SEQ ID NO: 146 SEQ ID NO: 146
(-75M-67) 1 8/12 19 0.272 (-78M-70) 28 225 8/12 AILDFLLLQ (67%) LLAAILDFL (67%)
SEQ ID NO: 147 SEQ ID NO: 184
(-47M-39) 2 2/12 0 0 (-45M-36) 21 70 2/12 PGCLQQPEQ (16%) CLQQPEQQGV (16%)
SEQ ID NO: 148 SEQ ID NO: 185
(-27M-19) 1 8/12 19 17 (-27M- 19 17 8/12 KLGAAEASA (67%) 19)KLGA AE AS A (67%) SEQ ID NO: 149 SEQ ID NO: 150
7-15 27 12 3/12
DLNALLPAV (25%)
6-15
3/12 SEQ ID NO: 186
RDLNALLPAV 1 18 0.2
(25%) 10-18 33 181 3/12
SEQ ID NO:152
ALLPAVPSL (25%)
SEQ ID NO: 187
22-31 9/12 13 0.003 22-31 13 0.003 9/12
GGCALPVSGA (75%) GGCALPVSGA (75%)
SEQ ID NO:153 3 SEQ ID NO: 153
38-46 2 8/12 11 0 37-45 16 4 0/12
LDFAPPGAS (67%) VLDFAPPGA (0%)
SEQ ID NO:155 SEQ ID NO: 188
126-134 1 5/12 22 313 126-134 22 313 5/12 PvMFPNAPYL (42%) RMFPNAPYL (42%) SEQ ID NO:161 SEQ ID NO: 161
238-246 2 3/12 19 0.3 235-243 17 1.5 0/8
WNQMNLGAT (25%) CMTWNQMNL
SEQ ID NO:171 SEQ ID NO: 189
total pool 13/27 8/12
(48%) (67%)
A0301 126-134 1 2/8 10 4.5 124-133 14 0.001 0/8 PvMFPNAPYL (25%) QARMFPNAPY
SEQ ID NO:161 SEQ ID NO: 190
total pool 1/8 2/8
(12%) (25%)
A2402 239-248 1 4/6 10 7.2 235-243 10 4 1/6
NQMNLGATL (60%) CMTWNQMNL (17%)
SEQ ID NO:172 SEQ ID NO: 189
total pool 2/6 6/6
(33%) (100%)
B0702 (-125M-117) 1 4/8 15 40 (-125M-117) 15 40 4/8 PvQPvPHPGAL (50%) RQRPHPGAL (50%) SEQ ID NO: 142 SEQ ID NO: 142
(-119H-111) 1 5/8 2 0.3 (-118M-109) 15 120 5/8 GALRNPTAC (63%) ALRNPTACPL (63%)
SEQ ID NO: 143 SEQ ID NO: 191 323-332 1 3/8 1 0.015 327-335 17 0.4 4/8
FMCAYPGCNK (38%) YPGCNKRYF (50%) SEQ ID NO:179 SEQ ID NO: 192
Total pool 2/8 3/8
(25%) (38%)
DRBl 38-48 1 0/2 NA NA 35-49 20 NA ND 0402 LDFAPPGASAY (tested on APVLDFAPPGAS
SEQ ID NO:156 CAMs not AYG
on AAPC) SEQ ID NO: 193
EXAMPLE 6
Comparison of responses to peptides identified by mapping responses to pooled WT-1 15-mers with responses to previously reported WT-1 peptides predicted by binding
5 algorithms to be immunogenic.
[0295] Primary responses by normal donor T-cells were compared to individual WT-
1 peptides identified by the mapping strategy to responses against other WT-1 peptides containing flanking sequences predicted to have a higher binding index for the presenting 1 0 HLA allele using binding algorithms previously described (44,45). As shown in Table 4 above, the predicted binding indices for 8/12 mapped epitopes were only somewhat lower than those for the most studied WT-1 peptide, RMF, presented by HLA A0201. However, their dissociation times were markedly lower. Nevertheless, T-cell responses to each of these peptides were elicited in a high proportion of normal donors.
15 [0296] In five instances, the mapped peptide specificity (i.e. (_99)_(_9i)THS, (-22M- i9)KLG, 22-3 IGGC, 126-134RMF and (_i25)-(-i i7)RQR) was identical to the peptide with the highest affinity for the presenting HLA allele predicted by the binding algorithm within the stimulating 15-mer. In those instances in which the mapped sequences and the sequences predicted to have the highest binding index differed, the proportion of donors responding to
20 individual mapped peptides were equal or greater than those generated in response to the neighboring epitopes predicted to have higher affinity. For example, IFNy+ T-cell responses were generated to the 38-46LDF peptide in 8/12 (67%) of HLA A0201 donors tested, while none responded to the predicted and previously reported (46) epitope 37.45 VLDFAPPGA. Similarly, among HLA A2402+ donors, 4/6 donors (60%) responded to the 239-
25 24sNQMNLGATL peptide while only 1/6 responded to the 235-243 CMTWNQMNL peptide previously reported to be presented by this allele (29).
[0297] To directly compare peptides presented by HLA A0201 that were identified by matrix mapping with flanking peptides with higher predicted binding indices, the peptides, mixed at equal concentration, were loaded on HLA A0201+ AAPCs and used to sensitize T- cells from 8 of the HLA A0201+ normal donors. After 35 days of sensitization, the T-cells were then washed and secondarily restimulated for 24 hours with aliquots of irradiated autologous PBMC loaded with each individual peptide. Responding IFNy-f- T-cells were then quantitated by FACS. The results, presented in Figure 5, demonstrate that although the 22- 31GGC peptide has the lowest binding index and the shortest predicted dissociation time, it induced strong IFNy-f- T-cell responses in 7/8 donors. Furthermore, although 3/8 donors responded to the 6-15RDL, ιο-isALL and 7_isDLN peptides, 6-15RDL peptides identified by response mapping elicited higher numbers of IFNy-f- T-cells. In comparisons of the (-75 (- 67)AILDFLLLQ with flanking (_78 (-70)LLAAILDFL sequence, the AIL peptide elicited superior responses and in a higher proportion of donors (6/8 vs. 3/8 donors). Similarly, in comparisons of the mapped 38_46LDFAPPGAS peptide with the previously reported 37_ 45VLDFAPPGA peptide (46) the LDF peptide induced strong responses in 5 of the 8 donors while the VLD peptide induced low responses in only 2 of these donors.
[0298] Detailed description of Figure 5.IFNy+ T-cell responses to equimolar mixtures of 9-mer peptides identified by epitope mapping of in vitro responses and peptides within the same 15-mer or adjacent overlapping 15-mer peptides predicted to have higher binding affinity and immunogenicity. A. Responses to a mixture of nonamers spanning amino acids +2 to +31 including the 6-15RDL and 22-31GGC peptides to which HLA A0201+ donors responded in epitope mapping studies. B. Responses to the in vitro mapped (-75 (- 67)AILDFLLLQ epitope and a flanking peptide (_78 (-70)LLAAILDFL with higher predicted binding affinity. C. Responses to the in vitro mapped 38-46LDFAPPGAS epitope and the overlapping 37_4sVLDFAPPGA predicted to have higher binding affinity. [0299] Figure 5 presents maps of the WT-1 protein. Fig. 5C defines the localization of all previously reported antigenic epitopes presented by HLA class I and II alleles; Fig 5D depicts the location of immunogenic peptides identified in this report. As can be seen, the 11 epitopes previously reported to be presented by class I and 10 presented by class II HLA alleles are principally clustered in sequences encoded by exons 1, 7 and 10, while the epitopes recognized by normal T-cells sensitized with the WT- 1 peptide pool are principally clustered in sequences encoded by the first 5 exons. Thus, 26 of the new epitopes are included in each of the four major isoforms of WT-1 resulting from splice variants that do or do not include the 17 amino acid sequence (aas 250-266) in exon 5 or the three amino acid sequence (400-410KTS) between zinc fingers 3 and 4. While the epitopes are broadly distributed, clusters of epitopes were detected in the RNA recognition domain in exon 1 and the activation domain (aa 181-250) (Fig. 5F) proximal to the spliced 17aa segment in exon 5. The latter area also contained those epitopes most frequently recognized by multiple donors (Fig. 5E). Nine newly identified epitopes map to a 126 amino acid sequence at the N terminus encoded by a segment of the WT-1 gene initially described by Gessler et al37 that is centromeric to exon 1 of the (Exon 5+, KTS+) isoform of WT-1 and includes the long isoform of WT-1 initiated at a CUG codon upstream of the AUG initiator for exon l .50 Strikingly, each of the epitopes identified in this sequence elicits IFNy+T-cells that are cytolytic against leukemic blasts coexpressing WT-1 and the T-cells' restricting HLA allele.
EXAMPLE 7
