Multi-valent immunotherapy composition, and use and method for treating WT1-positive cancers

Abstract

To provide methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer.SOLUTION: An immunotherapy composition comprises: (a) a combination of at least seven isolated peptides consisting of YMFPNAPYL, RSDELVRHHNMHQRNMTKL, PGCNKRYFKLSHLQMHSRKHTG, SGQAYMFPNAPYLPSCLES, NLMNLGATL, WNLMNLGATLKGVAA, and WNYMNLGATLKGVAA; (b) a nucleic acid encoding the combination of the at least seven isolated peptides; (c) immune cells comprising the nucleic acid and / or comprising or presenting the at least seven isolated peptides; (d) cytotoxic T cells (CTLs) induced by the combination of the at least seven isolated peptides; or (e) a combination of any two, three, or all four of the components (a) to (d).SELECTED DRAWING: None

Description

Technical Field

[0001] Cross-reference to related applications This application claims the benefit of priority to U.S. Provisional Application No. 62 / 832,244, filed Apr. 10, 2019, and is incorporated by reference in its entirety, including drawings, nucleic acid sequences, and amino acid sequences.

[0002] The Sequence Listing for this application was created on Apr. 9, 2020, and is labeled “Seq-List.txt” and is 47 KB in size. The entire contents of the Sequence Listing are hereby incorporated by reference in their entirety.

Background Art

[0003] The present invention provides methods for treating WT1-expressing cancer, reducing its incidence, and inducing an immune response thereto, and compositions useful for the same purpose.

Prior Art Documents

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Summary of the Invention

Means for Solving the Problems

[0005] The present invention provides a method for treating WT1-expressing cancer, reducing its incidence, and inducing an immune response thereto, and a composition comprising an immunogenic composition useful for the same purpose. In one embodiment, the present invention provides a method for such use comprising administering to a subject in need thereof a combination of at least the following 7 WT1 peptides: YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26), and WNYMNLGATLKGVAA (SEQ ID NO: 205). In some embodiments, all 7 peptides are present in the composition administered to the subject as a multivalent (heptavalent) immunotherapy composition.

[0006] The combination of at least 7 WT1 peptides can be administered to a subject by administering to the subject one or more WT1 delivery agents, as a result of which delivery of the combination of WT1 peptides and an immune response against WT1-expressing cancer are induced. Examples of these WT1 delivery agents used include (i) one or more isolated WT1 peptides themselves, (ii) one or more nucleic acids encoding WT1 peptides, and (iii) one or more immune cells comprising or presenting a combination of nucleic acids encoding WT1 peptides or a combination of WT1 peptides, or any combination of two or three of (i), (ii) or (iii).

[0007] Administration of one or more of at least seven WT1 peptides as peptides, via other forms of WT1 delivery agents such as nucleic acids encoding the WT1 peptides, or via immune cells that contain or present the WT1 peptides, and administration of others of at least seven WT1 peptides is desired. Thus, at least seven WT1 peptides are administered in the form of peptides, or in the form of nucleic acids encoding the peptides, or in the form of immune cells that contain or present the WT1 peptides or the encoding nucleic acids, or in two or all three of these forms (i.e., peptide and nucleic acid, peptide and immune cell, nucleic acid and immune cell, or peptide, nucleic acid, and immune cell). As such, the combination of at least seven WT1 peptides may be administered (i) individually or in any combination of one or more, as seven or more peptides, or (ii) individually or in any combination of one or more, as nucleic acids encoding combinations of at least seven WT1 peptides, or (iii) individually or in any combination of one or more, as immune cells that contain or present combinations of nucleic acids encoding at least seven WT1 peptides or combinations of at least seven or more peptides. In some embodiments, at least seven WT1 peptides are administered as combinations of two or all three of these forms (i.e., (i) and (ii), or (i) and (iii), or (ii) and (iii), or (i), (ii), and (iii))).

[0008] Optionally, instead of or in addition to a WT1 delivery agent (peptide, nucleic acid encoding a peptide, and immune cells) for WT1-expressing cancer, cytotoxic T cells (CTLs) against WT1-expressing cancer can be administered to a subject, and the CTLs are induced by a combination of at least seven isolated peptides: YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26), and WNYMNLGATLKGVAA (SEQ ID NO: 205).

[0009] Optionally, in addition to a combination of seven WT1 peptides, a nucleic acid encoding seven WT1 peptides, immune cells containing or presenting seven WT1 peptides, and / or immune cells containing and / or presenting a nucleic acid encoding a WT1 peptide, or CTLs induced by seven WT1 peptides, one or more additional WT1 peptides, or CTLs induced by one or more additional WT1 peptides may be included in the composition or administered to the subject. The one or more additional WT1 peptides may be natural peptides that are fragments of the WT1 protein, or they may be peptides having one or more modifications that enhance their immunogenicity, or they may be a mixture. Such modifications are amino acid changes (e.g., heteroclitic peptides), or any other modification. The one or more additional WT1 peptides may be administered to the subject via a WT1 delivery agent, or CTLs against WT1-expressing cancer that are induced by one or more additional WT1 peptides may be administered.

[0010] Optionally, one or more checkpoint inhibitors may be administered to the subject before, during, or after administration of the WT1 delivery agent and / or CTL. One or more checkpoint inhibitors (also known as immune checkpoint inhibitors) are compounds or agents that block or inhibit immune checkpoint proteins. Examples of compounds or agents that are checkpoint inhibitors include, but are not limited to, small molecules, peptides, and antibodies. Examples of antibodies include nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011), MEDI680 (AMP-514), AMP-224, AUNP-12, BMS936559, atezolizumab (MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS93559 (MDX-1105), rHIgM12B7, BMS-986016, GSK2831781, IMP321, rilumab (BMS-986015), IPH2101 (1-7F9), indoximod (NLG9189), NLG919, INCB024360, PF-05082566, urelumab (BMS-6635, but are not limited to these.

[0011] In one embodiment, a method is embodied in which one or more WT1 delivery agents or CTLs, and one or more checkpoint inhibitors are each administered to a subject according to a schedule that provides maximum benefit to the subject. Thus, the one or more WT1 delivery agents or CTLs and the one or more checkpoint inhibitors are not necessarily administered simultaneously, or in the same composition, or each over the same period of time, or each by the same route. Each WT1 peptide, like each checkpoint inhibitor, can be administered according to a specific schedule. In one embodiment, the dosing schedules of at least one WT1 peptide and at least one checkpoint inhibitor are simultaneous. In one embodiment, the dosing schedules of at least one WT1 peptide and at least one checkpoint inhibitor overlap. In one embodiment, at least one WT1 delivery agent or CTL and at least one checkpoint inhibitor are present in the same composition. In one embodiment, the methods embodied herein provide an enhanced or increased ability to treat WT1-expressing cancers, reduce their incidence, and induce an immune response thereto, compared to WT1 delivery agents or CTLs and checkpoint inhibitors alone. In one embodiment, the ability to treat WT1-expressing cancers, reduce their incidence, and induce an immune response thereto, provided by the methods described herein, is greater than the combination of the effects of WT1 delivery agents or CTLs alone and checkpoint inhibitors alone.

[0012] The dosage levels and dosing schedules of the WT1 delivery agent, or CTL, the dosage levels and dosing schedules of the checkpoint inhibitor, the route of administration, and other modes of administration are optimized for maximum benefit to the subject. Embodiments herein provide improved methods of treating WT1-expressing cancers, reducing their incidence, and inducing an immune response thereto, and improved compositions useful for the same purpose.

[0013] The cancer according to the method embodied in this specification is any cancer that expresses the WT1 protein or a fragment thereof. In one embodiment, the cancer is ovarian cancer. In other embodiments, the cancer is breast cancer. In other embodiments, the cancer is colon cancer or colorectal cancer. In other embodiments, the cancer is mesothelioma. In other embodiments, the cancer is leukemia. In other embodiments, the cancer is Wilms tumor, acute myeloid leukemia (AML), multiple myeloma, chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), melanoma, mesothelioma (e.g., malignant pleural mesothelioma), gastric cancer, prostate cancer, biliary tract cancer, urinary system cancer, glioblastoma, soft tissue sarcoma, osteosarcoma, or non-small cell lung cancer (NSCLC).

Embodiments for Carrying Out the Invention

[0014] The present invention provides a method for treating WT1-expressing cancer, reducing its incidence, and inducing an immune response thereto, and a composition comprising an immunogenic composition useful for the same purpose. In one embodiment, the present invention provides a method for such use, comprising administering to a subject in need thereof a combination of at least seven WT1 peptides or cytotoxic T lymphocytes (CTLs) thereto. Here, the combination includes each of YMFPNAPYL (also called SEQ ID NO: 124 and WT1-A1), RSDELVRHHNMHQRNMTKL (also called SEQ ID NO: 1 and WT1-427 long), PGCNKRYFKLSHLQMHSRKHTG (also called SEQ ID NO: 2 and WT1-331 long), SGQAYMFPNAPYLPSCLES (also called SEQ ID NO: 125 and WT-122A1 long), NLMNLGATL (also called SEQ ID NO: 21 and NLM short), WNLMNLGATLKGVAA (also called SEQ ID NO: 26 and WNLM or NLM long), and WNYMNLGATLKGVAA (also called SEQ ID NO: 205 and WNYM or NYM long). The combination of seven WT1 peptides is administered with or without one or more checkpoint inhibitors. In some embodiments, the immunotherapy composition is used to treat WT1-expressing tumors or to induce the formation and proliferation of T cells specific for WT1-expressing cancer in vitro, ex vivo, or in vivo, and the combination has a synergistic effect on one or more of the foregoing.

[0015] Combinations of at least seven WT1 peptides can be administered to a subject by administering to the subject one or more agents that effect delivery of the combination of at least seven WT1 peptides and induction of an immune response against WT1-expressing cancer (i.e., one or more WT1 delivery agents). Each of the at least seven WT1 peptides is delivered in one or more of the same or different WT1 delivery agents and in one or more combinations thereof. Examples of such WT1 delivery agents that can be used include: (i) isolated WT1 peptides, (ii) nucleic acids encoding at least one WT1 peptide, and (iii) immune cells that contain or present at least one WT1 peptide or nucleic acid encoding at least one WT1 peptide. Thus, in some embodiments, the combination of at least seven WT1 peptides is administered in the form of seven isolated WT1 peptides. In some embodiments, the composition is administered to a subject, and the composition contains all seven isolated WT1 peptides.

[0016] Optionally, instead of or in addition to the WT1 delivery agents (peptides, nucleic acids encoding peptides, and immune cells), cytotoxic T lymphocytes (CTLs) against WT1-expressing cancer can be administered to the subject, and the CTLs are induced by a combination of at least seven isolated peptides: YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26), and WNYMNLGATLKGVAA (SEQ ID NO: 205). The CTLs are made in vitro or ex vivo, or are made in vivo in a donor, and contain WT1-specific CTLs obtained from the donor.

[0017] The WT1 delivery agent or CTL may be provided in a composition containing a carrier, excipient, or diluent, which may be an adjuvant therein. The selection of peptide components used in the methods and compositions embodied herein will be described hereinafter, but is not limited thereto.

[0018] Ovarian cancer is one of the most common gynecological malignancies and is the fifth leading cause of cancer death in women in the United States. More than 22,000 cases are diagnosed each year, and an estimated 15,500 deaths occur annually [1]. The majority of patients have extensive lesions at the time of onset [2]. The 5-year survival rate for advanced cases remains less than 30% [1]. A complete clinical remission after initial chemotherapy is expected in many patients, but a review of second-look laparotomies, which are often performed as a routine care issue, has shown that less than 50% of patients are actually disease-free [3]. Furthermore, nearly half of the patients with a negative second-look surgery relapse and require additional treatment [4]. Many patients achieve a second clinical complete response with additional chemotherapy. However, almost all patients relapse after a short remission interval of 9 to 11 months [5]. Subsequent remissions are gradually shorter in duration until chemoresistance is widely manifested, so effective strategies to prolong the remission period or prevent relapse are needed [2].

[0019] Both antibodies and T cell effectors have been shown to confer benefits in ovarian cancer models. Antibodies have been noted to suppress early tissue infiltration [6]. In preclinical models, the use of both passively administered antibodies and vaccine-induced antibodies has demonstrated clearance of circulating tumor cells and elimination of systemic micrometastases. With regard to T cell effectors, it has been shown that a generally activated immune response is associated with improved clinical outcomes in patients with progressive ovarian cancer. Zhang et al. showed that the presence of tumor-infiltrating T cells within tumor cell islands was associated with improved progression-free survival and overall survival [7]. Conversely, infiltration of regulatory T cells worsens the prognosis [8].