INHIBITORY EFFECT OF PEPTIDES IN OVARIAN CARCINOMA [0300] The utility of peptides described herein in treating ovarian cancer was evaluated in two studies. In the first study, the inhibitory effect on ovarian tumor engraftment of T- cells specific for different WT-1 peptides was evaluated by pre-incubatingT cells at different doses with SKOV3-A2 ovarian carcinoma cells before injection into NOD/SCID mice. T cell cultures specific for the following immunodominant epitopes were prepared using methods described above: A0201 restricted WT- 1 peptide LKTHTTRTHT (SEQ ID NO: 182) specific T cells; A0301 restricted WT-1 peptide RQRPHPGAL (SEQ ID NO: 142) specific T cells, and A0201 restricted WT-1 peptide HFPPSLPPT (SEQ ID NO: 145) T cells. T cells to tumor cell ratios tested were 50: 1, 10: 1, 5: 1 and control (no T cells). Following tumor injection, the tumor burden was monitored by bioluminescent imaging. For all three T cell lines at each dose, a significant reduction in tumor burden was observed over timevs. control. Furthermore, mouse survival was prolonged by pre-incubation of tumor cells with WT-1 peptide specific T cells. In control groups, all mice were dead by 70 days post tumor injection. Increased survival was seen dose-responsively with the T cell: tumor cell dose, which for the 50: 1 dose for all T cell lines still had some animals alive at 96 days, and also at 10 : 1 for the LKTHTTRTHT specific line.
[0301] In a second experiment, WT-1 peptide specific T cells were administered intravenously to NOD/SCID mice bearing pre-established ovarian carcinoma SKOV3-A2 xenografts. T cell lines evaluated were: A0201 restricted WT-1 peptide LKTHTTRTHT (SEQ ID NO: 182) specific T cells and A0301 restricted WT-1 peptide RQRPHPGAL (SEQ ID NO: 142) specific T cells. Tumor burden was monitored by bioluminescence, tumor infiltration by human CD3+ cells evaluated and survival recorded. In both cases, the WT-1 specific T cells afforded reduced tumor burden vs. control, increased tumor infiltration by human CD3+ cells and increased survival.
EXAMPLE 8
RECOGNITION OF EPITOPES BY LEUKEMIA PATIENT T CELLS [0302] A phase I clinical trial was conducted using transplant donor-derived T-cells sensitized with the full pool of WT-1 derived pentadecapaptidesdescribed above, in the adoptive therapy of patients who have relapsed following an allogeneic marrow transplant from a normal related or unrelated donor. The HLA restricting alleles and corresponding immunodominant WT-1 epitopes are as follows: A0201 , SEQ ID NO: 147; A0203, SEQ ID NOs: 176 and 183; B3503 and C0401, SEQ ID NOs: 161 and 169; A6901, SEQ ID NO: 177; A0201 , SEQ ID NO: 182; B4701 and DRBj 0102, SEQ ID NOs: 148 and 149; A3101 , SEQ ID NO: 145; B4402, SEQ ID NOs: 158 and 166; B3503, SEQ ID NOs: 146 and 162; DRBj 1104, SEQ ID NO: 149. It is noted that several of the immunodominant epitopes eliciting the WT- 1 specific T-cells that were used were directed against epitopes in the N-terminal region of the gene, upstream from exon 1 , i.e., SEQ ID NOs: 145, 147, 148 and 149. Two of the donors responded to PGCLQQPEQQG, SEQ ID NO: 149, and both treatedpatients had temporary clearance of WT-1 + leukemic cells following adoptive transfer.
EXAMPLE 9
Pentadecapeptides [0303] The following pentadecapeptides were synthesized. H2N refers to the N- terminal end of the peptide, and -COOH the C-terminus.
Table 5.Sequence of pentadecapeptides
SEQ ID NO: l.H2N-SRQRP HPGAL RNPTA -COOH
SEQ ID NO:2.H2N-PHPGA LRNPT ACPLP -COOH
SEQ ID NO:3. H2N-ALRNP TACPL PHFPP -COOH SEQ ID NO:4. H2N-PTACP LPHFP PSLPP -COOH SEQ ID NO:5. H2N-PLPHF PPSLP PTHSP -COOH SEQ ID NO:6. H2N-FPPSL PPTHS PTHPP -COOH SEQ ID NO:7. H2N-LPPTH SPTHP PRAGT -COOH SEQ ID NO:8. H2N-HSPTH PPRAG TAAQA -COOH SEQ ID NO:9. H2N-HPPRA GTAAQ APGPR -COOH SEQ ID NO: 10. H2N-AGTAA QAPGP RRLLA-COOH SEQ ID NO: l l . H2N-AQAPG PRRLL AAILD-COOH SEQ ID NO: 12. H2N-GPRRL LAAIL DFLLL-COOH SEQ ID NO: 13. H2N-LLAAI LDFLL LQDPA -COOH SEQ ID NO: 14. H2N-ILDFL LLQDP ASTCV -COOH SEQ ID NO: 15. H2N-LLLQD PASTC VPEPA -COOH SEQ ID NO: 16. H2N-DPAST CVPEP ASQHT -COOH SEQ ID NO: 17. H2N-TCVPE PASQH TLRSG -COOH SEQ ID NO: 18. H2N-EPASQ HTLRS GPGCL -COOH SEQ ID NO: 19. H2N-QHTLR SGPGC LQQPE -COOH SEQ ID NO:20. H2N-RSGPG CLQQP EQQGV -COOH SEQ ID NO:21. H2N-GCLQQ PEQQG VRDPG -COOH SEQ ID NO:22. H2N-QPEQQ GVRDP GGIWA -COOH SEQ ID NO:23.H2N-QGVRD PGGIW AKLGA -COOH SEQ ID NO:24. H2N-DPGGI WAKLG AAEAS -COOH SEQ ID NO:25. H2N-IWAKL GAAEA SAERL -COOH SEQ ID NO:26. H2N-LGAAE ASAER LQGRR -COOH SEQ ID NO:27. H2N-EASAE RLQGR RSRGA -COOH SEQ ID NO:28.H2N-ERLQG RRSRG ASGSE -COOH
SEQ ID NO:29.H2N-GRRSR GASGS EPQQM -COOH SEQ ID NO:30.H2N-RGASG SEPQQ MGSDV -COOH SEQ ID NO:31.H2N-GSEPQ QMGSD VRDLN -COOH SEQ ID NO:32. H2N-QQMGS DVRDL NALLP -COOH SEQ ID NO:33. H2N-SDVRD LNALL PA VPS -COOH
SEQ ID NO:34. H2N-DLNAL LP A VP SLGGG -COOH SEQ ID NO:35. H2N-LLPAV PSLGG GGGCA -COOH SEQ ID NO:36. H2N-VPSLG GGGGC ALPVS -COOH SEQ ID NO:37. H2N-GGGGG CALPV SGAAQ -COOH SEQ ID NO:38. H2N-GCALP VSGAA QWAPV -COOH
SEQ ID NO:39. H2N-PVSGA AQWAP VLDFA -COOH SEQ ID NO:40. H2N-AAQWA PVLDF APPGA -COOH SEQ ID NO:41. H2N-APVLD FAPPG ASAYG -COOH SEQ ID NO:42. H2N-DFAPP GASAY GSLGG -COOH SEQ ID NO:43. H2N-PGASA YGSLG GPAPP -COOH
SEQ ID NO:44. H2N-AYGSL GGPAP PPAPP -COOH SEQ ID NO:45. H2N-LGGPA PPPAP PPPPP -COOH SEQ ID NO:46. H2N-APPPA PPPPP PPPPH -COOH SEQ ID NO:47. H2N-APPPP PPPPP HSFIK -COOH SEQ ID NO:48. H2N-PPPPP PHSFI KQEPS -COOH
SEQ ID NO:49. H2N-PPHSF IKQEP SWGGA -COOH SEQ ID NO:50. H2N-FIKQE PSWGG AEPHE -COOH SEQ ID NO:51. H2N-EPSWG GAEPH EEQCL -COOH SEQ ID NO:52. H2N-GGAEP HEEQC LSAFT -COOH SEQ ID NO:53. H2N-PHEEQ CLSAF TVHFS -COOH SEQ ID NO: :54. H2N-QCLSA FTVHF SGQFT -COOH
SEQ ID NO: :55. H2N-AFTVH FSGQF TGTAG -COOH
SEQ ID NO: :56. H2N-HFSGQ FTGTA GACRY -COOH
SEQ ID NO: :57. H2N-QFTGT AGACR YGPFG -COOH
SEQ ID NO: :58. H2N-TAGAC RYGPF GPPPP -COOH
SEQ ID NO: :59. H2N-CRYGP FGPPP PSQAS -COOH
SEQ ID NO: :60. H2N-PFGPP PPSQA SSGQA -COOH
SEQ ID NO: :61. H2N-PPPSQ ASSGQ ARMFP -COOH
SEQ ID NO: :62. H2N-QASSG QARMF PNAPY -COOH
SEQ ID NO: :63. H2N-GQARM FPNAP YLPSC -COOH
SEQ ID NO: :64. H2N-MFPNA PYLPS CLESQ -COOH
SEQ ID NO: :65. H2N-APYLP SCLES QPAIR -COOH
SEQ ID NO: :66. H2N-PSCLE SQPAI RNQGY -COOH
SEQ ID NO: :67. H2N-ESQPA IRNQG YSTVT -COOH
SEQ ID NO: :68. H2N-AIRNQ GYSTV TFDGT -COOH
SEQ ID NO: :69. H2N-QGYST VTFDG TPSYG -COOH
SEQ ID NO: :70. H2N-TVTFD GTPSY GHTPS -COOH
SEQ ID NO: :71. H2N-DGTPS YGHTP SHHAA -COOH
SEQ ID NO: :72. H2N-SYGHT PSHHA AQFPN -COOH
SEQ ID NO: :73. H2N-TPSHH AAQFP NHSFK -COOH
SEQ ID NO: :74. H2N-HAAQF PNHSF KHEDP -COOH
SEQ ID NO: :75. H2N-FPNHS FKHED PMGQQ -COOH
SEQ ID NO: 16. H2N-SFKHE DPMGQ QGSLG -COOH
SEQ ID NO: 11. H2N-EDPMG QQGSL GEQQY -COOH
SEQ ID NO: 18. H2N-GQQGS LGEQQ YSVPP -COOH
SEQ ID NO: 19. H2N-SLGEQ QYSVP PPVYG-COOH
SEQ ID NO: :80. H2N-QQYSV PPPVY GCHTP-COOH
SEQ ID NO: :81. H2N-VPPPV YGCHT PTDSC -COOH
SEQ ID NO: :82. H2N-VYGCH TPTDS CTGSQ -COOH
SEQ ID NO: :83. H2N-HTPTD SCTGS QALLL -COOH
SEQ ID NO: :84. H2N-DSCTG SQALL LRTPY -COOH
SEQ ID NO: :85. H2N-GSQAL LLRTP YSSDN -COOH
SEQ ID NO: :86. H2N-LLLRT PYSSD NLYQM -COOH
SEQ ID NO: :87. H2N-TPYSS DNLYQ MTSQL -COOH
SEQ ID NO: :88. H2N-SDNLY QMTSQ LECMT -COOH
SEQ ID NO: :89. H2N-YQMTS QLECM TWNQM -COOH
SEQ ID NO: :90. H2N-SQLEC MTWNQ MNLGA -COOH
SEQ ID NO: :91. H2N-CMTWN QMNLG ATLKG -COOH
SEQ ID NO: :92. H2N-NQMNL GATLK GVAAG -COOH
SEQ ID NO: :93. H2N-LGATL KGVAA GSSSS -COOH
SEQ ID NO: :94. H2N-LKGVA AGSSS SVKWT -COOH
SEQ ID NO: :95. H2N-AAGSS SSVKW TEGQS -COOH
SEQ ID NO: :96. H2N-SSSVK WTEGQ SNHST -COOH
SEQ ID NO: :97. H2N-KWTEG QSNHS TGYES -COOH
SEQ ID NO: :98. H2N-GQSNH STGYE SDNHT -COOH
SEQ ID NO: :99. H2N-HSTGY ESDNH TTPIL -COOH
SEQ ID NO: : 100. H2N-YESDN HTTPI LCGAQ -COOH
SEQ ID NO: : 101. H2N-NHTTP ILCGA QYRIH -COOH
SEQ ID NO: Λ02. H2N-PILCG AQYRI HTHGV -COOH
SEQ ID NO: : 103. H2N-GAQYR IHTHG VFRGI -COOH SEQ ID NO 104. H2N- RIHTH GVFRG IQDVR -COOH
SEQ ID NO 105. H2N- HGVFR GIQDV RRVPG -COOH
SEQ ID NO 106. H2N- RGIQD VRRVP GVAPT -COOH
SEQ ID NO 107. H2N- DVRRV PGVAP TLVRS -COOH
SEQ ID NO 108. H2N- VPGVA PTLVR SASET -COOH
SEQ ID NO 109. H2N- APTLV RSASE TSEKR -COOH
SEQ ID NO 110. H2N- VRSAS ETSEK RPFMC -COOH
SEQ ID NO 111. H2N- SETSE KRPFM CAYPG -COOH
SEQ ID NO 112. H2N- EKRPF MCAYP GCNKR -COOH
SEQ ID NO 113. H2N- FMCAY PGCNK RYFKL -COOH
SEQ ID NO 114. H2N- YPGCN KRYFK LSHLQ -COOH
SEQ ID NO 115. H2N- NKRYF KLSHL QMHSR -COOH
SEQ ID NO 116. H2N- FKLSH LQMHS RKHTG -COOH
SEQ ID NO 117. H2N- HLQMH SRKHT GEKPY -COOH
SEQ ID NO 118. H2N- HSRKH TGEKP YQCDF -COOH
SEQ ID NO 119. H2N- HTGEK PYQCD FKDCE -COOH
SEQ ID NO 120. H2N- KPYQC DFKDC ERRFS -COOH
SEQ ID NO 121. H2N- CDFKD CERRF SRSDQ-COOH
SEQ ID NO 122. H2N- DCERR FSRSD QLKRH-COOH
SEQ ID NO 123 H2N-RFSRS DQLKR HQRRH-COOH
SEQ ID NO 124. H2N- SDQLK RHQRR HTGVK-COOH
SEQ ID NO 125. H2N- KRHQR RHTGV KPFQC-COOH
SEQ ID NO 126. H2N- RRHTG VKPFQ CKTCQ-COOH
SEQ ID NO 127. H2N- GVKPF QCKTC QRKFS-COOH
SEQ ID NO 128. H2N- FQCKT CQRKF SRSDH-COOH
SEQ ID NO 129. H2N- TCQRK FSRSD HLKTH-COOH
SEQ ID NO 130. H2N- KFSRS DHLKT HTRTH-COOH
SEQ ID NO 131. H2N- SDHLK THTRT HTGKT-COOH
SEQ ID NO 132. H2N- KTHTR THTGK TSEKP-COOH
SEQ ID NO 133. H2N- RTHTG KTSEK PFSCR-COOH
SEQ ID NO 134. H2N- GKTSE KPFSC RWPSC-COOH
SEQ ID NO 135. H2N- EKPFS CRWPS CQKKF-COOH
SEQ ID NO 136. H2N- SCRWP SCQKK FARSD-COOH
SEQ ID NO 137. H2N- PSCQK KFARS DELVR-COOH
SEQ ID NO 138. H2N- KKFAR SDELV RHHNM-COOH
SEQ ID NO 139. H2N- RSDEL VRHHN MHQRN-COOH
SEQ ID NO 140. H2N- LVRHH NMHQR NMTKL-COOH
SEQ ID NO 141. H2N- HNMHQ RNMTK LQLAL-COOH
References
1. Kolb HJ, Mittermuller J, Clemm C, et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood. 1990;76(12):2462-2465. Prepublished on 1990/12/15 as DOI.
2. Papadopoulos EB, Ladanyi M, Emanuel D, et al. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med. 1994;330(17): 1185-1191. Prepublished on 1994/04/28 as DOI 10.1056/NEJM199404283301703.
3. Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science. 2002;298(5594):850-854. Prepublished on 2002/09/21 as DOI 10.1126/science.1076514. 4. Rosenberg SA, Yang JC, Sherry RM, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res. 2011 ;17(13):4550-4557. Prepublished on 2011/04/19 as DOI 10.1158/1078- 0432.CCR-11-0116.
5. Yee C, Thompson JA, Byrd D, et al. Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci U S A. 2002;99(25): 16168-16173. Prepublished on 2002/11/13 as DOI 10.1073/pnas.242600099.
6. Mackensen A, Meidenbauer N, Vogl S, Laumer M, Berger J, Andreesen R. Phase I study of adoptive T-cell therapy using antigen-specific CD8+ T cells for the treatment of patients with metastatic melanoma. J Clin Oncol. 2006;24(31):5060-5069. Prepublished on 2006/11/01 as DOI 10.1200/JCO.2006.07.1100.
7. Robbins PF, Morgan RA, Feldman SA, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol. 2011 ;29(7):917-924. Prepublished on 2011/02/02 as DOI 10.1200/JCO.2010.32.2537.
8. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006;314(5796): 126-129. Prepublished on 2006/09/02 as DOI 10.1126/science.1129003.
9. Pule MA, Savoldo B, Myers GD, et al. Virus-specific T cells engineered to coexpress tumor- specific receptors: persistence and antitumor activity in individuals with neuroblastoma. Nat Med. 2008;14(11): 1264-1270. Prepublished on 2008/11/04 as DOI 10.1038/nm. l882.
10. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor- modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011 ;365(8):725-733. Prepublished on 2011/08/13 as DOI 10.1056/NEJMoal 103849.
11. Brentjens RJ, Riviere I, Park JH, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood. 2011 ;118(18):4817-4828. Prepublished on 2011/08/19 as DOI 10.1182/blood-2011-04-348540.
12. Gao L, Xue SA, Hasserjian R, et al. Human cytotoxic T lymphocytes specific for Wilms' tumor antigen- 1 inhibit engraftment of leukemia-initiating stem cells in non-obese diabetic-severe combined immunodeficient recipients. Transplantation. 2003;75(9): 1429- 1436. Prepublished on 2003/06/07 as DOI 10.1097/01.TP.0000061516.57346.E8.
13. Gerber JM, Qin L, Kowalski J, et al. Characterization of chronic myeloid leukemia stem cells. Am J Hematol. 2011 ;86(l):31-37. Prepublished on 2010/12/07 as DOI 10.1002/ajh.21915. 14. Greiner J, Bullinger L, Guinn BA, Dohner H, Schmitt M. Leukemia-associated antigens are critical for the proliferation of acute myeloid leukemia cells. Clin Cancer Res. 2008;14(22):7161-7166. Prepublished on 2008/11/18 as DOI 10.1158/1078-0432.CCR-08- 1102.
15. Call KM, Glaser T, Ito CY, et al. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor locus. Cell. 1990;60(3):509- 520. Prepublished on 1990/02/09 as DOI.
16. Haber DA, Sohn RL, Buckler AJ, Pelletier J, Call KM, Housman DE. Alternative splicing and genomic structure of the Wilms tumor gene WT-1. Proc Natl Acad Sci U S A. 1991 ;88(21):9618-9622. Prepublished on 1991/11/01 as DOI.
17. Kreidberg JA, Sariola H, Loring JM, et al. WT-1 is required for early kidney development. Cell. 1993;74(4):679-691. Prepublished on 1993/08/27 as DOI.
18. Scharnhorst V, van der Eb AJ, Jochemsen AG. WT-1 proteins: functions in growth and differentiation. Gene. 2001 ;273(2): 141-161. Prepublished on 2001/10/12 as DOI.
19. Ellisen LW, Carlesso N, Cheng T, Scadden DT, Haber DA. The Wilms tumor suppressor WT-1 directs stage- specific quiescence and differentiation of human hematopoietic progenitor cells. EMBO J. 2001 ;20(8): 1897-1909. Prepublished on 2001/04/11 as DOI 10.1093/emboj/20.8.1897.
20. Hosen N, Sonoda Y, Oji Y, et al. Very low frequencies of human normal CD34+ haematopoietic progenitor cells express the Wilms' tumour gene WT-1 at levels similar to those in leukaemia cells. Br J Haematol. 2002;116(2):409-420. Prepublished on 2002/02/14 as DOI.
21. Yang L, Han Y, Suarez Saiz F, Minden MD. A tumor suppressor and oncogene: the WT-1 story. Leukemia. 2007;21(5):868-876. Prepublished on 2007/03/16 as DOI 10.1038/sj.leu.2404624.
22. Bergmann L, Miething C, Maurer U, et al. High levels of Wilms' tumor gene (WT-1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome. Blood. 1997;90(3): 1217-1225. Prepublished on 1997/08/01 as DOI.
23. Lapillonne H, Renneville A, Auvrignon A, et al. High WT-1 expression after induction therapy predicts high risk of relapse and death in pediatric acute myeloid leukemia.
J Clin Oncol. 2006;24(10): 1507-1515. Prepublished on 2006/04/01 as DOI 10.1200/JCO.2005.03.5303.
24. Chen ZX, Kaeda J, Saunders S, Goldman JM. Expression patterns of WT-1 and Bcr- Abl measured by TaqMan quantitative real-time RT-PCR during follow-up of leukemia patients with the Ph chromosome. Chin Med J (Engl). 2004;117(7):968-971. Prepublished on 2004/07/22 as DOI.
25. Cilloni D, Gottardi E, Messa F, et al. Significant correlation between the degree of WT-1 expression and the International Prognostic Scoring System Score in patients with myelodysplastic syndromes. J Clin Oncol. 2003;21(10): 1988-1995. Prepublished on 2003/05/14 as DOI 10.1200/JCO.2003.10.503. 26. Tamaki H, Ogawa H, Ohyashiki K, et al. The Wilms' tumor gene WT-1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Leukemia. 1999;13(3):393-399. Prepublished on 1999/03/23 as DOI.
27. Keilholz U, Letsch A, Busse A, et al. A clinical and immunologic phase 2 trial of Wilms tumor gene product 1 (WT-1) peptide vaccination in patients with AML and MDS.
Blood. 2009; 113(26):6541-6548. Prepublished on 2009/04/25 as DOI 10.1182/blood-2009- 02-202598.
28. Tatsumi N, Oji Y, Tsuji N, et al. Wilms' tumor gene WT-l-shRNA as a potent apoptosis-inducing agent for solid tumors. Int J Oncol. 2008;32(3):701-711. Prepublished on 2008/02/23 as DOI.
29. Ohminami H, Yasukawa M, Fujita S. HLA class I-restricted lysis of leukemia cells by a CD8(+) cytotoxic T-lymphocyte clone specific for WT-1 peptide. Blood. 2000;95(1):286- 293. Prepublished on 1999/12/23 as DOI.
30. Oka Y, Elisseeva OA, Tsuboi A, et al. Human cytotoxic T-lymphocyte responses specific for peptides of the wild-type Wilms' tumor gene (WT-1 ) product. Immunogenetics.
2000;51(2):99-107. Prepublished on 2000/02/09 as DOI.
31. Rezvani K, Brenchley JM, Price DA, et al. T-cell responses directed against multiple HLA- A*0201 -restricted epitopes derived from Wilms' tumor 1 protein in patients with leukemia and healthy donors: identification, quantification, and characterization. Clin Cancer Res. 2005;11(24 Pt l):8799-8807. Prepublished on 2005/12/20 as DOI 10.1158/1078- 0432.CCR-05-1314.
32. Doubrovina ES, Doubrovin MM, Lee S, et al. In vitro stimulation with WT-1 peptide- loaded Epstein-Barr virus-positive B cells elicits high frequencies of WT-1 peptide-specific T cells with in vitro and in vivo tumoricidal activity. Clin Cancer Res. 2004;10(21):7207-7219. Prepublished on 2004/11/10 as DOI 10.1158/1078-0432.CCR-04-1040.