[0020] Data from ovarian cancer patients in remission for two or more times have confirmed that recurrence occurs in a predictable manner [9]. In recent years, ovarian cancer has been targeted by various novel immune-based approaches. Antibody therapies include oregovomab, a monoclonal antibody therapy targeting the CA125 antigen

[10] , abagovomab, an anti-idiotype antibody targeting CA-125

[11] , and trastuzumab, a monoclonal humanized anti-HER2 antibody

[12] . Other strategies include cytokine therapies such as interferon-γ [13, 14] and IL-2

[15] . Active immunization with other antigens such as Lewis y

[16] , MUC1

[17] , HLA-restricted peptide NY-ESO-1b

[18] , and KH-1-KLH conjugate has also been evaluated. The strategies so far have been ineffective, and new therapies are needed not only for ovarian cancer that has not been effectively treated with currently available therapies but also to enhance the effectiveness of therapies for many other cancers.

[0021] WT1 refers to the gene product of Wilms tumor 1 or the WT1 gene. The Wilms tumor suppressor gene WT1 was first identified in pediatric kidney tumors, but WT1 is also highly expressed in multiple other hematological malignancies and solid tumors including mesothelioma [19, 20]. WT1 was originally identified by cDNA mapping to the region of chromosome 11p13. WT1 cDNA encodes a protein containing four Kruppel zinc fingers and includes a complex pattern of alternative splicing that gives rise to four different transcription factors. Each WT1 isoform has different DNA binding and transcriptional activities

[21] and can positively or negatively regulate various genes involved in cell proliferation, differentiation, apoptosis, organogenesis, and sex determination. WT1 is normally expressed in tissues of mesodermal origin during embryonic development, including the kidney, gonads, heart, mesothelium, and spleen

[22] . In normal adult tissues, WT1 expression is usually limited to low levels in the nuclei of CD34+ hematopoietic stem cells, myoepithelial progenitor cells, renal podocytes, and some cells in the testis and ovary

[23] . WT1 is highly homologous in mice and humans (96% at the amino acid level) and has similar tissue distribution and functions [24, 25]. Although originally described as a tumor suppressor gene, the WT1 protein appears to be involved in tumor formation.

[0022] In combination with the proposed mechanism of action, the strong expression of WT1 protein in ovarian cancer, as well as in many other cancers where WT1 protein is also expressed, but not limited to, mesothelioma, leukemia, Wilms tumor, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), melanoma, gastric cancer, prostate cancer, cholangiocarcinoma, urinary system cancer, glioblastoma, soft tissue sarcoma, osteosarcoma, and non-small cell lung cancer (NSCLC), etc., is a reasonable target for immunotherapy. In ovarian cancer, due to its very high expression frequency, pathologists routinely use immunohistochemical staining of WT1 (using a standardized protocol to describe the expression and determine "positive" or "negative") to help distinguish epithelial ovarian cancer from other tumors. WT1 is particularly sensitive and specific as a marker for serous ovarian cancer

[26] . Ovarian tissue microarrays suggest that 70-80% of serous ovarian cancers express WT1, and as a result, the majority of patients have a target and are eligible to participate in the study.

[0023] One or more additional WT1 peptides used in combination with the seven WT1 peptides are natural peptides that are fragments of the WT1 protein. In one embodiment, the one or more additional WT1 peptides are LVRHHNMHQRNMTKL (SEQ ID NO: 3) or NKRYFKLSHLQMHSR (SEQ ID NO: 4). In other embodiments, the one or more additional peptides are SGQARMFPNAPYLPSCLES (SEQ ID NO: 5) or QARMFPNAPYLPSCL (SEQ ID NO: 6). In other embodiments, the one or more additional peptides are selected from RMFPNAPYL (SEQ ID NO: 7), SLGEQQYSV (SEQ ID NO: 8), ALLPAVPSL (SEQ ID NO: 9), NLGATLKGV (SEQ ID NO: 10), DLNALLPAV (SEQ ID NO: 11), GVFRGIQDV (SEQ ID NO: 12), KRYFKLSHL (SEQ ID NO: 13), ALLLRTPYS (SEQ ID NO: 14), CMTWMQMNL (SEQ ID NO: 15), NMHQRNMTK (SEQ ID NO: 16), QMNLGATLK (SEQ ID NO: 17), FMCAYPGCNK (SEQ ID NO: 18), or KLSHLQMHSR (SEQ ID NO: 19).

[0024] In other embodiments, one or more additional WT1 peptides are NQMNLGATL (SEQ ID NO: 20), NYMNLGATL (SEQ ID NO: 22), CMTWNQMNLGATLKG (SEQ ID NO: 23), CMTWNLMNLGATLKG (SEQ ID NO: 24), WNQMNLGATLKGVAA (SEQ ID NO: 25), MTWNQMNLGATLKGV (SEQ ID NO: 27), TWNQMNLGATLKGVA (SEQ ID NO: 28), CMTWNLMNLGATLKG (SEQ ID NO: 29), MTWNLMNLGATLKGV (SEQ ID NO: 30), TWNLMNLGATLKGVA (SEQ ID NO: 31), WNLMNLGATLKGVAA (SEQ ID NO: 32), MTWNYMNLGATLKGV (SEQ ID NO: 33), TWNYMNLGATLKGVA (SEQ ID NO: 34), CMTWNQMNLGATLKGVA (SEQ ID NO: 35), WNQMNLGAT (SEQ ID NO: 36), TWNQMNLGA (SEQ ID NO: 37), MTWNQMNLG (SEQ ID NO: 38), CMTWNLMNLGATLKGVA (SEQ ID NO: 39), WNLMNLGAT (SEQ ID NO: 40), MNLGATLKG (SEQ ID NO: 41), MTWNQMNLG (SEQ ID NO: 42), CMTWNYMNLGATLKGVA (SEQ ID NO: 43), MNLGATLKG (SEQ ID NO: 44), MTWNQMNLG (SEQ ID NO: 45), GALRNPTAC (SEQ ID NO: 46), GYLRNPTAC (SEQ ID NO: 47), GALRNPTAL (SEQ ID NO: 48), YALRNPTAC (SEQ ID NO: 49), GLLRNPTAC (SEQ ID NO: 50), RQRPHPGAL (SEQ ID NO: 51), RYRPHPGAL (SEQ ID NO: 52), YQRPHPGAL (SEQ ID NO: 53), RLRPHPGAL (SEQ ID NO: 54), RIRPHPGAL (SEQ ID NO: 55), GALRNPTAC (SEQ ID NO: 56), GALRNPTAL (SEQ ID NO: 57), RQRPHPGAL (SEQ ID NO: 58), RLRPHPGAL (SEQ ID NO: 59), RIRPHPGAL (SEQ ID NO: 60), QFPNHSFKHEDPMGQ (SEQ ID NO: 61), QFPNHSFKHEDPMGQ (SEQ ID NO: 62), HSFKHEDPM (SEQ ID NO: 63), HSFKHEDPY (SEQ ID NO: 64), HSFKHEDPK (SEQ ID NO: 65), KRPFMCAYPGCYKRY (SEQ ID NO: 66), SEKRPFMCAYPGCNK (SEQ ID NO: 67), KRPFMCAYPGCNK (SEQ ID NO: 68).It is selected from FMCAYPGCN (SEQ ID NO: 69), FMCAYPGCY (SEQ ID NO: 70) or FMCAYPGCK (SEQ ID NO: 71).

[0025] In other embodiments, one or more additional WT1 peptides are RQRPHPGAL (SEQ ID NO: 72), GALRNPTAC (SEQ ID NO: 73), PLPHFPPSL (SEQ ID NO: 74), HFPPSLPPT (SEQ ID NO: 75), THSPTHPPR (SEQ ID NO: 76), AILDFLLLQ (SEQ ID NO: 77), PGCLQQPEQ (SEQ ID NO: 78), PGCLQQPEQQG (SEQ ID NO: 79), KLGAAEASA (SEQ ID NO: 80), ASGSEPQQM (SEQ ID NO: 81), RDLNALLPAV (SEQ ID NO: 82), GGCALPVSGA (SEQ ID NO: 83), GAAQWAPVL (SEQ ID NO: 84), LDFAPPGAS (SEQ ID NO: 85), LDFAPPGASAY (SEQ ID NO: 86), SAYGSLGGP (SEQ ID NO: 87), PAPPPPPPP (SEQ ID NO: 88), ACRYGPFGP (SEQ ID NO: 89), SGQARMFPN (SEQ ID NO: 90), RMFPNAPYL (SEQ ID NO: 91), PSCLESQPA (SEQ ID NO: 92), NQGYSTVTF (SEQ ID NO: 93), HHAAQFPNH (SEQ ID NO: 94), HSFKHEDPM (SEQ ID NO: 9), CHTPTDSCT (SEQ ID NO: 96), CTGSQALLL (SEQ ID NO: 97), TDSCTGSQA (SEQ ID NO: 98), RTPYSSDNL (SEQ ID NO: 99), NLYQMTSQLE (SEQ ID NO: 100), WNQMNLGAT (SEQ ID NO: 101), NQMNLGATL (SEQ ID NO: 102), WNQMNLGATLK (SEQ ID NO: 103), CMTWNQMNLGATLKG (SEQ ID NO: 104), NLGATLKGV (SEQ ID NO: 105), LGATLKGVAA (SEQ ID NO: 106), TLGVAAGS (SEQ ID NO: 107), GYESDNHTT (SEQ ID NO: 108), FMCAYPGCNK (SEQ ID NO: 109), KRPFMCAYPGC (SEQ ID NO: 110), RKFSRSDHL (SEQ ID NO: 111), LKTHTTRTHT (SEQ ID NO: 112), NMHQRNHTKL (SEQ ID NO: 113), LLAAILDFL (SEQ ID NO: 114), CLQQPEQQGV (SEQ ID NO: 115), DLNALLPAV (SEQ ID NO: 116), ALLPAVPSL (SEQ ID NO: 117), VLDFAPPGA (SEQ ID NO: 118), CMTWNQMNL (SEQ ID NO: 119), QARMFPNAPY (SEQ ID NO: 120), ALRNPTACPL (SEQ ID NO: 121),It is selected from YPGCNKRYF (SEQ ID NO: 122) or APVLDFAPPGASAYG (SEQ ID NO: 123).

[0026] In other embodiments, one or more additional WT1 peptides are any peptides described in WO2017087857, WO2014113490, or WO2019006401. The above are incorporated herein by reference in their entirety.

[0027] In other embodiments, one or more additional WT1 peptides are any native WT1 peptides described in WO2005053618, WO2007047763, WO2007047764, WO2007120673, US20060084609, WO2014113490, and WO2013106834. The above are incorporated herein by reference in their entirety.

[0028] In other embodiments, one or more additional WT1 peptides are any native WT1 peptides described in US20110070251A1, US7,063,854B1, US7,063,854, US7901693, US7662386, US7,063,854, US7115272, US7368119, US7329410, US7144581, US7323181, US76555249, US7553494, US7608685, US7380871, US7030212, US7807792, US7517950, US2010 / 0166738, US2011 / 0070251, US2009 / 0143291, and WO2003037060. The above are incorporated herein by reference in their entirety.

[0029] In other embodiments, one or more additional WT1 peptides are any of the native WT1 peptides described in US76666985B2, US20080070835A1, US20070128207A1, US791539B2, US20110136141A1, US7598221B2, US20100111986A1, US20100092522A1, US2003008219494A1, and WO2001025273A2. The foregoing are incorporated herein by reference in their entirety.

[0030] One or more additional WT1 peptides may be modified WT1 peptide fragments that include one or more heteroclitic modifications to enhance immunogenicity against the native peptide sequence. In other embodiments, an additional WT1 peptide is QAYMFPNAPYLPSCL (SEQ ID NO: 126). In other embodiments, one or more additional peptides are any of YLGEQQYSV (SEQ ID NO: 127), YLLPAVPSL (SEQ ID NO: 128), YLGATLKGV (SEQ ID NO: 129), YLNALLPAV (SEQ ID NO: 130), GLRRGIQDV (SEQ ID NO: 131), KLYFKLSHL (SEQ ID NO: 132), ALLLRTPYV (SEQ ID NO: 133), YMTWNQMNL (SEQ ID NO: 134), NMYQRNMTK (SEQ ID NO: 135), NMHQRVMTK (SEQ ID NO: 136), NMYQRVMTK (SEQ ID NO: 137), QMYLGATLK (SEQ ID NO: 138), QMNLGVTLK (SEQ ID NO: 139), QMYLGVTLK (SEQ ID NO: 140), FMYAYPGCNK (SEQ ID NO: 141), FMCAYPFCNK (SEQ ID NO: 142), FMYAYPFCNK (SEQ ID NO: 143), KLYHLQMHSR (SEQ ID NO: 144), KLSHLQMHSK (SEQ ID NO: 145), and KLYHLQMHSK (SEQ ID NO: 146).