33. Xue SA, Gao L, Hart D, et al. Elimination of human leukemia cells in NOD/SCID mice by WT-l-TCR gene-transduced human T cells. Blood. 2005;106(9):3062-3067. Prepublished on 2005/07/16 as DOI 10.1182/blood-2005-01-0146.
34. Oka Y, Tsuboi A, Taguchi T, et al. Induction of WT-1 (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WT- 1 peptide vaccine and the resultant cancer regression. Proc
Natl Acad Sci U S A. 2004;101(38): 13885-13890. Prepublished on 2004/09/15 as DOI 10.1073/pnas.0405884101.
35. Pinilla-Ibarz J, May RJ, Korontsvit T, et al. Improved human T-cell responses against synthetic HLA-0201 analog peptides derived from the WT-1 oncoprotein. Leukemia. 2006;20(11):2025-2033. Prepublished on 2006/09/23 as DOI 10.1038/sj.leu.2404380.
36. Rezvani K, Yong AS, Mielke S, et al. Leukemia-associated antigen- specific T-cell responses following combined PRl and WT-1 peptide vaccination in patients with myeloid malignancies. Blood. 2008;l l l(l):236-242. Prepublished on 2007/09/19 as DOI 10.1182/blood-2007-08- 108241.
37. Gessler M, Poustka A, Cavenee W, Neve RL, Orkin SH, Bruns GA. Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping. Nature. 1990;343(6260):774-778. Prepublished on 1990/02/22 as DOI 10.1038/343774a0.
38. Koehne G, Smith KM, Ferguson TL, et al. Quantitation, selection, and functional characterization of Epstein-Barr virus-specific and alloreactive T cells detected by intracellular interferon-gamma production and growth of cytotoxic precursors. Blood. 2002;99(5): 1730-1740. Prepublished on 2002/02/28 as DOI.
39. Roskrow MA, Suzuki N, Gan Y, et al. Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for the treatment of patients with EBV-positive relapsed Hodgkin's disease. Blood. 1998;91(8):2925-2934. Prepublished on 1998/05/16 as DOI.
40. Waldrop SL, Pitcher CJ, Peterson DM, Maino VC, Picker LJ. Determination of antigen-specific memory /effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency. J Clin Invest. 1997;99(7): 1739-1750. Prepublished on 1997/04/01 as DOI 10.1172/JCIl 19338.
41. Trivedi D, Williams RY, O'Reilly RJ, Koehne G. Generation of CMV-specific T lymphocytes using protein-spanning pools of pp65 -derived overlapping pentadecapeptides for adoptive immunotherapy. Blood. 2005;105(7):2793-2801. Prepublished on 2004/10/30 as DOI 10.1182/blood-2003-05-1433.
42. Hasan AN, Kollen WJ, Trivedi D, et al. A panel of artificial APCs expressing prevalent HLA alleles permits generation of cytotoxic T cells specific for both dominant and subdominant viral epitopes for adoptive therapy. J Immunol. 2009;183(4):2837-2850. Prepublished on 2009/07/29 as DOI 10.4049/jimmunol.0804178.
43. Gao L, Bellantuono I, Elsasser A, et al. Selective elimination of leukemic CD34(+) progenitor cells by cytotoxic T lymphocytes specific for WT-1. Blood. 2000;95(7):2198- 2203. Prepublished on 2000/03/25 as DOI.
44. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999;50(3-4):213-219.
Prepublished on 1999/12/22 as DOI.
45. Parker KC, Bednarek MA, Coligan JE. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol. 1994;152(1): 163-175. Prepublished on 1994/01/01 as DOI.
46. Smithgall M, Misher L, Spies G, Cheever MA, Gaiger A. Identification of a novel
WT-1 HLA A*0201 -restricted CTL epitope using whole gene in vitro priming. . ASH. Orlando, FL: Blood; 2001 : 121a.
47. Cheever MA, Allison JP, Ferris AS, et al. The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research. Clin Cancer Res. 2009;15(17):5323-5337. Prepublished on 2009/09/03 as DOI 10.1158/1078- 0432.CCR-09-0737.
48. Kiecker F, Streitz M, Ay B, et al. Analysis of antigen-specific T-cell responses with synthetic peptides-what kind of peptide for which purpose? Hum Immunol. 2004;65(5):523- 536. Prepublished on 2004/06/03 as DOI 10.1016/j.humimm.2004.02.017.
49. Pelte C, Cherepnev G, Wang Y, Schoenemann C, Volk HD, Kern F. Random screening of proteins for HLA-A*0201 -binding nine-amino acid peptides is not sufficient for identifying CD8 T cell epitopes recognized in the context of HLA-A*0201. J Immunol. 2004;172(l l):6783-6789. Prepublished on 2004/05/22 as DOI.
50. Bruening W, Pelletier J. A non-AUG translational initiation event generates novel WT-1 isoforms. J Biol Chem. 1996;271(15):8646-8654. Prepublished on 1996/04/12 as DOI.
51. Pittet MJ, Valmori D, Dunbar PR, et al. High frequencies of naive Melan-A/MART- 1 -specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J Exp Med. 1999;190(5):705-715. Prepublished on 1999/09/08 as DOI.
52. Chen Q, Jackson H, Gibbs P, Davis ID, Trapani J, Cebon J. Spontaneous T cell responses to melanoma differentiation antigens from melanoma patients and healthy subjects. Cancer Immunol Immunother. 1998;47(4): 191-197. Prepublished on 1999/01/06 as DOI.
53. Pittet MJ, Zippelius A, Valmori D, Speiser DE, Cerottini JC, Romero P. Melan- A/MART-1 -specific CD8 T cells: from thymus to tumor. Trends Immunol. 2002;23(7):325- 328. Prepublished on 2002/07/10 as DOI.
54. Weber G, Karbach J, Kuci S, et al. WT-1 peptide-specific T cells generated from peripheral blood of healthy donors: possible implications for adoptive immunotherapy after allogeneic stem cell transplantation. Leukemia. 2009;23(9): 1634-1642. Prepublished on 2009/04/10 as DOI 10.1038/leu.2009.70.
55. Nagorsen D, Scheibenbogen C, Marincola FM, Letsch A, Keilholz U. Natural T cell immunity against cancer. Clin Cancer Res. 2003;9(12):4296-4303. Prepublished on 2003/10/14 as DOI.
56. Scheibenbogen C, Letsch A, Thiel E, et al. CD8 T-cell responses to Wilms tumor gene product WT-1 and proteinase 3 in patients with acute myeloid leukemia. Blood. 2002;100(6):2132-2137. Prepublished on 2002/08/30 as DOI 10.1182/blood-2002-01-0163.
57. Elisseeva OA, Oka Y, Tsuboi A, et al. Humoral immune responses against Wilms tumor gene WT-1 product in patients with hematopoietic malignancies. Blood. 2002;99(9):3272-3279. Prepublished on 2002/04/20 as DOI.
58. Tyler E, Jungbluth AA, O'Reilly RJ, Koehne G. WT-l-Specific Immune Responses in Patients with High-Risk Multiple Myeloma Undergoing Allogeneic T Cell-Depleted
Hematopoietic Stem Cell Transplantation Followed by Donor Lymphocyte Infusions. ASH Annual Meeting Abstracts. 2011 ;118(21): 1993-.
59. King JW, Thomas S, Corsi F, et al. IL15 can reverse the unresponsiveness of Wilms' tumor antigen-specific CTL in patients with prostate cancer. Clin Cancer Res. 2009;15(4): 1145-1154. Prepublished on 2009/02/21 as DOI 10.1158/1078-0432.CCR-08- 1821.
60. Gillmore R, Xue SA, Holler A, et al. Detection of Wilms' tumor antigen— specific CTL in tumor-draining lymph nodes of patients with early breast cancer. Clin Cancer Res. 2006;12(l):34-42. Prepublished on 2006/01/07 as DOI 10.1158/1078-0432.CCR-05-1483.
Murao A, Oka Y, Tsuboi A, et al. High frequencies of less differentiated and more proliferative WT-l-specific CD8+ T cells in bone marrow in tumor-bearing patients: an important role of bone marrow as a secondary lymphoid organ. Cancer Sci. 2010;101(4):848- 854. Prepublished on 2010/02/09 as DOI 10.1111/j. l349-7006.2009.01468.x.
62. van der Bruggen P, Stroobant V, Vigneron N, van den Eynde B. Database of T-cell defined tumor antigens. http://cancerimmunity.org/peptidedatabase/Tcellepitopes.htm.
Cancer Immunity. 2012.
63. Pospori C, Xue SA, Holler A, et al. Specificity for the tumor-associated self-antigen WT-1 drives the development of fully functional memory T cells in the absence of vaccination. Blood. 2011 ;117(25):6813-6824. Prepublished on 2011/03/31 as DOI 10.1182/blood-2010-08-304568.
64. Rezvani K, Yong AS, Mielke S, et al. Repeated PR1 and WT-1 peptide vaccination in Montanide-adjuvant fails to induce sustained high-avidity, epitope- specific CD8+ T cells in myeloid malignancies. Haematologica. 2011 ;96(3):432-440. Prepublished on 2010/12/08 as DOI 10.3324/haematol.2010.031674.
65. Lehe C, Ghebeh H, Al-Sulaiman A, et al. The Wilms' tumor antigen is a novel target for human CD4+ regulatory T cells: implications for immunotherapy. Cancer Res. 2008;68(15):6350-6359. Prepublished on 2008/08/05 as DOI 10.1158/0008-5472.CAN-08- 0050.