[0031] In other embodiments, one or more additional WT1 peptides are NQMNLGATL (SEQ ID NO: 147), NYMNLGATL (SEQ ID NO: 149), CMTWNQMNLGATLKG (SEQ ID NO: 150), CMTWNLMNLGATLKG (SEQ ID NO: 151), WNQMNLGATLKGVAA (SEQ ID NO: 152), MTWNQMNLGATLKGV (SEQ ID NO: 154), TWNQMNLGATLKGVA (SEQ ID NO: 155), CMTWNLMNLGATLKG (SEQ ID NO: 156), MTWNLMNLGATLKGV (SEQ ID NO: 157), TWNLMNLGATLKGVA (SEQ ID NO: 158), WNLMNLGATLKGVAA (SEQ ID NO: 159), MTWNYMNLGATLKGV (SEQ ID NO: 160), TWNYMNLGATLKGVA (SEQ ID NO: 161), CMTWNQMNLGATLKGVA (SEQ ID NO: 162), WNQMNLGAT (SEQ ID NO: 163), TWNQMNLGA (SEQ ID NO: 164), MTWNQMNLG (SEQ ID NO: 165), CMTWNLMNLGATLKGVA (SEQ ID NO: 166), WNLMNLGAT (SEQ ID NO: 167), MNLGATLKG (SEQ ID NO: 168), MTWNQMNLG (SEQ ID NO: 169), CMTWNYMNLGATLKGVA (SEQ ID NO: 170), MNLGATLKG (SEQ ID NO: 171), MTWNQMNLG (SEQ ID NO: 172), GALRNPTAC (SEQ ID NO: 173), GYLRNPTAC (SEQ ID NO: 174), GALRNPTAL (SEQ ID NO: 175), YALRNPTAC (SEQ ID NO: 176), GLLRNPTAC (SEQ ID NO: 177), RQRPHPGAL (SEQ ID NO: 178), RYRPHPGAL (SEQ ID NO: 179), YQRPHPGAL (SEQ ID NO: 180), RLRPHPGAL (SEQ ID NO: 181), RIRPHPGAL (SEQ ID NO: 182), GALRNPTAC (SEQ ID NO: 183), GALRNPTAL (SEQ ID NO: 184), RQRPHPGAL (SEQ ID NO: 185), RLRPHPGAL (SEQ ID NO: 186), RIRPHPGAL (SEQ ID NO: 187), QFPNHSFKHEDPMGQ (SEQ ID NO: 188), QFPNHSFKHEDPMGQ (SEQ ID NO: 189), HSFKHEDPM (SEQ ID NO: 190), HSFKHEDPY (SEQ ID NO: 191), HSFKHEDPK (SEQ ID NO: 192), KRPFMCAYPGCYKRY (SEQ ID NO: 193).SEKRPFMCAYPGCNK (SEQ ID NO: 194), KRPFMCAYPGCNK (SEQ ID NO: 195), FMCAYPGCN (SEQ ID NO: 196), and FMCAYPGCY (SEQ ID NO: 197) are any modified WT1 peptides of FMCAYPGCK (SEQ ID NO: 198).

[0032] In other embodiments, the WT1 peptide is any modified WT1 peptide described in WO2005053618, WO2007047763, WO2007047764, WO2007120673, US20060084609, WO2014113490, and WO2013106834. The above are hereby incorporated by reference in their entirety herein.

[0033] In other embodiments, the WT1 peptide is any modified WT1 peptide described in US20110070251A1, US7,063,854B1, US7,063,854, US7901693, US7662386, 7,063,854, US7115272, US7368119, US7329410, US7144581, US7323181, US76555249, US7553,494, US7608685, US7380871, US7030212, US7807792, US7517950, US2010 / 0166738, US2011 / 0070251, US2009 / 0143291, and WO2003037060. The above are hereby incorporated by reference in their entirety herein.

[0034] In other embodiments, the WT1 peptide is any modified WT1 peptide described in US76666985B2, US20080070835A1, US20070128207A1, US791539B2, US20110136141A1, US7598221B2, US20100111986A1, US20100092522A1, US2003008219494A1, and WO2001025273A2. The above are hereby incorporated by reference in their entirety herein.

[0035] One or more additional WT1 peptides useful for the purposes described herein are a single peptide or a combination of peptides. Each of the one or more additional WT1 peptides is either a native WT1 peptide or a modified WT1 peptide. When two or more peptides are used, they are each administered individually (in separate formulations) or in combination with one or more other peptides (in the same formulation). One or more peptides are administered in combination with a carrier, diluent, or excipient. In one embodiment, the peptide is administered in combination with an adjuvant. Each peptide may be administered with a different adjuvant or combination of adjuvants, or multiple peptides may be administered in combination as a combination of multiple adjuvants with a combination of two or more peptides. An immunogen or composition containing one or more peptides is referred to herein as a vaccine, peptide vaccine, WT1 vaccine, etc.

[0036] Adjuvants can be of any class, such as alum salts and other mineral adjuvants, bacterial products or bacteria-derived adjuvants, tensioactive agents (e.g., saponins), oil-in-water (o / w) and water-in-oil (w / o) emulsions, liposome adjuvants, cytokines (e.g., IL-2, GM-CSF, IL-12, and IFN-gamma), and alpha-galactosylceramide analogs. Examples of adjuvants include, but are not limited to, Montanide emulsions, QS21, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, Bacillus Calmette-Guerin (BCG), and alum. In one embodiment, the adjuvant is an agent that enhances the CTL response of the immune system to a WT1 peptide, such as the surfactant manidomonooleate (Montanide ISA 51 VG w / o emulsion) containing plant-grade (VG) oleic acid derived from olive oil. The adjuvant is administered in the same composition as one or more WT1 peptides, or in the same composition as one or more checkpoint inhibitors, or in the same composition as both one or more WT1 peptides and one or more checkpoint inhibitors, or in a composition separate from one or more WT1 peptides and one or more checkpoint inhibitors.

[0037] In other embodiments, any of the aforementioned peptides (the seven WT1 peptides, and optionally, combinations of one or more additional WT1 peptides) have one or more point mutations in the primary or secondary anchor residues of the HLA class I binding motif. In one embodiment, the peptide has a point mutation at position 2 or 9 of the class I binding motif, or at positions 1, 3, 4, 5, 6, 7 or 8 of the secondary anchor residues of the class I binding motif. In one embodiment, position 1 of the peptide, HLA class I binding motif is changed to glycine, threonine or phenylalanine. In one embodiment, position 2 of the HLA class I binding motif is changed to leucine or isoleucine. In one embodiment, position 6 of the HLA class I binding motif is changed to valine, glutamine or histidine. In one embodiment, position 9 of the HLA class I binding motif is changed to valine, alanine, threonine, isoleucine, or cysteine.

[0038] Optionally, the combination of seven WT1 peptides is one or more natural or modified WT1 peptides disclosed in WO2014113490, for example, NQMNLGATL (SEQ ID NO: 147), NLMNLGATL (SEQ ID NO:), NYMNLGATL (SEQ ID NO: 149), CMTWNQMNLGATLKG (SEQ ID NO: 150), CMTWNLMNLGATLKG (SEQ ID NO: 151), WNQMNLGATLKGVAA (SEQ ID NO: 152), MTWNQMNLGATLKGV (SEQ ID NO: 154), TWNQMNLGATLKGVA (SEQ ID NO: 155), CMTWNLMNLGATLKG (SEQ ID NO: 156), MTWNLMNLGATLKGV (SEQ ID NO: 157), TWNLMNLGATLKGVA (SEQ ID NO: 158), WNLMNLGATLKGVAA (SEQ ID NO: 159), MTWNYMNLGATLKGV (SEQ ID NO: 1260), TWNYMNLGATLKGVA (SEQ ID NO: 161), CMTWNQMNLGATLKGVA (SEQ ID NO: 162), WNQMNLGAT (SEQ ID NO: 163), TWNQMNLGA (SEQ ID NO: 164), MTWNQMNLG (SEQ ID NO: 165), CMTWNLMNLGATLKGVA (SEQ ID NO: 166), WNLMNLGAT (SEQ ID NO: 167), MNLGATLKG (SEQ ID NO: 168), MTWNQMNLG (SEQ ID NO: 169), CMTWNYMNLGATLKGVA (SEQ ID NO: 170), MNLGATLKG (SEQ ID NO: 171), MTWNQMNLG (SEQ ID NO: 172), GALRNPTAC (SEQ ID NO: 173), GYLRNPTAC (SEQ ID NO: 174), GALRNPTAL (SEQ ID NO: 175), YALRNPTAC (SEQ ID NO: 176), GLLRNPTAC (SEQ ID NO: 177), RQRPHPGAL (SEQ ID NO: 178), RYRPHPGAL (SEQ ID NO: 179), YQRPHPGAL (SEQ ID NO: 180), RLRPHPGAL (SEQ ID NO: 181), RIRPHPGAL (SEQ ID NO: 182), GALRNPTAC (SEQ ID NO: 183), GALRNPTAL (SEQ ID NO: 184), RQRPHPGAL (SEQ ID NO: 185), RLRPHPGAL (SEQ ID NO: 186), RIRPHPGAL (SEQ ID NO: 187), QFPNHSFKHEDPMGQ (SEQ ID NO: 188), QFPNHSFKHEDPMGQ (SEQ ID NO: 189), HSFKHEDPM (SEQ ID NO: 190),It further comprises HSFKHEDPY (SEQ ID NO: 191), HSFKHEDPK (SEQ ID NO: 192), KRPFMCAYPGCYKRY (SEQ ID NO: 194), SEKRPFMCAYPGCNK (SEQ ID NO: 194), KRPFMCAYPGCNK (SEQ ID NO: 195), FMCAYPGCN (SEQ ID NO: 196), FMCAYPGCY (SEQ ID NO: 197) or FMCAYPGCK (SEQ ID NO: 198).

[0039] Each peptide of the combination may be administered separately within its own formulation, or two, three, four, five, six, or seven or more peptides of the combination may be administered together within the same formulation. In one embodiment, a combination of at least seven WT1 peptides is administered within the same formulation.

[0040] The dosage level of each peptide, the individual dosing frequency, or the dosing frequency, dosing period, and other aspects of immunization with one or more combinations of up to seven or more peptides, are optimized according to the patient's clinical profile, disease duration or course, co-morbidities, and other aspects of clinical care. The present invention is not limited with respect to particular aspects of the immunizing components of the methods embodied herein.

[0041] In one embodiment, the multivalent immunotherapy composition comprises 280 mcg each of the seven aforementioned peptides YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26), and WNYMNLGATLKGVAA (SEQ ID NO: 205). In one embodiment, the composition further comprises one or more additional WT1 peptides. In one embodiment, the composition does not comprise an additional WT1 peptide. In one embodiment, the composition does not comprise an additional peptide.

[0042] In one embodiment, each peptide of 200 mcg is administered at each dose (0.5 ml). In one embodiment, each peptide of 100 - 2000 mcg is administered at each dose. In one embodiment, the aforementioned doses are administered once every week over a period of 10 weeks (i.e., 6 administrations). In one embodiment, the administration is subcutaneous. In one embodiment, the adjuvant is mixed (emulsified) with the vaccine prior to administration. In one embodiment, 0.5 mL of the immunotherapy composition (i.e., 200 mcg of each peptide) is emulsified with 1.0 mL of the adjuvant prior to administration. In other embodiments, the adjuvant is injected at the same site as the vaccine either before or after the immunotherapy composition is injected. In one embodiment, the adjuvant is an emulsion. In one embodiment, the emulsion is a Montanide emulsion. In one embodiment, the Montanide emulsion is Montanide ISA51VG, which is an immunological adjuvant. Optionally, in the practice of the present invention, one or more checkpoint inhibitors are also administered to the subject together with the immunotherapy composition as further described below.

[0043] As described above, an immunotherapeutic composition comprising or consisting of a combination of seven WT1 peptides may be administered as an immunogenic composition for inducing an immune response against WT1-expressing cancer, or in other embodiments, the combination of WT1 peptides may be used to prepare WT1-specific CTLs using in vitro or ex vivo methods, and the CTLs, upon administration to a patient, are directed against WT1-expressing cancer. In one embodiment, a combination of at least seven WT1 peptides is used, for example, using cells from a cell line, to induce the generation of CTLs in vitro. In other embodiments, a combination of at least seven WT1 peptides is used to induce the generation of CTLs in a sample of cells collected from a patient, and the ex vivo-induced CTLs are injected back into the same patient in need thereof. In other embodiments, a combination of at least seven WT1 peptides is used to induce the generation of CTLs in a sample of cells collected from a donor, where the ex vivo-induced CTLs are injected into a patient in need thereof who is not the donor. In other embodiments, a combination of more than seven WT1 peptides described herein is administered to a subject who is not a patient in need of treatment to induce the formation of CTLs, and then this is transferred from the donor to the patient. Each of these embodiments is another aspect of the invention and, as described herein, a source of WT1-specific cells useful in treating cancer or reducing the incidence or recurrence thereof. In any of the foregoing embodiments, CTLs against each of at least seven WT1 peptides are prepared individually or in combination. CTLs prepared individually can be administered separately to a subject or combined prior to administration to a subject.

[0044] In any of the foregoing methods, whether immunotherapy is performed on a patient to induce a CTL response against WT1-expressing cancer, or WT1-specific CTLs are obtained from a donor by an in vitro or ex vivo method using immune cells from a cell line of the patient or a donor who is not the patient, the combined use of checkpoint inhibitors can be optionally embodied herein. A method for reducing cancer treatment, cancer incidence, or its recurrence rate, whether by immunizing a subject in need thereof with a combination of seven or more WT1 peptides, or by generating CTLs in vitro, ex vivo, or in a donor subject, is independent of this. In any of these methods, the combined use of one or more checkpoint inhibitors may be optionally embodied herein. One or more checkpoint inhibitors may be administered to a patient immunized with one or more WT1 peptides. Checkpoint inhibitors are used in vitro or ex vivo to enhance the formation of WT1-specific CTLs subsequently infused into the patient. One or more checkpoint inhibitors are used in a donor subject to enhance the formation of WT1-specific CTLs that are then transferred to the patient. Checkpoint inhibitors are used in a patient receiving CTLs prepared in vitro, ex vivo, or in a donor, regardless of whether the donor was also administered a checkpoint inhibitor. In the latter embodiment, the same or different one or more checkpoint inhibitors may be used in vitro, ex vivo, or in a donor subject, and in the patient.

[0045] Immune checkpoints regulate T cell function in the immune system. T cells play a central role in cellular immunity. Checkpoint proteins send signals to T cells and interact with specific ligands that essentially turn off or inhibit T cell function. Cancer cells utilize this system by driving the high-level expression of checkpoint proteins on their surface, thereby controlling T cells expressing checkpoint proteins on the surface of T cells that have entered the tumor microenvironment and suppressing the anti-cancer immune response. Thus, inhibition of checkpoint proteins restores T cell function and brings about an immune response against cancer cells. An immune checkpoint inhibitor (or checkpoint inhibitor) is a compound or agent that blocks or inhibits an immune checkpoint protein (i.e., blocks or inhibits a checkpoint receptor or a checkpoint receptor ligand). Examples of checkpoint proteins include CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4 (which belongs to the CD2 family of molecules and is present on all NK cells and memory CD8 +Those expressed in T cells include, but are not limited to, CD160 (also called BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. Programmed death-1 (PD-1) is a member of the immunoglobulin superfamily (IGSF) of molecules involved in the regulation of T cell activation. PD-1 was named "programmed death" when it was identified in 1992 as a gene upregulated in T cell hybridomas that undergo cell death. The structure of PD-1 consists of one IGSF domain, a transmembrane domain, and an intracellular domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM)

[38] . PD-1 has two binding partners, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). PD-L1 is widely expressed in both hematopoietic and non-hematopoietic lineages [39, 40]. In addition to T cells, B cells, macrophages, NK cells, DCs, and mast cells, it is also found in peripheral tissues [41, 42]. PD-1 involvement represents one means by which tumors evade immune surveillance and clearance

[43] . Blockade of the PD-1 pathway has been demonstrated by nivolumab, which shows activity in immune-competent mouse cancer models

[44] .

[0046] Examples of checkpoint inhibitors include, but are not limited to, small molecules, peptides, and antibodies. Examples of antibodies include nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011), MEDI680 (AMP-514), AMP-224, AUNP-12, BMS 936559, atezolizumab (MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS93559 (MDX-1105), rHIgM12B7, BMS-986016, GSK2831781, IMP321, rilumab (BMS-986015), IPH2101 (1-7F9), indoximod (NLG 9189), NLG919, INCB024360, PF-05082566, urelumab (BMS-6635, but are not limited to these.

[0047] Nivolumab (OPDIVO) is a fully human IgG4 monoclonal antibody that targets the PD-1 receptor on activated T and B lymphocytes

[47] . Pembrolizumab (KEYTRUDA) is another non-limiting example of an antibody that targets PD-1. Other compounds and agents that block, inhibit, or target checkpoint proteins include compounds that are under investigation and are not yet commercially available. The present invention is not limited to specific checkpoint inhibitors. Examples of checkpoint inhibitors that may be used are listed in Table 1, but are not limited to these.

[0048] [Table 1] [Table 2]

[0049] In one embodiment, a combination of two or more checkpoint inhibitors is administered to a subject. In one embodiment, the combination of checkpoint inhibitors is selected from those in Table 1. The two or more checkpoint inhibitors can be administered simultaneously or sequentially with respect to each other and with respect to one or more WT1 peptides. In a further embodiment, the combination of two or more checkpoint inhibitors targets two different checkpoint proteins, e.g., PD-1 (e.g., nivolumab or other PD-1 inhibitor) and CTLA-4 (e.g., ipilimumab or other CTLA-4 inhibitor), and is administered to the subject simultaneously or sequentially with respect to each other and with respect to one or more WT1 peptides. In one embodiment, the combination of two or more checkpoint inhibitors targets two or more different checkpoint proteins of CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1 kinase, CHK2 kinase, A2aR, and B-7 family ligands. In one embodiment, the combination of two or more checkpoint inhibitors targeting two or more different checkpoint proteins is selected from those in Table 1.

[0050] The dosage level, dosing frequency, dosing period, and other aspects of administration of the checkpoint inhibitor are optimized according to the clinical symptoms of the patient, the duration or course of the disease, complications, and other aspects of clinical care. The present invention is not limited with respect to particular aspects of the checkpoint inhibitor component of the methods embodied herein.

[0051] In one embodiment, the dose of nivolumab and the schedule of 3 mg / kg are administered every two weeks over a 12-week course. In one embodiment, the administration is intravenous. In one embodiment, the process of checkpoint inhibitor administration is simultaneous with the process of WT1 vaccine administration. In one embodiment, the process of checkpoint inhibitor administration overlaps with the process of WT1 vaccine administration. In one embodiment, the process of checkpoint inhibitor administration is initiated substantially simultaneously with the process of WT1 vaccine administration.

[0052] In one embodiment, the immunotherapy composition comprises 200 mcg of peptide YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26), and WNYMNLGATLKGVAA (SEQ ID NO: 205), combined in a total volume of 0.5 mL emulsified in 1.0 mL of Montanide ISA51VG and administered subcutaneously every two weeks for six administrations, and nivolumab 3 mg / kg is initiated simultaneously with the WT1 immunotherapy and administered intravenously by infusion over 60 minutes every two weeks for seven administrations.

[0053] In one embodiment, a method is embodied herein in which a combination of seven or more WT1 peptides, and optionally one or more checkpoint inhibitors, are each administered to a subject according to a schedule that provides the maximum benefit to the patient. Thus, one or more WT1 peptides and one or more checkpoint inhibitors are not necessarily administered simultaneously, or in the same composition, or over the same period of time. Each WT1 peptide, or combination of WT1 peptides, is administered according to a specific schedule, as is each checkpoint inhibitor. In one embodiment, the combination of seven or more WT1 peptides and one or more checkpoint inhibitors is present in the same composition, but is not limited thereto.

[0054] As described herein, the dosage levels and dosing schedules include the frequency and duration of the WT1 peptides (administered separately or together), the dosage levels and dosing schedules of one or more checkpoint inhibitors (administered separately or together), the route of administration, and other modes of administration, and are optimized for maximum benefit to the patient subject. The same considerations apply if the donor subject is the recipient of the WT1 peptides and checkpoint inhibitors for the purpose of generating WT1-specific CTLs for administration to the patient.

[0055] In one embodiment, a composition is provided that comprises a combination of at least seven WT1 peptides and at least one checkpoint inhibitor. In one embodiment, the WT1 peptides in the composition are those disclosed herein. In one embodiment, the checkpoint inhibitor is one disclosed herein. In one embodiment, the composition comprises the checkpoint inhibitor nivolumab, pembrolizumab, or a combination thereof. The composition may further comprise an excipient, diluent, or carrier. The composition may also comprise one or more adjuvants.

[0056] The foregoing embodiments provide improved methods of treating WT1-expressing cancers, reducing their incidence, and inducing an immune response thereto, and compositions useful for the same purpose. Other aspects of the invention are further described below.

[0057] In one embodiment, the modified WT1 peptide has one or more modified amino acids and is referred to herein as a mutant WT1 peptide. In one embodiment, the mutant WT1 peptide comprises (a) a binding motif for a human leukocyte antigen (HLA) class II molecule and (b) a binding motif for an HLA class I molecule that contains point mutations in one or more anchor residues of the HLA class I molecule binding motif. In other embodiments, the peptide is 11 or more amino acids in length. In certain other embodiments, the peptide is 11-22, 11-30, 16-22, or 16-30 amino acids in length. In other embodiments, the point mutation is in 1-3 anchor residues of the HLA class I molecule binding motif. In other embodiments, the point mutation is in one anchor residue of the HLA class I molecule binding motif. In other embodiments, the point mutation is in two anchor residues of the HLA class I molecule binding motif. In other embodiments, the point mutation is in 1-2 anchor residues of the HLA class I molecule binding motif. In other embodiments, the point mutation is in 2-3 anchor residues of the HLA class I molecule binding motif. In other embodiments, the point mutation is in 1-4 anchor residues of the HLA class I molecule binding motif. Each possibility corresponds to a separate embodiment of the invention.

[0058] In other embodiments, the invention provides a method of treating a subject having a WT1-expressing cancer, the method comprising administering to the subject a combination of at least seven WT1 peptides and optionally at least one checkpoint inhibitor, thereby treating the subject having a WT1-expressing cancer.

[0059] In other embodiments, the invention provides a method of reducing the incidence of WT1-expressing cancer or its recurrence in a subject, the method comprising administering to the subject a combination of at least seven WT1 peptides and optionally at least one checkpoint inhibitor, thereby reducing the incidence of WT1-expressing cancer or its recurrence in the subject.

[0060] In other embodiments, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs, the method comprising contacting a lymphocyte population with a combination of at least seven WT1 peptides and optionally at least one checkpoint inhibitor, thereby inducing the formation and proliferation of WT1 protein-specific CTLs.

[0061] In other embodiments, the present invention provides a method for inducing the formation and proliferation of (a) WT1 protein-specific CD8 + lymphocytes and (b) CD4 + lymphocytes specific for the WT1 protein, the method comprising contacting a lymphocyte population with a combination of at least seven WT1 peptides and optionally at least one checkpoint inhibitor, thereby inducing the formation and proliferation of (a) WT1 protein-specific CD8 + lymphocytes and (b) CD4 + lymphocytes specific for the WT1 protein.

[0062] In one embodiment, the aforementioned method of treating WT1-expressing cancer, reducing the incidence of WT1-expressing cancer, or inducing the formation and proliferation of WT1 protein-specific T cell responses achieves a greater effect than when such a method uses only a combination of at least seven WT1 peptides alone or checkpoint inhibitors alone. In one embodiment, the administration process of the WT1 immunotherapy and the administration process of one or more checkpoint inhibitors are simultaneous, overlapping, or concurrent such that the biological response to the vaccine is enhanced by the administration of one or more checkpoint inhibitors. Concurrent administration includes a course of WT1 immunotherapy for inducing WT1-specific CTLs and the administration of one or more checkpoint inhibitors for enhancing the activity of CTLs against cancer. In one embodiment, the course of WT1 vaccine administration can be completed before the course of checkpoint inhibitor therapy begins, as long as the effectiveness of the CTLs induced by WT1 immunotherapy administration is enhanced by checkpoint inhibitor therapy. In one embodiment, the first administration of checkpoint inhibitor therapy is on the same day as the last WT1 immunotherapy administration. In one embodiment, the end of WT1 immunotherapy and the start of checkpoint inhibitor therapy are separated by 1 to 7 days or 1 to 4 weeks.

[0063] As described herein, one or more additional WT1 peptides are natural fragments or contiguous amino acid sequences of the WT1 protein, or they may have one or more modifications of the amino acid sequence to enhance immunogenicity or any other beneficial property to the peptide and the generation of immunity against WT1-expressing cancer. In certain embodiments, one or more amino acids are altered to enhance immunogenicity. In one embodiment, the method of use employs an isolated mutant WT1 peptide that includes (a) a binding motif for human leukocyte antigen (HLA) class II molecules and (b) a binding motif for HLA class I molecules having point mutations in one or more anchor residues of the HLA class I molecule binding motif. In other embodiments, the peptide is 11 aa or longer in length. Each possibility corresponds to a separate embodiment of the invention.

[0064] In other embodiments, the "point mutation" indicates that the fragment is mutated with respect to the native sequence of the protein and thus creates an HLA class I molecule binding motif. In other embodiments, the "point mutation" enhances the binding ability of the HLA class I molecule binding motif present in the native sequence. Each possibility corresponds to a separate embodiment of the methods of use of the invention.