Claims

WHAT IS CLAIMED IS:
1. An isolated WT- 1 peptide consisting ofan amino acid sequence selected from among AILDFLLLQ (SEQ ID NO: 147), RQRPHPGAL (SEQ ID NO: 142), GALRNPTAC (SEQ ID NO: 143), THSPTHPPR (SEQ ID NO: 146), ASGSEPQQM (SEQ ID NO: 151), WNQMNLGATLK (SEQ ID NO: 173), PGCLQQPEQQG (SEQ ID
NO: 149), and LDFAPPGASAY (SEQ ID NO: 156).
2. An isolated WT- 1 peptide consisting ofan amino acid sequence selected from among PLPHFPPSL (SEQ ID NO: 144), HFPPSLPPT (SEQ ID NO: 145), PGCLQQPEQ (SEQ ID NO: 148),KLGAAEASA (SEQ ID NO: 150), LLAAILDFL (SEQ ID NO: 184), CLQQPEQQGV (SEQ ID NO: 185) and ALRNPTACPL (SEQ ID
NO: 191).
3. An isolated WT-1 peptide consisting of an amino acid sequence selected from amongRDLNALLPAV (SEQ ID NO: 152), GGCALPVSGA (SEQ ID NO: 153), GAAQWAPVL (SEQ ID NO: 154), LDFAPPGAS (SEQ ID NO: 155), SAYGSLGGP (SEQ ID NO: 157), PAPPPPPPP (SEQ ID NO: 158), ACRYGPFGP (SEQ ID
NO: 159), SGQARMFPN (SEQ ID NO: 160), PSCLESQPA (SEQ ID NO: 162), NQGYSTVTF (SEQ ID NO: 163), HHAAQFPNH (SEQ ID NO: 164), HSFKHEDPM (SEQ ID NO: 165), CHTPTDSCT (SEQ ID NO: 166), CTGSQALLL (SEQ ID NO: 167), TDSCTGSQA (SEQ ID NO: 168), RTPYSSDNL (SEQ ID NO: 169), NLYQMTSQLE (SEQ ID NO: 170), WNQMNLGAT (SEQ ID NO: 171),
WNQMNLGATLK (SEQ ID NO: 173), CMTWNQMNLGATLKG (SEQ ID NO: 174), NLGATLKGV (SEQ ID NO: 175), LGATLKGVAA (SEQ ID NO: 176), TLGVAAGS (SEQ ID NO: 177), GYESDNHTT (SEQ ID NO: 178), FMCAYPGCNK (SEQ ID NO: 179), KRPFMCAYPGC (SEQ ID NO: 180), RKFSRSDHL (SEQ ID NO: 181), LKTHTTRTHT (SEQ ID NO: 182), NMHQRNHTKL (SEQ ID NO: 183),
QARMFPNAPY (SEQ ID NO: 190), ALRNPTACPL (SEQ ID NO: 191) and APVLDFAPPGASAYG (SEQ ID NO: 193).
4. An isolated WT- 1 peptide consisting ofan amino acid sequence selected from among SEQ ID NO: l-141.
5. An isolated WT-1 peptide consisting of 8-30 amino acids comprising an amino acid sequence selected from SEQ ID NO: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 184 and 185.
6. An isolated WT-1 peptide consisting of 16-30 amino acids comprising an amino acid sequence selected from SEQ ID NO: 1-141.
7. The isolated WT-1 peptide of any one of claims 1-6, wherein said isolated WT-1 peptide binds to an HLA class I molecule, an HLA class II molecule, or the combination thereof.
8. A pharmaceutical composition comprising a peptide of any one of claims 1-6 and a pharmaceutically acceptable carrier, vehicle or excipient.
9. A vaccine comprising (a) one or more isolated WT-1 peptides of claims 1-6 and (b) an adjuvant or a carrier.
10. The vaccine of claim 9, wherein said adjuvant is QS21, Freund's incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG, alum, a growth factor, a cytokine, a chemokine, an interleukin, Montanideor GM-CSF.
11. A method of treating a subject with a WT-1 -expressing cancer or reducing an incidence of a WT-1 -expressing cancer, or its relapse, the method comprising administering to said subject the vaccine of claim 9, thereby treating a subject with a WT-1 -expressing cancer, reducing an incidence of a WT-1 -expressing cancer or its relapse therein.
12. The method of claim 11, wherein said WT-1 -expressing cancer is a leukemia, a desmoplastic small round cell tumor, a gastric cancer, a colon cancer, a lung cancer, a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, a renal cancer, a Kaposi's sarcoma, a sarcoma, a hepatocellular carcinoma, a Wilms' tumor, an acute myelogenous leukemia (AML), a myelodysplastic syndrome (MDS), or a non-small cell lung cancer (NSCLC).
13. A method of inducing the formation and proliferation of CTL specific for cells of a WT-1 -expressing cancer, the method comprising administering to said subject the vaccine of claim 9, thereby inducing the formation and proliferation of CTL specific for cells of a WT-1 -expressing cancer.
14. The method of claim 13, wherein said WT-1 -expressing cancer is a leukemia, a desmoplastic small round cell tumor, a gastric cancer, a colon cancer, a lung cancer, a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, a renal cancer, a kaposi's sarcoma, a sarcoma, a hepatocellular carcinoma, a Wilms' tumor, an acute myelogenous leukemia (AML), a myelodysplastic syndrome (MDS), or a non-small cell lung cancer (NSCLC).
15. A composition comprising (a) an antigen-presenting cell and (b) a peptide of any one of claims 1-6.
16. The method of any one of claims 11-14 wherein the cancer is mesothelioma.
PCT/US2013/021448 2012-01-13 2013-01-14 Immunogenic wt-1 peptides and methods of use thereof WO2013106834A2 (en)

Priority Applications (11)