[0065] In other embodiments, the point mutation is in one to three anchor residues of the HLA class I molecule binding motif. In other embodiments, the point mutation is in one anchor residue of the HLA class I molecule binding motif. In other embodiments, the point mutation is in two anchor residues of the HLA class I molecule binding motif. In other embodiments, the point mutation is in one to two anchor residues of the HLA class I molecule binding motif. In other embodiments, the point mutation is in two to three anchor residues of the HLA class I molecule binding motif. In other embodiments, the point mutation is in one to four anchor residues of the HLA class I molecule binding motif. Each possibility corresponds to a separate embodiment of the invention.

[0066] In other embodiments, the peptides of the invention are from 11 to 453 amino acids (AA) in length. In other embodiments, the length is from 12 to 453 AA. In other embodiments, the length is from 13 to 453 AA. In other embodiments, the length is from 14 to 453 AA. In other embodiments, the length is from 15 to 453 AA. In other embodiments, the length is from 16 to 453 AA. In other embodiments, the length is from 17 to 453 AA. In other embodiments, the length is from 18 to 453 AA. In other embodiments, the length is from 19 to 453 AA. In other embodiments, the length is from 20 to 453 AA.

[0067] In other embodiments, the length is 11 - 449 amino acids (AA). In other embodiments, the length is 12 - 449 AA. In other embodiments, the length is 13 - 449 AA. In other embodiments, the length is 14 - 449 AA. In other embodiments, the length is 15 - 449 AA. In other embodiments, the length is 16 - 449 AA. In other embodiments, the length is 17 - 449 AA. In other embodiments, the length is 18 - 449 AA. In other embodiments, the length is 19 - 449 AA. In other embodiments, the length is 20 - 449 AA.

[0068] In other embodiments, the length is 11 - 30 AA. In other embodiments, the length is 16 - 22 AA. In other embodiments, the length is 19 AA. In other embodiments, the length of the peptide is 15 - 23 AA. In other embodiments, the length is 15 - 24 AA. In other embodiments, the length is 15 - 25 AA. In other embodiments, the length is 15 - 26 AA. In other embodiments, the length is 15 - 27 AA. In other embodiments, the length is 15 - 28 AA. In other embodiments, the length is 14 - 30 AA. In other embodiments, the length is 14 - 29 AA. In other embodiments, the length is 14 - 28 AA. In other embodiments, the length is 14 - 26 AA. In other embodiments, the length is 14 - 24 AA. In other embodiments, the length is 14 - 22 AA. In other embodiments, the length is 14 - 20 AA. In other embodiments, the length is 16 - 30 AA. In other embodiments, the length is 16 - 28 AA. In other embodiments, the length is 16 - 26 AA. In other embodiments, the length is 16 - 24 AA. In other embodiments, the length is 16 - 22 AA. In other embodiments, the length is 18 - 30 AA. In other embodiments, the length is 18 - 28 AA. In other embodiments, the length is 18 - 26 AA. In other embodiments, the length is 18 - 24 AA. In other embodiments, the length is 18 - 22 AA. In other embodiments, the length is 18 - 20 AA. In other embodiments, the length is 20 - 30 AA. In other embodiments, the length is 20 - 28 AA. In other embodiments, the length is 20 - 26 AA. In other embodiments, the length is 20 - 24 AA. In other embodiments, the length is 22 - 30 AA. In other embodiments, the length is 22 - 28 AA. In other embodiments, the length is 22 - 26 AA. In other embodiments, the length is 24 - 30 AA. In other embodiments, the length is 24 - 28 AA. In other embodiments, the length is 24 - 26 AA.

[0069] In other embodiments, the peptides useful in the methods and compositions of the invention are longer than the minimum length for binding to HLA class II molecules (about 12 AA in other embodiments). In other embodiments, increasing the length of the HLA class II binding peptide allows binding to two or more HLA class II molecules. In other embodiments, increasing the length allows binding to HLA class II molecules for which the binding motif is unknown. In other embodiments, increasing the length allows binding to HLA class I molecules. In other embodiments, the binding motif of the HLA class I molecule is known. In other embodiments, the binding motif of the HLA class I molecule is unknown. Each possibility corresponds to a separate embodiment of the invention.

[0070] Each of the above peptide lengths represents a separate embodiment of the invention.

[0071] In other embodiments, HLA molecules, known as major histocompatibility complex (MHC) molecules, bind to peptides and present them to immune cells. Thus, in other embodiments, the immunogenicity of a peptide is partially determined by its affinity for HLA molecules. HLA class I molecules typically interact with CD8 molecules present on cytotoxic T lymphocytes (CTLs). HLA class II molecules typically interact with CD4 molecules present on helper T lymphocytes.

[0072] In other embodiments, the peptides of the invention are immunogenic. In other embodiments, the term "immunogenic" refers to the ability to stimulate, induce, or participate in an immune response. In other embodiments, the induced immune response is a cell-mediated immune response. In other embodiments, the immune response is a combination of a cellular response and a humoral response.

[0073] In other embodiments, T cells that bind to the HLA molecule-peptide complex are activated and induced to proliferate and lyse cells that express a protein containing the peptide. T cells are typically first activated by "professional" antigen-presenting cells ("APCs", e.g., dendritic cells, monocytes, and macrophages), which present co-stimulatory molecules that promote T cell activation rather than anergy or apoptosis. In other embodiments, the response is heteroclitic as described herein, such that CTLs lyse neoplastic cells that express a protein having an AA sequence homologous to the peptide of the invention, or a peptide different from the peptide used to initially stimulate the T cells.

[0074] In other embodiments, the encounter of T cells with the peptides of the invention induces their differentiation into effector and / or memory T cells. Subsequent encounters with the same peptide as the effector or memory T cell, or in other embodiments, with the heteroclitic peptides of the invention, result in a faster and more robust immune response. Such responses are measured in other embodiments by measuring the degree of proliferation of the T cell population exposed to the peptide. In other embodiments, such responses are measured by any of the methods listed below herein.

[0075] In other embodiments, as described herein, a subject is exposed to a peptide of the invention that is different from the native protein being expressed, or a composition / cell population containing the peptide, and thereafter a host immune response that cross-reacts with the native protein / antigen occurs.

[0076] In other embodiments, the peptides, compositions, and vaccines of the invention stimulate an immune response that results in tumor cell lysis. In all of the foregoing embodiments, the concomitant use of checkpoint inhibitors enhances the immune response against the tumor.

[0077] In other embodiments, the HLA class I molecule binding motif of the peptide of the present invention is contained within the HLA class II molecule binding motif of the peptide. In other embodiments, the HLA class I molecule binding motif overlaps with the HLA class II molecule binding motif. In other embodiments, the HLA class I molecule binding motif does not overlap with the HLA class II molecule binding motif. Each possibility corresponds to a separate embodiment of the present invention.

[0078] In other embodiments, the HLA class II molecule in which the binding motif is contained in the peptide of the present invention is an HLA-DR molecule. In other embodiments, the HLA class II molecule is an HLA-DP molecule. In other embodiments, the HLA class II molecule is an HLA-DQ molecule.

[0079] In other embodiments, the HLA class II molecule is an HLA-DRB molecule. In other embodiments, the HLA class II molecule is DRB101. In other embodiments, the HLA class II molecule is DRB301. In other embodiments, the HLA class II molecule is DRB401. In other embodiments, the HLA class II molecule is DRB701. In other embodiments, the HLA class II molecule is DRB1101. In other embodiments, the HLA class II molecule is DRB1501. In other embodiments, the HLA class II molecule is any other HLA-DRB molecule known in the art. In other embodiments, the HLA class II molecule is an HLA-DRA molecule. In other embodiments, the HLA class II molecule is an HLA-DQA1 molecule. In other embodiments, the HLA class II molecule is an HLA-DQB1 molecule. In other embodiments, the HLA class II molecule is an HLA-DPA1 molecule. In other embodiments, the HLA class II molecule is an HLA-DPB1 molecule. In other embodiments, the HLA class II molecule is an HLA-DMA molecule. In other embodiments, the HLA class II molecule is an HLA-DMB molecule. In other embodiments, the HLA class II molecule is an HLA-DOA molecule. In other embodiments, the HLA class II molecule is an HLA-DOB molecule. In other embodiments, the HLA class II molecule is any other HLA class II molecule known in the art.

[0080] In other embodiments, the peptide of the present invention binds to two different HLA class II molecules. In other embodiments, the peptide binds to three different HLA class II molecules. In other embodiments, the peptide binds to four different HLA class II molecules. In other embodiments, the peptide binds to five different HLA class II molecules. In other embodiments, the peptide binds to six different HLA class II molecules. In other embodiments, the peptide binds to more than six distinct HLA class II molecules.

[0081] In other embodiments, the HLA class II molecules bound by the peptides of the invention are encoded by two or more different alleles at a given HLA class II locus. In other embodiments, the HLA class II molecules are encoded by three different alleles at the locus. In other embodiments, the HLA class II molecules are encoded by four different alleles at the locus. In other embodiments, the HLA class II molecules are encoded by five different alleles at the locus. In other embodiments, the HLA class II molecules are encoded by six different alleles at the locus. In other embodiments, the HLA class II molecules are encoded by more than six distinct alleles at the locus.

[0082] In other embodiments, the HLA class II molecules bound by the peptides are encoded by HLA class II genes at two different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at two or more different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at three different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at three or more different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at four different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at four or more different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at five different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at five or more different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at six different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at six or more different loci. In other embodiments, the HLA class II molecules are encoded by HLA class II genes at separate loci exceeding six. Each possibility corresponds to a separate embodiment of the invention.

[0083] In other embodiments, the peptides of the invention bind to two different HLA-DRB molecules. In other embodiments, the peptides bind to three different HLA-DRB molecules. In other embodiments, the peptides bind to four different HLA-DRB molecules. In other embodiments, the peptides bind to five different HLA-DRB molecules. In other embodiments, the peptides bind to six different HLA-DRB molecules. In other embodiments, the peptides bind to separate HLA-DRB molecules exceeding six.

[0084] In other embodiments, the HLA class II molecules bound by the WT1 peptide are encoded by HLA class II genes at two different loci. In other embodiments, the bound HLA molecules are encoded by HLA class II genes at two or more different loci. In other embodiments, the bound HLA molecules are encoded by HLA class II genes at three different loci. In other embodiments, the bound HLA molecules are encoded by HLA class II genes at three or more different loci. In other embodiments, the bound HLA molecules are encoded by HLA class II genes at four different loci. In other embodiments, the bound HLA molecules are encoded by HLA class II genes at four or more different loci. In other embodiments, the bound HLA molecules are encoded by HLA class II genes at more than four distinct loci. In other embodiments, the locus is selected from the HLA-DRB locus. In other embodiments, the HLA class II binding peptide is an HLA-DRA binding peptide. In other embodiments, the peptide is an HLA-DQA1 binding peptide. In other embodiments, the peptide is an HLA-DQB1 binding peptide. In other embodiments, the peptide is an HLA-DPA1 binding peptide. In other embodiments, the peptide is an HLA-DPB1 binding peptide. In other embodiments, the peptide is an HLA-DMA binding peptide. In other embodiments, the peptide is an HLA-DMB binding peptide. In other embodiments, the peptide is an HLA-DOA binding peptide. In other embodiments, the peptide is an HLA-DOB binding peptide. In other embodiments, the peptide binds to any other HLA class II molecule known in the art. Each possibility corresponds to a separate embodiment of the invention.

[0085] In other embodiments, the peptide of the invention binds to HLA-DRB molecules encoded by two different HLA-DRB alleles selected from DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. In other embodiments, the peptide binds to HLA-DRB molecules encoded by three different HLA-DRB alleles selected from DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. In other embodiments, the peptide binds to HLA-DRB molecules encoded by four different HLA-DRB alleles selected from DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. In other embodiments, the peptide binds to HLA-DRB molecules encoded by five different HLA-DRB alleles selected from DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. In other embodiments, the peptide binds to HLA-DRB molecules encoded by each of the following HLA-DRB alleles, DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. Each possibility corresponds to a separate embodiment of the invention.

[0086] Each of the above HLA class II molecules, types, classes, and combinations thereof represents a separate embodiment of the invention.

[0087] The HLA class I molecules containing the binding motif in the peptides of the present invention are HLA-A molecules in other embodiments. In other embodiments, the HLA class I molecule is an HLA-B molecule. In other embodiments, the HLA class I molecule is an HLA-C molecule. In other embodiments, the HLA class I molecule is an HLA-A0201 molecule. In other embodiments, the molecule is HLA A1. In other embodiments, the HLA class I molecule is HLA A2. In other embodiments, the HLA class I molecule is HLA A2.1. In other embodiments, the HLA class I molecule is HLA A3. In other embodiments, the HLA class I molecule is HLA A3.2. In other embodiments, the HLA class I molecule is HLA A11. In other embodiments, the HLA class I molecule is HLA A24. In other embodiments, the HLA class I molecule is HLA B7. In other embodiments, the HLA class I molecule is HLA B27. In other embodiments, the HLA class I molecule is HLA B8. Each possibility corresponds to a separate embodiment of the present invention.

[0088] In other embodiments, the HLA class I molecule-binding WT1 peptides of the methods and compositions of the present invention bind to the superfamily of HLA class I molecules. In other embodiments, the superfamily is the A2 superfamily. In other embodiments, the superfamily is the A3 superfamily. In other embodiments, the superfamily is the A24 superfamily. In other embodiments, the superfamily is the B7 superfamily. In other embodiments, the superfamily is the B27 superfamily. In other embodiments, the superfamily is the B44 superfamily. In other embodiments, the superfamily is the C1 superfamily. In other embodiments, the superfamily is the C4 superfamily. In other embodiments, the superfamily is any other superfamily known in the art. Each possibility corresponds to a separate embodiment of the present invention.

[0089] In other embodiments, the HLA class I molecule binding motif of the peptides of the invention exhibits an increased affinity for HLA class I molecules as compared to the non-mutated counterparts of the peptides. In other embodiments, the point mutations increase the affinity of the isolated mutant WT1 peptides for HLA class I molecules. In other embodiments, the increased affinity is related to the affinity of the isolated non-mutated WT1 peptides (for the same HLA class I molecule) induced by the isolated mutant WT1 peptides. Each possibility corresponds to a separate embodiment of the invention.

[0090] In other embodiments, the HLA class I molecule binding WT peptides of the methods and compositions of the invention have a length of 9 - 13 AA. In other embodiments, the length is 8 - 13 AA. In other embodiments, the peptides have any of the lengths of the peptides of the invention recited herein.

[0091] In other embodiments, the HLA class I molecule binding WT peptides have a length of 8 AA. In other embodiments, the peptides have a length of 9 AA. In other embodiments, the peptides have a length of 10 AA. As provided herein, natural and heteroclitic peptides of 9 - 10 AA have shown the ability to induce substantial binding to HLA class I molecules and cytokine secretion and cell lysis by CTLs.

[0092] In other embodiments, the HLA class I molecule binding WT1 peptides embedded within the WT1 peptides of the invention have one of the lengths described above. Each possibility corresponds to a separate embodiment of the invention. In one embodiment, the WT1 peptide is a peptide of a length longer than the HLA class I molecule binding WT1 peptide. The longer length peptide is degraded by the cell to the appropriate length presented by the HLA class 1 molecule.

[0093] In other embodiments, the HLA class I molecule bound by the HLA class I molecule-binding WT1 peptide is an HLA-A molecule. In other embodiments, the HLA class I molecule is an HLA-A2 molecule. In other embodiments, the HLA class I molecule is an HLA-A3 molecule. In other embodiments, the HLA class I molecule is an HLA-A11 molecule. In other embodiments, the HLA class I molecule is an HLA-B8 molecule. In other embodiments, the HLA class I molecule is an HLA-0201 molecule. In other embodiments, the HLA class I molecule binds to any other HLA class I molecule known in the art. Each possibility corresponds to a separate embodiment of the invention.

[0094] In other embodiments, the peptide of the invention retains the ability to bind to multiple HLA class II molecules, as shown by the isolated WT1 peptide from which the peptide of the invention was derived.

[0095] In all aspects herein, in the vaccines herein, or for generating CTLs in vitro, ex vivo or in a donor, the selection of one or more WT1 peptides useful for generating CTLs is embodied herein such that they match the HLA type(s) of the patient or donor, whether natural or modified.

[0096] The WT1 molecule from which the peptide of the present invention can be derived, in other embodiments, has the sequence MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO: 199, GenBank accession number AY245105).

[0097] In other embodiments, the WT1 molecule has the following sequence It has AAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO: 200, GenBank accession number NM_000378).

[0098] In other embodiments, the WT1 molecule has the following sequence It has MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO: 201, GenBank accession number NP_077742).

[0099] In other embodiments, the WT1 molecule has the following sequence MGHHHHHHHHHHSSGHIEGRHMRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFFRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO: 202).

[0100] In other embodiments, the WT1 protein comprises one of the sequences defined by one of the following GenBank accession numbers: NM_024426, NM_024425, NM_024424, NM_000378, S95530, D13624, D12496, D12497, AH003034, or X77549. In other embodiments, the WT1 protein has one of the sequences defined by one of the above GenBank accession numbers. In other embodiments, the WT1 protein is any WT1 protein known in the art. In other embodiments, the WT1 protein has any other WT1 sequence known in the art.

[0101] In other embodiments, the peptide useful for the purposes of the present invention is derived from a fragment of the WT1 protein. In other embodiments, the induction process includes the introduction of point mutations at the anchor residues of the HLA class I molecule binding motif. In other embodiments, the induction process consists of the introduction of point mutations at the anchor residues of the HLA class I molecule binding motif. In other embodiments, the peptide of the present invention differs from the corresponding fragment of the WT1 protein only by point mutations at the HLA class I molecule binding motif anchor residues. In other embodiments, the HLA class I molecule binding motif of the peptide of the present invention differs from the corresponding WT1 sequence only by point mutations at the anchor residues. Each possibility corresponds to a separate embodiment of the present invention.

[0102] In other embodiments, the induction process of the peptide of the present invention further includes one or more modifications of amino acids (AAs) to AA analogs. In other embodiments, the induction process further includes one or more modifications of peptide bonds that link two or more AAs. In other embodiments, the AA analog or peptide bond modification is one of the AA analogs or peptide bond modifications listed below. Each possibility corresponds to a separate embodiment of the present invention.

[0103] The non-mutated fragment of the WT1 protein from which the peptide of the present invention (the "counterpart" in the wild-type sequence) is derived has, in other embodiments, the sequence SGQARMFPNAPYLPSCLES (SEQ ID NO: 5). In other embodiments, the non-mutated WT1 fragment has the sequence QARMFPNAPYLPSCL (SEQ ID NO: 6). In other embodiments, the non-mutated WT1 fragment has the sequence LVRHHNMHQRNMTKL (SEQ ID NO: 3). In other embodiments, the non-mutated WT1 fragment has the sequence RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1). In other embodiments, the non-mutated WT1 fragment has the sequence NKRYFKLSHLQMHSR (SEQ ID NO: 4). In other embodiments, the non-mutated WT1 fragment has the sequence PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2). In other embodiments, the non-mutated WT1 fragment is any other WT1 fragment that includes an HLA class II molecule binding motif. In other embodiments, the non-mutated WT1 fragment is any other WT1 fragment that includes an HLA-DR molecule binding motif. In other embodiments, the non-mutated WT1 fragment includes a plurality of HLA-DR molecule binding motifs. In other embodiments, the non-mutated WT1 fragment is any other WT1 fragment that includes an HLA-DRB molecule binding motif. In other embodiments, the non-mutated WT1 fragment includes a plurality of HLA-DRB molecule binding motifs. In other embodiments, the peptide of the present invention differs from its counterpart only at the point mutations it contains. In other embodiments, the peptide of the present invention differs from its counterpart only at the mutations in the HLA class I anchor residues. Each possibility corresponds to a separate embodiment of the present invention.

[0104] In other embodiments, the peptides of the invention retain the ability to bind to HLA class II molecules, as shown by the non-mutated WT1 fragment from which the peptide was derived. In other embodiments, the peptides of the invention retain the ability to bind to multiple HLA class II molecules, as shown by the non-mutated WT1 fragment. Each possibility corresponds to a separate embodiment of the invention.

[0105] In other embodiments, the invention provides isolated peptides comprising the AA sequences GATLKGVAAGSSSSVKWT (SEQ ID NO: 203) and LKGVAAGSSSSVKWT (SEQ ID NO: 204).

[0106] "Peptide" in other embodiments of the methods and compositions of the invention refers to a compound of subunits AA linked by peptide bonds. In other embodiments, the peptide comprises AA analogs. In other embodiments, the peptide is a peptidomimetic. In other embodiments, the peptides of the invention comprise one of the AA analogs listed below. The subunits are linked by peptide bonds in other embodiments. In other embodiments, the subunits are linked by another type of bond, such as an ester, ether, etc. In other embodiments, the peptides of the invention are one of the types of peptidomimetics listed below. Each possibility corresponds to a separate embodiment of the invention.

[0107] In other embodiments, the peptides of the methods and compositions of the invention bind with high affinity to HLA class I molecules that contain their binding motif therein. In other embodiments, the HLA class I molecule is any HLA class I molecule listed herein. In other embodiments, the peptide binds to the HLA class I molecule with moderate affinity. In other embodiments, the peptide binds to the HLA class I molecule with great affinity. In other embodiments, the peptide binds to the HLA class I molecule with measurable affinity. In other embodiments, the peptide exhibits stable binding to the HLA class I molecule. Each possibility corresponds to a separate embodiment of the invention.

[0108] In other embodiments, the peptides of the methods and compositions of the invention bind with high affinity to HLA class II molecules that contain their binding motif therein. In other embodiments, the HLA class II molecule is any HLA class II molecule listed herein. In other embodiments, the peptide binds with high affinity to more than one HLA class II molecule. In other embodiments, the peptide binds to HLA class II molecules with moderate affinity. In other embodiments, the peptide binds to more than one HLA class II molecule with moderate affinity. In other embodiments, the peptide binds to HLA class II molecules with great affinity. In other embodiments, the peptide binds to more than one HLA class II molecule with great affinity. In other embodiments, the peptide binds to HLA class II molecules with measurable affinity. In other embodiments, the peptide binds to more than one HLA class II molecule with measurable affinity. In other embodiments, the peptide exhibits stable binding to HLA class II molecules. In other embodiments, the peptide exhibits stable binding to more than one HLA class II molecule. Each possibility corresponds to a separate embodiment of the invention.

[0109] In other embodiments, the peptides of the methods and compositions of the invention bind with great affinity to both HLA class I and HLA class II molecules. In other embodiments, the peptide binds with high affinity to both HLA class I and HLA class II molecules. In other embodiments, the peptide binds to both HLA class I and HLA class II molecules with moderate affinity. In other embodiments, the peptide binds to both HLA class I and HLA class II molecules with measurable affinity. Each possibility corresponds to a separate embodiment of the invention.

[0110] In other embodiments, a "fragment" refers to a peptide of AA with a length of 11 or more. In other embodiments, the peptide fragment of the present invention has an AA length of 16 or more. In other embodiments, the fragment has an AA length of 12 or more. In other embodiments, the fragment is 13 or more AAs. In other embodiments, the fragment is 14 or more AAs. In other embodiments, the fragment is 15 or more AAs. In other embodiments, the fragment is 17 or more AAs. In other embodiments, the fragment is 18 or more AAs. In other embodiments, the fragment is 19 or more AAs. In other embodiments, the fragment is 22 or more AAs. In other embodiments, the fragment is 8 - 12 AAs. In other embodiments, the fragment is about 8 - 12 AAs. In other embodiments, the fragment is 16 - 19 AAs. In other embodiments, the fragment is about 16 - 19 AAs. In other embodiments, the fragment is 10 - 25 AAs. In other embodiments, the fragment is about 10 - 25 AAs. In other embodiments, the fragment has any other length. Each possibility corresponds to a separate embodiment of the present invention.

[0111] In one embodiment, the present invention YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26) and a combination (a) of at least seven isolated peptides consisting of WNYMNLGATLKGVAA (SEQ ID NO: 205), or (b) a nucleic acid encoding the combination of at least seven isolated peptides of (a), or An immunocyte (c) comprising a nucleic acid encoding a combination of at least 7 peptides of (a), and / or comprising and / or presenting at least 7 peptides of (a), or A cytotoxic T lymphocyte (CTL) (d) induced by a combination of at least 7 isolated peptides of (a), or Provided is a composition comprising a combination (e) of any two, three, or four of (a), (b), (c), and (d).

[0112] In one embodiment, the composition comprises a combination of WT1 peptides each comprising YMFPNAPYL (also called SEQ ID NO: 124, WT1-A1), RSDELVRHHNMHQRNMTKL (also called SEQ ID NO: 1, WT1-427 long), PGCNKRYFKLSHLQMHSRKHTG (also called SEQ ID NO: 2, also called SEQ ID NO: 125, WT1-122A1 long), NLMNLGATL (also called SEQ ID NO: 21, NLM short), WNLMNLGATLKGVAA (also called SEQ ID NO: 26, WNLM or NLM long), and WNYMNLGATLKGVAA (also called SEQ ID NO: 205, WNYM or NYM long). Optionally, the composition further comprises at least one additional WT1 peptide. In certain embodiments, provided are compositions comprising at least two different isolated peptides of the invention. In certain embodiments, provided are compositions comprising at least three or at least four different isolated peptides of the invention. Each possibility corresponds to a separate embodiment of the invention. In certain embodiments, the composition of the invention is a vaccine.