Application Number Priority Date Filing Date Title
EP18215878.2A EP3520810A3 (en) 2012-01-13 2013-01-14 Immunogenic wt1 peptides and use thereof
JP2014552368A JP6282598B2 (en) 2012-01-13 2013-01-14 Immunogenic WT1 peptides and methods of use thereof
CA2861206A CA2861206C (en) 2012-01-13 2013-01-14 Immunogenic wt-1 peptides and methods of use thereof
AU2013207669A AU2013207669C1 (en) 2012-01-13 2013-01-14 Immunogenic WT-1 peptides and methods of use thereof
CN201380014022.7A CN104684577B (en) 2012-01-13 2013-01-14 Immunogenicity WT-1 peptides and its application method
EP13736176.2A EP2802347B1 (en) 2012-01-13 2013-01-14 Immunogenic wt-1 peptides and methods of use thereof
US14/372,174 US20150104413A1 (en) 2012-01-13 2013-01-14 Immunogenic wt-1 peptides and methods of use thereof
HK15104710.4A HK1204261A1 (en) 2012-01-13 2015-05-18 Immunogenic wt-1 peptides and methods of use thereof wt-1
US15/608,964 US10100087B2 (en) 2012-01-13 2017-05-30 Immunogenic WT-1 peptides and methods of use thereof
AU2018201210A AU2018201210B2 (en) 2012-01-13 2018-02-20 Immunogenic WT-1 peptides and methods of use thereof
US16/125,213 US10815274B2 (en) 2012-01-13 2018-09-07 Immunogenic WT-1 peptides and methods of use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201261586177P 2012-01-13 2012-01-13
US61/586,177 2012-01-13
US201261647207P 2012-05-15 2012-05-15
US61/647,207 2012-05-15

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US14/372,174 A-371-Of-International US20150104413A1 (en) 2012-01-13 2013-01-14 Immunogenic wt-1 peptides and methods of use thereof
US15/608,964 Continuation US10100087B2 (en) 2012-01-13 2017-05-30 Immunogenic WT-1 peptides and methods of use thereof

Publications (2)

Publication Number Publication Date
WO2013106834A2 true WO2013106834A2 (en) 2013-07-18
WO2013106834A3 WO2013106834A3 (en) 2015-01-22

Family

ID=48782099

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/021448 WO2013106834A2 (en) 2012-01-13 2013-01-14 Immunogenic wt-1 peptides and methods of use thereof

Country Status (8)

Country Link
US (3) US20150104413A1 (en)
EP (2) EP3520810A3 (en)
JP (2) JP6282598B2 (en)
CN (2) CN104684577B (en)
AU (2) AU2013207669C1 (en)
CA (1) CA2861206C (en)
HK (1) HK1204261A1 (en)
WO (1) WO2013106834A2 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016208332A1 (en) * 2015-06-25 2016-12-29 国立大学法人神戸大学 Oral tumor vaccine
WO2017044678A1 (en) * 2015-09-10 2017-03-16 Memorial Sloan Kettering Cancer Center Methods of treating multiple myeloma and plasma cell leukemia by t cell therapy
JP2017518270A (en) * 2014-04-24 2017-07-06 ロード アイランド ホスピタル Aspartate-β-hydroxylase induces epitope-specific T cell responses in tumors
WO2019094607A3 (en) * 2017-11-08 2019-06-20 Advaxis, Inc. Immunogenic heteroclitic peptides from cancer-associated proteins and methods of use thereof
EP3520810A2 (en) 2012-01-13 2019-08-07 Memorial Sloan-Kettering Cancer Center Immunogenic wt1 peptides and use thereof
US11033613B2 (en) 2015-11-20 2021-06-15 Memorial Sloan Kettering Cancer Center Methods and compositions for treating cancer
US11859004B2 (en) 2017-01-06 2024-01-02 Eutilex Co., Ltd. Anti-human 4-1BB antibodies and uses thereof
US11897927B2 (en) 2016-11-30 2024-02-13 Advaxis, Inc. Immunogenic compositions targeting recurrent cancer mutations and methods of use thereof

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102208505B1 (en) 2012-12-11 2021-01-27 앨버트 아인슈타인 컬리지 오브 메디신 Methods for high throughput receptor:ligand identification
CN108604257B (en) 2015-10-12 2022-12-13 南托米克斯有限责任公司 Methods of producing specific immunotherapeutic compositions and related nucleic acid constructs
WO2017201210A1 (en) 2016-05-18 2017-11-23 Cue Biopharma, Inc. T-cell modulatory multimeric polypeptides and methods of use thereof
US11339201B2 (en) 2016-05-18 2022-05-24 Albert Einstein College Of Medicine Variant PD-L1 polypeptides, T-cell modulatory multimeric polypeptides, and methods of use thereof
AU2017379900A1 (en) 2016-12-22 2019-06-13 Cue Biopharma, Inc. T-cell modulatory multimeric polypeptides and methods of use thereof
EP3565829A4 (en) 2017-01-09 2021-01-27 Cue Biopharma, Inc. T-cell modulatory multimeric polypeptides and methods of use thereof
EP3596118B1 (en) 2017-03-15 2024-08-21 Cue Biopharma, Inc. Combination of multimeric fusion polypeptides and immune checkpoint inhibitor for treating hpv-associated cancer
CA3074839A1 (en) 2017-09-07 2019-03-14 Cue Biopharma, Inc. T-cell modulatory multimeric polypeptide with conjugation sites and methods of use thereof
WO2019139896A1 (en) 2018-01-09 2019-07-18 Cue Biopharma, Inc. Multimeric t-cell modulatory polypeptides and methods of use thereof
WO2019227106A1 (en) * 2018-05-25 2019-11-28 The Wistar Institute Tumor-specific neoantigens and methods of using the same
WO2020132366A2 (en) * 2018-12-19 2020-06-25 Cue Biopharma, Inc. T-cell modulatory multimeric polypeptides with conjugation sites and methods of use thereof
CN110423723A (en) * 2019-07-18 2019-11-08 南方医科大学南方医院 A kind of construction method of peripheral blood B cell system and its application
CA3162690A1 (en) 2019-12-31 2021-07-08 Minoru S. H. Ko Temperature-based transient delivery of nucleic acids and proteins to cells and tissues
IL296209A (en) 2020-05-12 2022-11-01 Cue Biopharma Inc Multimeric t-cell modulatory polypeptides and methods of use thereof
JP2023541366A (en) 2020-09-09 2023-10-02 キュー バイオファーマ， インコーポレイテッド MHC class II T cell modulating multimeric polypeptides and methods of use thereof to treat type 1 diabetes mellitus (T1D)

Family Cites Families (118)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA8748A (en) 1878-05-04 George W. Ainsworth Improvements on clothes dryers
CA23766A (en) 1886-04-06 Albert M. Todd Process of, and appartus for treating essential oils to obtain the concrete or crystalline part thereof separate and apart from the liquid portion
CA59350A (en) 1898-02-26 1898-03-19 Thomas Henry Simmonds Brake for velocipede, tricycle, etc.
US4722848A (en) 1982-12-08 1988-02-02 Health Research, Incorporated Method for immunizing animals with synthetically modified vaccinia virus
US5837249A (en) 1985-04-19 1998-11-17 The Wistar Institute Method for generating an immunogenic T cell response protective against a virus
US5726288A (en) * 1989-11-13 1998-03-10 Massachusetts Institute Of Technology Localization and characterization of the Wilms' tumor gene
US5229115A (en) 1990-07-26 1993-07-20 Immunex Corporation Adoptive immunotherapy with interleukin-7
EP0573608A1 (en) 1991-02-07 1993-12-15 Molecumetics, Ltd. Conformationally restricted mimetics of beta turns and beta bulges and peptides containing the same
CA2158281A1 (en) 1993-03-15 1994-09-29 Jay A. Berzofsky Peptide coated dendritic cells as immunogens
AU2392795A (en) 1994-04-22 1995-11-16 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The Isolation and characterization of a novel primate t-cell lymphotropic virus and the use of this virus or components thereof in diagnostics assays and vaccines
US5622835A (en) 1994-04-28 1997-04-22 The Wistar Institute Of Anatomy & Biology WT1 monoclonal antibodies
US5643786A (en) 1995-01-27 1997-07-01 The United States Of America As Represented By The Department Of Health And Human Services Method for isolating dendritic cells
US6156316A (en) 1995-05-08 2000-12-05 Sloan-Kettering Institute For Cancer Research Oncogene fusion protein peptide vaccines
JPH11171896A (en) 1995-09-19 1999-06-29 Kirin Brewery Co Ltd New peptide compound and its pharmaceutical composition
US5981217A (en) 1995-12-11 1999-11-09 Mayo Foundation For Medical Education And Research DNA encoding TGF-β inducible early factor-1 (TIEF-1), a gene expressed by osteoblasts
PT879282E (en) 1996-01-17 2003-11-28 Imp College Innovations Ltd IMMUNOTHERAPY USING CITOTOXIC T-LYMPHOCYTES (CTL)
US5853719A (en) 1996-04-30 1998-12-29 Duke University Methods for treating cancers and pathogen infections using antigen-presenting cells loaded with RNA
US6207375B1 (en) 1996-12-11 2001-03-27 Mayo Foundation For Medical Educational & Research TGF-β inducible early factor-1 (TIEF-1) and a method to detect breast cancer
ID27813A (en) 1998-01-28 2001-04-26 Corixa Corp COMPOUNDS FOR THERAPY AND DIAGNOSIS OF LUNG CANCER AND METHODS FOR USE
BRPI9912663B8 (en) 1998-07-31 2021-07-06 Int Inst Cancer Immunology Inc vaccine against cancer and polypeptide.
US20030072767A1 (en) 1998-09-30 2003-04-17 Alexander Gaiger Compositions and methods for WT1 specific immunotherapy
US7144581B2 (en) 2000-10-09 2006-12-05 Corixa Corporation Compositions and methods for WT1 specific immunotherapy
US7115272B1 (en) 1998-09-30 2006-10-03 Corixa Corporation Compositions and methods for WT1 specific immunotherapy
US20030235557A1 (en) 1998-09-30 2003-12-25 Corixa Corporation Compositions and methods for WT1 specific immunotherapy
US7655249B2 (en) 1998-09-30 2010-02-02 Corixa Corporation Compositions and methods for WT1 specific immunotherapy
US7329410B1 (en) 1998-09-30 2008-02-12 Corixa Corporation Compositions and method for WT1 specific immunotherapy
US7063854B1 (en) * 1998-09-30 2006-06-20 Corixa Corporation Composition and methods for WTI specific immunotherapy
US7901693B2 (en) 1998-09-30 2011-03-08 Corixa Corporation Compositions and methods for WT1 specific immunotherapy
US20120301492A1 (en) 1998-09-30 2012-11-29 Corixa Corporation Compositions and methods for wt1 specific immunotherapy
GB9823897D0 (en) 1998-11-02 1998-12-30 Imp College Innovations Ltd Immunotherapeutic methods and molecules
EP1144444A3 (en) 1998-12-22 2002-01-02 Genset Dnas encoding proteins with signal sequences
AU3395900A (en) 1999-03-12 2000-10-04 Human Genome Sciences, Inc. Human lung cancer associated gene sequences and polypeptides
CA2367692A1 (en) 1999-03-15 2000-09-21 Introgen Therapeutics, Inc. Dendritic cells transduced with a wild-type self gene elicit potent antitumor immune responses
US6593299B1 (en) * 1999-04-21 2003-07-15 University Of Florida Research Foundation, Inc. Compositions and methods for controlling pests
AU7859900A (en) 1999-10-04 2001-05-10 Corixa Corporation Compositions and methods for wt1 specific immunotherapy
US20030082194A1 (en) 2000-02-22 2003-05-01 Alexander Gaiger Compositions and methods for diagnosis and therapy of malignant mesothelioma
AU2001247220A1 (en) * 2000-02-22 2001-09-03 Corixa Corporation Compositions and methods for diagnosis and therapy of malignant mesothelioma
US20040002068A1 (en) 2000-03-01 2004-01-01 Corixa Corporation Compositions and methods for the detection, diagnosis and therapy of hematological malignancies
US6861234B1 (en) 2000-04-28 2005-03-01 Mannkind Corporation Method of epitope discovery
EP1209226A3 (en) 2000-11-07 2002-06-05 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Maturation of dendritic cells by recombinant heat shock protein 70 (hsp70)
TWI318630B (en) 2001-03-22 2009-12-21 Int Inst Cancer Immunology Inc Wt1 modified peptide
US8771702B2 (en) 2001-03-26 2014-07-08 The Trustees Of The University Of Pennsylvania Non-hemolytic LLO fusion proteins and methods of utilizing same
DE60238958D1 (en) 2001-06-29 2011-02-24 Chugai Pharmaceutical Co Ltd A cancer vaccine with a cancer antigen based on the product of the tumor suppressing gene WT1 and a cationic liposome
US7553494B2 (en) 2001-08-24 2009-06-30 Corixa Corporation WT1 fusion proteins
US20050002951A1 (en) 2001-09-28 2005-01-06 Haruo Sugiyama Novel method of inducing antigen-specific t cells
EP1447091A4 (en) 2001-09-28 2008-02-13 Institute Of Can International Novel method of inducing antigen-specific t cells
US20030072761A1 (en) 2001-10-16 2003-04-17 Lebowitz Jonathan Methods and compositions for targeting proteins across the blood brain barrier
US20030175272A1 (en) 2002-03-07 2003-09-18 Medcell Biologics, Inc. Re-activated T-cells for adoptive immunotherapy
CN101113163B (en) 2002-06-12 2010-10-06 株式会社国际癌症免疫研究所 Hla-a24-restricted cancer antigen peptides
KR101173871B1 (en) 2003-02-06 2012-08-16 앤저 테라퓨틱스 인코퍼레이티드 Modified free-living microbes vaccine compositions and methods of use thereof
US20050008618A1 (en) 2003-02-27 2005-01-13 Howard Kaufman Composition for delivering an agent to a target cell and uses thereof
WO2004111075A2 (en) * 2003-03-05 2004-12-23 Dendreon Corporation Alternative reading frame polypeptides for treatment
KR101431312B1 (en) 2003-06-27 2014-08-20 인터내셔널 인스티튜트 오브 캔서 이무놀로지 인코퍼레이티드 Method of selecting wt1 vaccine adaptive patient
US20050147621A1 (en) 2003-10-10 2005-07-07 Higgins Darren E. Use of bacterial 5' untranslated regions for nucleic acid expression
ES2378264T3 (en) 2003-11-05 2012-04-10 International Institute Of Cancer Immunology, Inc. HLA-DR binding antigenic peptide derived from WT1
US7488718B2 (en) 2003-12-01 2009-02-10 Sloan Kettering Institue For Cancer Research Synthetic HLA binding peptide analogues and uses thereof
ATE426617T1 (en) 2003-12-05 2009-04-15 Multimmune Gmbh THERAPEUTIC AND DIAGNOSTIC ANTI-HSP 70- ANTIBODIES
EP1550458A1 (en) 2003-12-23 2005-07-06 Vectron Therapeutics AG Synergistic liposomal adjuvants
EP1720565A1 (en) 2004-03-04 2006-11-15 Corixa Corporation Co-encapsulated wt1 polypeptide and immunostimulant microsphere formulations and methods thereof
US20050214268A1 (en) 2004-03-25 2005-09-29 Cavanagh William A Iii Methods for treating tumors and cancerous tissues
US20050221481A1 (en) 2004-03-30 2005-10-06 Istituto Superiore Di Sanita' Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2
ATE462003T1 (en) 2004-03-31 2010-04-15 Int Inst Cancer Immunology Inc CANCER ANTIGEN PEPTIDES DERIVED FROM WT1
US9629927B2 (en) 2005-07-29 2017-04-25 Sloan-Kettering Institute For Cancer Research Single wall nanotube constructs and uses thereof
EP1951281B1 (en) 2005-10-17 2015-04-15 Sloan Kettering Institute For Cancer Research Wt1 hla class ii-binding peptides and compositions and methods comprising same
KR101385805B1 (en) * 2005-11-30 2014-04-16 인터내셔널 인스티튜트 오브 캔서 이무놀로지 인코퍼레이티드 Novel peptide compound
PL1988163T3 (en) 2006-02-22 2012-11-30 Int Inst Cancer Immunology Inc Hla-a*3303-restricted wt1 peptide and pharmaceutical composition comprising the same
WO2007119564A1 (en) 2006-03-29 2007-10-25 International Institute Of Cancer Immunology, Inc. siRNA SPECIFIC TO WT1 17AA(-)ISOFORM AND USE THEREOF
EP3117836A1 (en) 2006-04-10 2017-01-18 Sloan Kettering Institute For Cancer Research Immunogenic wt-1 peptides and uses thereof
WO2007120603A2 (en) 2006-04-10 2007-10-25 Sloan Kettering Institute For Cancer Research Immunogenic bcr-abl peptides and methods of use thereof
CN101663323A (en) 2006-12-27 2010-03-03 埃默里大学 The composition and the method that are used for the treatment of transmissible disease and tumour
DK2341142T3 (en) 2006-12-28 2014-12-08 Int Inst Cancer Immunology Inc HLA-A * 1101-restricted WT1 peptide or pharmaceutical composition comprising this
WO2008096831A1 (en) 2007-02-07 2008-08-14 The Research Foundation For Microbial Diseases Of Osaka University Therapeutic agent for cancer
CN103103246B (en) 2007-02-27 2018-05-01 株式会社癌免疫研究所 The method of activation of helper t cell and the composition for this method
KR20110091817A (en) 2007-05-24 2011-08-12 글락소스미스클라인 바이오로지칼즈 에스.에이. Lyophilised antigen composition
KR100995340B1 (en) 2007-11-19 2010-11-19 재단법인서울대학교산학협력재단 Vaccine comprising Monocyte or immature myeloid cellsIMC which was loaded with the ligand of natural killer T cell and antigen
KR101592855B1 (en) 2007-12-05 2016-02-12 인터내셔널 인스티튜트 오브 캔서 이무놀로지 인코퍼레이티드 Cancer vaccine composition
KR100900837B1 (en) 2007-12-07 2009-06-04 (주)두비엘 A powerful vaccine composition comprising lipopeptide and poly(i:c) as an adjuvant
CA2724649A1 (en) 2008-05-19 2009-11-26 Anza Therapeutics, Inc. Compositions comprising prfa* mutant listeria and methods of use thereof
EP2337795A2 (en) * 2008-10-01 2011-06-29 Dako Denmark A/S Mhc multimers in cancer vaccines and immune monitoring
AR076349A1 (en) 2009-04-23 2011-06-01 Int Inst Cancer Immunology Inc CANCER ANTIGEN AUXILIARY PEPTIDE
WO2011059835A2 (en) 2009-11-11 2011-05-19 Quest Diagnostics Investments Incorporated Wt1 mutations for prognosis of myeloproliferative disorders
US20110136141A1 (en) 2009-12-03 2011-06-09 Abbott Laboratories Peptide reagents and method for inhibiting autoantibody antigen binding
US20110223187A1 (en) 2010-02-15 2011-09-15 Vafa Shahabi Live listeria-based vaccines for central nervous system therapy
MX353165B (en) 2010-08-24 2017-12-20 Univ Of Pittsburgh Of The Commonwealth System Of Higher Education Star Interleukin-13 receptor alpha 2 peptide-based brain cancer vaccines.
WO2012046730A1 (en) 2010-10-05 2012-04-12 国立大学法人大阪大学 Method for activating helper t cell
AP2013007158A0 (en) 2011-03-11 2013-10-31 Gilead Calistoga Llc Combination therapies for hematologic malignancies
CA2839663A1 (en) 2011-06-28 2013-01-03 International Institute Of Cancer Immunology, Inc. Receptor gene for peptide cancer antigen-specific t cell
EP2738253A4 (en) 2011-07-29 2015-04-22 Riken Cell for use in immunotherapy which contains modified nucleic acid construct encoding wilms tumor gene product or fragment thereof, method for producing said cell, and said nucleic acid construct
US9539299B2 (en) 2011-10-27 2017-01-10 International Institute Of Cancer Immunology, Inc. Combination therapy with WT1 peptide vaccine and temozolomide
CN104136609B (en) 2011-12-14 2016-12-07 国立大学法人高知大学 The modification of helper T lymphocyte inducing polypeptide
CN104684577B (en) 2012-01-13 2018-05-08 纪念斯隆凯特林癌症中心 Immunogenicity WT-1 peptides and its application method
US9303079B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9751928B2 (en) 2012-05-03 2017-09-05 Fred Hutchinson Cancer Research Center Enhanced affinity T cell receptors and methods for making the same
JP6163486B2 (en) 2012-07-02 2017-07-12 大日本住友製薬株式会社 Cancer antigen peptide transdermal agent
EP2911683B1 (en) 2012-10-23 2019-09-25 Ubivac, LLC Allogeneic autophagosome-enriched composition for the treatment of disease
EP2933261B1 (en) 2012-12-17 2020-06-10 Otsuka Pharmaceutical Co., Ltd. Method for activating helper t cell
WO2014103310A1 (en) 2012-12-26 2014-07-03 バイオコモ株式会社 Vaccine prepared utilizing human parainfluenza virus type 2 vector
PT2945647T (en) 2013-01-15 2020-11-26 Memorial Sloan Kettering Cancer Center Immunogenic wt-1 peptides and methods of use thereof
KR20140100419A (en) 2013-02-05 2014-08-14 닛토덴코 가부시키가이샤 Wt1 peptide cancer vaccine composition for transdermal administration
CA2840988A1 (en) 2013-02-05 2014-08-05 Nitto Denko Corporation Wt1 peptide cancer vaccine composition for mucosal administration
RU2685933C2 (en) 2013-02-05 2019-04-23 Нитто Денко Корпорейшн Anticancer vaccine composition containing peptide wt1 for transdermal administration
JP6512569B2 (en) 2013-02-05 2019-05-15 日東電工株式会社 WT1 peptide cancer vaccine tape for transdermal administration
EP2956544B1 (en) 2013-02-14 2017-11-01 Immunocellular Therapeutics Ltd. Cancer vaccines and vaccination methods
ES2694328T3 (en) 2013-03-12 2018-12-19 Sumitomo Dainippon Pharma Co., Ltd. Liquid aqueous composition
US11419925B2 (en) 2013-03-15 2022-08-23 The Trustees Of The University Of Pennsylvania Cancer vaccines and methods of treatment using the same
MX364459B (en) 2013-03-29 2019-04-26 Sumitomo Dainippon Pharma Co Ltd Wt1-antigen peptide conjugate vaccine.
MX364922B (en) 2013-04-25 2019-05-14 Vaximm Ag Salmonella-based vectors for cancer immunotherapy targeting wilms' tumor gene wt1.
KR20160007579A (en) 2013-05-13 2016-01-20 인터내셔널 인스티튜트 오브 캔서 이무놀로지 인코퍼레이티드 Method for predicting clinical effect of immunotherapy
US10898555B2 (en) 2013-07-02 2021-01-26 Japanese Foundation For Cancer Research Cellular immunity inducing vaccine
PL3071593T3 (en) 2013-11-22 2019-11-29 Univ Illinois Engineered high-affinity human t cell receptors
RU2016129044A (en) 2013-12-18 2018-01-23 Факсимм Гмбх NEW DNA VACCINE TARGETED ON MESOTELIN FOR CANCER IMMUNOTHERAPY
CA2935375C (en) 2014-01-06 2023-08-08 The Trustees Of The University Of Pennsylvania Pd1 and pdl1 antibodies and vaccine combinations and use of same for immunotherapy
EP3104878B1 (en) 2014-02-14 2019-05-22 Immune Design Corp. Immunotherapy of cancer through combination of local and systemic immune stimulation
WO2015129790A1 (en) 2014-02-26 2015-09-03 株式会社バイオイミュランス Wt1 antigenic polypeptide, and anti-tumor agent containing said polypeptide
US10023841B2 (en) 2014-05-23 2018-07-17 Baylor Research Institute Methods and compositions for treating breast cancer with dendritic cell vaccines
US10525096B2 (en) 2014-12-11 2020-01-07 International Institute Of Cancer Immunology, Inc. Immunotherapy for angiogenic disease
US10695385B2 (en) 2015-06-25 2020-06-30 National University Corporation Kobe University Oral cancer vaccine
US20190054112A1 (en) 2015-09-18 2019-02-21 Moderna Therapeutics, Inc. Polynucleotide formulations for use in the treatment of renal diseases