[0113] In other embodiments, each peptide of the methods and compositions of the invention binds independently with high affinity to HLA class II molecules, while each peptide derived from the original peptide binds independently with high affinity to HLA class I molecules, and is independently related to each peptide comprising the combination.

[0114] In other embodiments, "affinity" refers to the concentration of peptide required to inhibit 50% of the binding of a standard peptide to the indicated MHC molecule. In other embodiments, "high affinity" refers to an affinity such that a peptide at a concentration of about 500 nanomolar (nM) or less is required to inhibit 50% of the binding of the standard peptide. In other embodiments, a peptide at a concentration of about 400 nM or less is required. In other embodiments, the binding affinity is 300 nM. In other embodiments, the binding affinity is 200 nM. In other embodiments, the binding affinity is 150 nM. In other embodiments, the binding affinity is 100 nM. In other embodiments, the binding affinity is 80 nM. In other embodiments, the binding affinity is 60 nM. In other embodiments, the binding affinity is 40 nM. In other embodiments, the binding affinity is 30 nM. In other embodiments, the binding affinity is 20 nM. In other embodiments, the binding affinity is 15 nM. In other embodiments, the binding affinity is 10 nM. In other embodiments, the binding affinity is 8 nM. In other embodiments, the binding affinity is 6 nM. In other embodiments, the binding affinity is 4 nM. In other embodiments, the binding affinity is 3 nM. In other embodiments, the binding affinity is 2 nM. In other embodiments, the binding affinity is 1.5 nM. In other embodiments, the binding affinity is 1 nM. In other embodiments, the binding affinity is 0.8 nM. In other embodiments, the binding affinity is 0.6 nM. In other embodiments, the binding affinity is 0.5 nM. In other embodiments, the binding affinity is 0.4 nM. In other embodiments, the binding affinity is 0.3 nM. In other embodiments, the binding affinity is less than 0.3 nM.

[0115] In other embodiments, "affinity" refers to a measure of the binding strength to an MHC molecule. In other embodiments, the affinity is measured using methods known in the art to measure competitive binding affinity. In other embodiments, the affinity is measured using methods known in the art to measure relative binding affinity. In other embodiments, the method is a competitive binding assay. In other embodiments, the method is a radioimmunoassay or RIA. In other embodiments, the method is a BiaCore analysis. In other embodiments, the method is any other method known in the art. In other embodiments, the method yields an IC50 in relation to the IC50 of a reference peptide of known affinity.

[0116] Each type of affinity and method of measuring affinity represents a separate embodiment of the invention.

[0117] In other embodiments, "high affinity" refers to an IC50 of 0.5 to 100 nM. In other embodiments, the IC50 is 1 to 100 nM. In other embodiments, the IC50 is 1.5 to 200 nM. In other embodiments, the IC50 is 2 to 100 nM. In other embodiments, the IC50 is 3 to 100 nM. In other embodiments, the IC50 is 4 to 100 nM. In other embodiments, the IC50 is 6 to 100 nM. In other embodiments, the IC50 is 10 to 100 nM. In other embodiments, the IC50 is 30 to 100 nM. In other embodiments, the IC50 is 3 to 80 nM. In other embodiments, the IC50 is 4 to 60 nM. In other embodiments, the IC50 is 5 to 50 nM. In other embodiments, the IC50 is 6 to 50 nM. In other embodiments, the IC50 is 8 to 50 nM. In other embodiments, the IC50 is 10 to 50 nM. In other embodiments, the IC50 is 20 to 50 nM. In other embodiments, the IC50 is 6 to 40 nM. In other embodiments, the IC50 is 8 to 30 nM. In other embodiments, the IC50 is 10 to 25 nM. In other embodiments, the IC50 is 15 to 25 nM. Each affinity and range of affinities represents a separate embodiment of the present invention.

[0118] In other embodiments, "moderate affinity" refers to an IC50 of 100 to 500 nM. In other embodiments, the IC50 is 100 to 300 nM. In other embodiments, the IC50 is 100 to 200 nM. In other embodiments, the IC50 is 50 to 100 nM. In other embodiments, the IC50 is 50 to 80 nM. In other embodiments, the IC50 is 50 to 60 nM. Each affinity and range of affinities represents a separate embodiment of the present invention.

[0119] As used in other embodiments, "high affinity" refers to an affinity sufficient to mediate recognition of a target cell by a T cell having a T cell receptor (TCR) that recognizes an MHC molecule-peptide complex. In other embodiments, the term refers to an affinity sufficient to mediate recognition of a cancer cell by a T cell having a TCR that recognizes an MHC molecule-peptide complex. In other embodiments, the term refers to an affinity sufficient to mediate activation of naive T cells by dendritic cells presenting a peptide. In other embodiments, the term refers to an affinity sufficient to mediate activation of naive T cells by an APC presenting a peptide. In other embodiments, the term refers to an affinity sufficient to mediate reactivation of memory T cells by dendritic cells presenting a peptide. In other embodiments, the term refers to an affinity sufficient to mediate reactivation of memory T cells by an APC presenting a peptide. In other embodiments, the term refers to an affinity sufficient to mediate reactivation of memory T cells by somatic cells presenting a peptide. Each possibility corresponds to a separate embodiment of the invention.

[0120] As used in other embodiments, "measurable affinity" refers to an affinity sufficient to be measurable by an immunological assay. In other embodiments, the immunological assay is any assay listed herein. Each possibility corresponds to a separate embodiment of the invention.

[0121] In other embodiments, the peptides of the methods and compositions of the invention bind to the superfamily of HLA molecules. The superfamily of HLA molecules share very similar or identical binding motifs. In other embodiments, the superfamily is the HLA class I superfamily. In other embodiments, the superfamily is the HLA class II superfamily. Each possibility corresponds to a separate embodiment of the invention.

[0122] The terms "HLA-binding peptide", "HLA class I molecule-binding peptide", and "HLA class II molecule-binding peptide" refer, in other embodiments, to peptides that bind to HLA molecules with measurable affinity. In other embodiments, the term refers to peptides that bind to HLA molecules with high affinity. In other embodiments, the term refers to peptides that bind to HLA molecules with sufficient affinity to activate T cell precursors. In other embodiments, the term refers to peptides that bind to HLA molecules with sufficient affinity to mediate recognition by T cells. HLA molecules are, in other embodiments, any of the HLA molecules listed herein. Each possibility corresponds to a separate embodiment of the invention.

[0123] In other embodiments, the peptides of the methods and compositions of the invention are heteroclitic. "Heteroclitic", in other embodiments, refers to a peptide that elicits an immune response that recognizes the original peptide from which the heteroclitic peptide is derived (e.g., a peptide that does not contain an anchor residue mutation). In other embodiments, the "original peptide" was generated from the wild-type WT1 peptide SGQARMFPNAPYLPSCLES (SEQ ID NO: 5) by mutation of the arginine at residue 5 to the "WT1122A1" peptide having the sequence SGQAYMFPNAPYLPSCLES (SEQ ID NO: 124). The heteroclitic mutation is CD8 +The WT1 peptide RMFPNAPYL (SEQ ID NO: 7) was introduced to generate the peptide YMFPNAPYL (SEQ ID NO: 124). In other embodiments, "heteroclitic" refers to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide is derived, where the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide. In other embodiments, a "heteroclitic" immune response refers to an immune response that recognizes the original peptide from which the improved peptide is derived (e.g., a peptide that does not contain an anchor residue mutation). In other embodiments, a "heteroclitic" immune response refers to an immune response that recognizes the original peptide from which the heteroclitic peptide is derived, where the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide. In other embodiments, the magnitude of the immune response generated by vaccination with a heteroclitic peptide is greater than an immune response that is substantially equal to the response to vaccination with the original peptide. In other embodiments, the magnitude of the immune response generated by vaccination with a heteroclitic peptide is greater than an immune response that is less than the response to vaccination with the original peptide. Each possibility corresponds to a separate embodiment of the invention.

[0124] In other embodiments, the heteroclitic peptide of the invention induces an immune response that is at least 2-fold increased compared to the WT1 peptide (the "native peptide") from which the heteroclitic peptide is derived. In other embodiments, the increase is 3-fold relative to the native peptide. In other embodiments, the increase is 5-fold relative to the native peptide. In other embodiments, the increase is 7-fold relative to the native peptide. In other embodiments, the increase is 10-fold relative to the native peptide. In other embodiments, the increase is 15-fold relative to the native peptide. In other embodiments, the increase is 20-fold relative to the native peptide. In other embodiments, the increase is 30-fold relative to the native peptide. In other embodiments, the increase is 50-fold relative to the native peptide. In other embodiments, the increase is 100-fold relative to the native peptide. In other embodiments, the increase is 150-fold relative to the native peptide. In other embodiments, the increase is 200-fold relative to the native peptide. In other embodiments, this increase is 300-fold relative to the native peptide. In other embodiments, the increase is 500-fold relative to the native peptide. In other embodiments, the increase is 1000-fold relative to the native peptide. In other embodiments, the increase exceeds 1000-fold relative to the native peptide. Each possibility represents a separate embodiment of the invention, and in combination, each heteroclitic, and the native peptide derived therefrom, is related independently.

[0125] In other embodiments, the heteroclitic peptide of the invention is an HLA class I heteroclitic peptide. In other embodiments, the heteroclitic peptide of the invention is an HLA class II heteroclitic peptide. In other embodiments, the heterologous class II peptide of the invention is mutated at the class II binding residues. In other embodiments, the heteroclitic class II peptide of the invention is identified and tested in a manner similar to the identification and testing of HLA class I heteroclitic peptides, as exemplified herein. Each possibility corresponds to a separate embodiment of the invention.

[0126] In other embodiments, the "anchor motif" or "anchor residue" refers to one or a set of preferred residues at specific positions in the HLA binding sequence. For example, the residues at positions 1, 2, 3, 6, and 9 are used as anchor residues. In other embodiments, the HLA binding sequence is an HLA class II binding sequence. In other embodiments, the HLA binding sequence is an HLA class I binding sequence. In other embodiments, the positions corresponding to the anchor motif are positions that play an important role in the binding of the HLA molecule. In other embodiments, the anchor residue is a primary anchor motif. In other embodiments, the anchor residue is a secondary anchor motif. Each possibility corresponds to a separate embodiment of the present invention.

[0127] In other embodiments, the "anchor residue" is the residue at positions 1, 3, 6, and 9 of the HLA class I binding motif. In other embodiments, the term refers to positions 1, 2, 6, and 9 of the HLA class I binding motif. In other embodiments, the term refers to positions 1, 6, and 9 of the HLA class I binding motif. In other embodiments, the term refers to positions 1, 2, and 9 of the HLA class I binding motif. In other embodiments, the term refers to positions 1, 3, and 9 of the HLA class I binding motif. In other embodiments, the term refers to positions 2 and 9 of the HLA class I binding motif. In other embodiments, the term refers to positions 6 and 9 of the HLA class I binding motif. Each possibility corresponds to a separate embodiment of the present invention.

[0128] Methods for identifying MHC class II epitopes are well known in the art. In other embodiments, MHC class II epitopes are predicted using TEPITOPE (Meister GE, Roberts CG et al., Vaccine 1995 13:581-91). In other embodiments, MHC class II epitopes are identified using EpiMatrix (De Groot AS, Jesdale BM et al., AIDS Res Hum Retroviruses 1997 13:529-31). In other embodiments, MHC class II epitopes are identified using the Predict Method (Yu K, Petrovsky N et al., Mol Med. 2002 8:137-48). In other embodiments, MHC class II epitopes are identified using the SYFPEITHI epitope prediction algorithm. SYFPEITHI is a database containing over 4500 peptide sequences known to bind to class I and class II MHC molecules. SYFPEITHI provides scores based on the presence of specific amino acids at certain positions along the MHC binding groove. Ideal amino acid anchors are scored 10, abnormal anchors are worth 6 - 8 points, auxiliary anchors are worth 4 - 6 points, preferred residues are worth 1 - 4 points, and negative amino acid effects on binding scores between -1 and -3. The maximum score for HLA-A*0201 is 36.

[0129] In other embodiments, MHC class II epitopes are identified using Rankpep. Rankpep uses a position-specific scoring matrix (PSSM) or a profile from a set of aligned peptides known to bind to a given MHC molecule as predictors of MHC-peptide binding. Rankpep uses the selected profile to provide information on the score of a peptide and the optimal % or percentile value score of the predicted peptide relative to the score of the consensus sequence that yields the maximum score. Rankpep includes a selection of 102 and 80 PSSMs for the prediction of peptide binding to MHC I and MHC II molecules, respectively. Several PSSMs for the prediction of peptide binders of different sizes are usually available for each MHC I molecule.