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Bioinformatics & Molecular Analysis Section", NATIONAL INSTITUTES OF HEALTH
PARKER KC ET AL.: "Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains.", J IMMUNOL, vol. 152, 1994, pages 163 - 175
RAMMENSEE HG ET AL.: "SYFPEITHI: database for MHC ligands and peptide motifs", IMMUNOGENETICS, vol. 50, 1999, pages 213 - 219
See also references of EP2802347A4

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3520810A2 (en) 2012-01-13 2019-08-07 Memorial Sloan-Kettering Cancer Center Immunogenic wt1 peptides and use thereof
US10813984B2 (en) 2014-04-24 2020-10-27 Rhode Island Hospital Aspartate-β-hydroxylase induces epitope-specific T cell responses in tumors
JP7025119B2 (en) 2014-04-24 2022-02-24 ロード アイランド ホスピタル Aspartic acid-β-hydroxylase induces an epitope-specific T cell response in tumors
JP2017518270A (en) * 2014-04-24 2017-07-06 ロード アイランド ホスピタル Aspartate-β-hydroxylase induces epitope-specific T cell responses in tumors
JP2020125325A (en) * 2014-04-24 2020-08-20 ロード アイランド ホスピタル ASPARTATE-β-HYDROXYLASE INDUCES EPITOPE-SPECIFIC T CELL RESPONSES IN TUMORS
JPWO2016208332A1 (en) * 2015-06-25 2018-06-28 国立大学法人神戸大学 Oral tumor vaccine
WO2016208332A1 (en) * 2015-06-25 2016-12-29 国立大学法人神戸大学 Oral tumor vaccine
CN108350411A (en) * 2015-06-25 2018-07-31 白川利朗 Oral tumor vaccine
CN108350411B (en) * 2015-06-25 2021-06-22 白川利朗 Oral tumor vaccine
US10695385B2 (en) 2015-06-25 2020-06-30 National University Corporation Kobe University Oral cancer vaccine
CN108348552A (en) * 2015-09-10 2018-07-31 纪念斯隆-凯特林癌症中心 The method for treating Huppert's disease and plasma cell leukemia by T cell therapy
RU2743381C2 (en) * 2015-09-10 2021-02-17 Мемориал Слоан-Кеттеринг Кэнсер Сентер Methods of multiple myeloma and plasma cell leukemia treatment by t-cell therapy
WO2017044678A1 (en) * 2015-09-10 2017-03-16 Memorial Sloan Kettering Cancer Center Methods of treating multiple myeloma and plasma cell leukemia by t cell therapy
US11033613B2 (en) 2015-11-20 2021-06-15 Memorial Sloan Kettering Cancer Center Methods and compositions for treating cancer
EP4130026A1 (en) 2015-11-20 2023-02-08 Memorial Sloan-Kettering Cancer Center Methods and compositions for treating cancer
US11897927B2 (en) 2016-11-30 2024-02-13 Advaxis, Inc. Immunogenic compositions targeting recurrent cancer mutations and methods of use thereof
US11859004B2 (en) 2017-01-06 2024-01-02 Eutilex Co., Ltd. Anti-human 4-1BB antibodies and uses thereof
WO2019094607A3 (en) * 2017-11-08 2019-06-20 Advaxis, Inc. Immunogenic heteroclitic peptides from cancer-associated proteins and methods of use thereof

Also Published As

Publication number Publication date
EP2802347A2 (en) 2014-11-19
AU2018201210A1 (en) 2018-03-08
HK1204261A1 (en) 2015-11-13
JP2015512866A (en) 2015-04-30
WO2013106834A3 (en) 2015-01-22
US10815274B2 (en) 2020-10-27
CN104684577A (en) 2015-06-03
EP2802347A4 (en) 2016-06-01
AU2018201210B2 (en) 2020-04-09
AU2013207669A1 (en) 2014-08-07
EP2802347B1 (en) 2019-01-09
CA2861206A1 (en) 2013-07-18
US20190092813A1 (en) 2019-03-28
JP6995645B2 (en) 2022-01-14
EP3520810A2 (en) 2019-08-07
US10100087B2 (en) 2018-10-16
EP3520810A3 (en) 2019-11-06
CN104684577B (en) 2018-05-08
JP2018104438A (en) 2018-07-05
CN108676069A (en) 2018-10-19
CA2861206C (en) 2021-07-06
AU2013207669B2 (en) 2017-11-23
AU2013207669C1 (en) 2018-05-31
US20170334951A1 (en) 2017-11-23
JP6282598B2 (en) 2018-02-21
AU2013207669B9 (en) 2017-12-14
US20150104413A1 (en) 2015-04-16

Similar Documents

Publication Publication Date Title
AU2018201210B2 (en) Immunogenic WT-1 peptides and methods of use thereof
US11548924B2 (en) WT1 HLA class II-binding peptides and compositions and methods comprising same
AU2020250313B2 (en) Immunogenic WT-1 peptides and methods of use thereof
US11859015B2 (en) Immunogenic WT-1 peptides and methods of use thereof
WO2007120603A2 (en) Immunogenic bcr-abl peptides and methods of use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13736176

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 2014552368

Country of ref document: JP

Kind code of ref document: A

Ref document number: 2861206

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 14372174

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2013207669

Country of ref document: AU

Date of ref document: 20130114

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2013736176

Country of ref document: EP