[0130] In other embodiments, MHC class II epitopes are identified using SVMHC (Donnes P, Elofsson A Prediction of MHC class I binding peptides, using SVMHC BMC Bioinformatics. 2002 Sep 11, 3:25). In other embodiments, MHC class II epitopes are identified using any other method known in the art. The above methods are utilized in other embodiments to identify that MHC class II binding is disrupted by the introduction of MHC class I anchor residue mutations into the WT1 sequence. Each possibility corresponds to a separate embodiment of the invention.

[0131] Methods for identifying MHC class I epitopes are well known in the art. In other embodiments, MHC class I epitopes are predicted using BIMAS software. The BIMAS score is based on the calculation of the theoretical half-life of the MHC-I / β2-microglobulin / peptide complex, which is a measure of peptide binding affinity. This program uses information on HLA-I peptides that are 8 to 10 amino acids in length. The higher the binding affinity of a peptide for MHC, the more likely that peptide is to represent an epitope. The BIMAS algorithm assumes that each amino acid in the peptide contributes independently to binding to the class I molecule. Dominant anchor residues that are important for binding have coefficients that are significantly higher than 1 in the table. Unfavorable amino acids have positive coefficients less than 1. When an amino acid is not known to make a favorable or unfavorable contribution to binding, a value of 1 is assigned. All the values assigned to the amino acids are multiplied together, and the resulting running score is multiplied by a constant to obtain an estimated value for the half-life of dissociation.

[0132] In other embodiments, the MHC class I epitopes are identified using SYFPEITHI. In other embodiments, the MHC class I epitopes are identified using SVMHC (Donnes P, Elofsson A. Prediction of MHC class I binding peptides, using SVMHC. BMC Bioinformatics. 2002 Sep 11;3:25). In other embodiments, the MHC class I epitopes are identified using NetMHC-2.0 ("Sensitive quantitative prediction of peptide-MHC binding by a 'Query by Committee' artificial neural network approach", Buus S, Lauemoller SL, Worning P, Kesmir C, Frimurer T, Corbet S, Fomsgaard A, Hilden J, Holm A, Brunak S. Tissue Antigens, 62:378-84, 2003). In other embodiments, the MHC class I epitopes are identified using any other method known in the art. The above methods are utilized in other embodiments to identify MHC class I epitopes that can be generated by introducing anchor residue mutations into the WT1 sequence. Each possibility corresponds to a separate embodiment of the invention.

[0133] In other embodiments, the mutation that enhances MHC binding is at the residue at position 1 of the HLA class I binding motif. In other embodiments, the residue is changed to tyrosine. In other embodiments, the residue is changed to glycine. In other embodiments, the residue is changed to threonine. In other embodiments, the residue is changed to phenylalanine. In other embodiments, the residue is changed to any other residue known in the art. In other embodiments, the substitution at position 1 (e.g., to tyrosine) stabilizes the binding of the anchor residue at position 2.

[0134] In other embodiments, the mutation is at position 2 of the HLA class I binding motif. In other embodiments, the residue is changed to leucine. In other embodiments, the residue is changed to valine. In other embodiments, the residue is changed to isoleucine. In other embodiments, the residue is changed to methionine. In other embodiments, the residue is changed to any other residue known in the art.

[0135] In other embodiments, the mutation is at position 6 of the HLA class I binding motif. In other embodiments, the residue is changed to valine. In other embodiments, the residue is changed to cysteine. In other embodiments, the residue is changed to glutamine. In other embodiments, the residue is changed to histidine. In other embodiments, the residue is changed to any other residue known in the art.

[0136] In other embodiments, the mutation is at position 9 of the HLA class I binding motif. In other embodiments, the mutation changes the residue at the C-terminal position. In other embodiments, the residue is changed to valine. In other embodiments, the residue is changed to threonine. In other embodiments, the residue is changed to isoleucine. In other embodiments, the residue is changed to leucine. In other embodiments, the residue is changed to alanine. In other embodiments, the residue is changed to cysteine. In other embodiments, the residue is changed to any other residue known in the art.

[0137] In other embodiments, the point mutation is in the primary anchor residue. In other embodiments, the HLA class I primary anchor residues are at positions 2 and 9. In other embodiments, the point mutation is in the secondary anchor residue. In other embodiments, the HLA class I secondary anchor residues are at positions 1 and 8. In other embodiments, the HLA class I secondary anchor residues are at positions 1, 3, 6, 7, and 8. In other embodiments, the point mutation is at a position selected from positions 4, 5, and 8. Each possibility corresponds to a separate embodiment of the invention.

[0138] In other embodiments, the point mutation is in one or more residues at positions selected from positions 1, 2, 8, and 9 of the HLA class I binding motif. In other embodiments, the point mutation is in one or more residues at positions selected from positions 1, 3, 6, and 9. In other embodiments, the point mutation is in one or more residues at positions selected from positions 1, 2, 6, and 9. In other embodiments, the point mutation is in one or more residues at positions selected from positions 1, 6, and 9. In other embodiments, the point mutation is in one or more residues at positions selected from positions 1, 2, and 9. In other embodiments, the point mutation is in one or more residues at positions selected from positions 1, 3, and 9. In other embodiments, the point mutation is in one or more residues at positions selected from positions 2 and 9. In other embodiments, the point mutation is in one or more residues at positions selected from positions 6 and 9. Each possibility corresponds to a separate embodiment of the invention. In other embodiments, the mutation is at position 4 of the HLA class I binding motif. In other embodiments, the mutation is at position 5 of the HLA class I binding motif. In other embodiments, the mutation is at position 7 of the HLA class I binding motif. In other embodiments, the mutation is at position 8 of the HLA class I binding motif. Each possibility corresponds to a separate embodiment of the invention.

[0139] Each of the above anchor residues and substitutions represents a separate embodiment of the invention.

[0140] In other embodiments, the HLA class II binding site in the peptides of the invention is created or improved by mutation of the HLA class II motif anchor residues. In other embodiments, the modified anchor residue is at the P1 position. In other embodiments, the anchor residue is at the P2 position. In other embodiments, the anchor residue is at the P6 position. In other embodiments, the anchor residue is at the P9 position. In other embodiments, the anchor residue is selected from the P1, P2, P6, and P9 positions. In other embodiments, the anchor residue is at the P3 position. In other embodiments, the anchor residue is at the P4 position. In other embodiments, the anchor residue is at the P5 position. In other embodiments, the anchor residue is at the P6 position. In other embodiments, the anchor residue is at the P8 position. In other embodiments, the anchor residue is at the P10 position. In other embodiments, the anchor residue is at the P11 position. In other embodiments, the anchor residue is at the P12 position. In other embodiments, the anchor residue is at the P13 position. In other embodiments, the anchor residue is at any other anchor residue of HLA class II molecules known in the art. In other embodiments, residues other than P1, P2, P6, and P9 act as secondary anchor residues. Thus, mutating them can improve HLA class II binding. In other embodiments, any combination of the above residues is mutated. Each possibility corresponds to a separate embodiment of the invention.

[0141] In other embodiments, the invention provides a method of inducing an anti-WT1-expressing cancer immune response in a subject, the method comprising administering to the subject an immunotherapeutic composition disclosed herein, optionally in combination with at least one checkpoint inhibitor, thereby inducing an anti-WT1-expressing cancer immune response in the subject.

[0142] In other embodiments, the invention provides a method of treating a subject having a WT1-expressing cancer, the method comprising administering to the subject an immunotherapeutic composition disclosed herein, optionally in combination with at least one checkpoint inhibitor, thereby treating the subject having a WT1-expressing cancer.

[0143] In other embodiments, the present invention provides a method for reducing the incidence of WT1-expressing cancer or its recurrence in a subject, the method comprising administering to the subject the immunotherapy composition disclosed herein, optionally together with at least one checkpoint inhibitor, thereby reducing the incidence of WT1-expressing cancer or its recurrence in the subject.

[0144] The terms "homology", "homologous", etc., with respect to any protein or peptide, in other embodiments, after aligning the sequences to achieve maximum percent homology and introducing gaps if necessary, refer to the percentage of AA residues in a candidate sequence that are identical to the residues of the corresponding native polypeptide, without considering any conservative substitutions as part of sequence identity. Methods and computer programs for alignment are well known in the art.

[0145] In other embodiments, the term "homology" with respect to any nucleic acid sequence similarly refers to the percentage of nucleotides in a candidate sequence that are identical to the nucleotides of the corresponding native nucleic acid sequence.

[0146] Homology is determined in other embodiments by a 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 use of any number of available software packages, such as, for example, BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT, and TREMBL packages.

[0147] The percent identity between two arrays is a function of the number of identical positions shared by the arrays, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two arrays (i.e., % identity = number of identical positions / total number of positions × 100). The comparison of arrays and the determination of the percent identity between two arrays are achieved using mathematical algorithms in array analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications including conservative amino acid substitutions.

[0148] The percent identity between two amino acid sequences is determined, for example, using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm incorporated in the GAP program in the GCG software package (available at www.gcg.com), either the Blossum62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. Polypeptide sequences are also compared using FASTA, applying default or recommended parameters. Programs such as GCG version 6.1, FASTA (e.g., FASTA2 and FASTA3) provide the alignment of the best overlapping regions and percent sequence identity between the query sequence and the search sequence (Pearson, Methods Enzymol. 1990;183:63-98; Pearson, Methods MolBiol. 2000;132:185-219). The percent identity between two amino acid sequences is also determined using the algorithm of EMeyers and WMiller (Comput. Appl. Biosci., 1988;11-17) incorporated in the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0149] Another algorithm for comparing an array with other arrays included in a database is the computer program BLAST, in particular blastp, which uses default parameters. See, for example, Altschul et al., J. Mol. Biol. 1990; 215: 403-410; Altschul et al., Nucleic Acids Res. 1997, 25: 3389-402 (1997), each of which is incorporated herein by reference. The protein sequences of the present invention can be used, for example, as "query sequences" for performing searches against public databases to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al., 1990 (supra). The BLAST protein search can be performed using the XBLAST program (score = 50, word length = 3) to obtain an amino acid sequence homologous to the WT1 peptide of the present invention. To obtain a gap alignment for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997 (supra). When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0150] In other embodiments, "homology" with respect to homologous sequences refers to percent identity to the sequences disclosed herein that is greater than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Each possibility corresponds to a separate embodiment of the present invention.

[0151] In other embodiments, the present invention provides a composition comprising one or more WT1 delivery agents for delivering at least seven WT1 peptides, or a combination of CTLs induced by at least seven WT1 peptides, and at least one checkpoint inhibitor. In other embodiments, the composition further comprises a pharmaceutically acceptable carrier. In other embodiments, the composition further comprises an adjuvant. In other embodiments, the composition comprises two or more peptides of the present invention. In other embodiments, the composition further comprises any of the additives, compounds, or excipients described below. In other embodiments, the adjuvant is alum salt or other mineral adjuvant, bacterial product or bacterium-derived adjuvant, tensioactive agent (e.g., saponin), o / w or w / o emulsion, liposomal adjuvant, cytokine (e.g., IL-2, GM-CSF, IL-12, and IFN-gamma), or alpha-galactosylceramide analog. In other embodiments, the adjuvant is QS21, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG or alum. In other embodiments, the carrier is any carrier listed herein. In other embodiments, the adjuvant is any adjuvant listed herein. Each possibility corresponds to a separate embodiment of the present invention.

[0152] In other embodiments, the present invention provides a vaccine comprising one or more WT1 delivery agents for delivering at least seven WT1 peptides or a combination of CTLs, and at least one checkpoint inhibitor. In other embodiments, the vaccine further comprises a carrier. In other embodiments, the vaccine further comprises an adjuvant. In other embodiments, the vaccine further comprises a combination of a carrier and an adjuvant. In other embodiments, the vaccine further comprises an APC. In other embodiments, the vaccine further comprises a combination of an APC and a carrier or an adjuvant. In other embodiments, the vaccine is a cell-based composition. Each possibility corresponds to a separate embodiment of the present invention.

[0153] In other embodiments, the present invention provides an immunogenic composition comprising a peptide of the present invention and at least one checkpoint inhibitor. In other embodiments, the immunogenic composition further comprises a carrier. In other embodiments, the immunogenic composition further comprises an adjuvant. In other embodiments, the immunogenic composition further comprises a combination of a carrier and an adjuvant. Each possibility corresponds to a separate embodiment of the present invention.

[0154] In other embodiments, the term "vaccine" refers to a material or composition that, when introduced into a subject, provides a prophylactic or therapeutic response against a particular disease, condition, or its symptoms. In other embodiments, the present invention includes a peptide-based vaccine, the peptide including any of the embodiments listed herein and optionally further including an immunomodulatory compound such as a cytokine, adjuvant, etc.

[0155] In other embodiments, the composition or vaccine of the method of the present invention, and the composition further comprises an adjuvant. In other embodiments, the adjuvant is Montanide ISA51. Montanide ISA51 contains a natural metabolizable oil and a purified emulsifier. In other embodiments, the adjuvant is GM-CSF. In other embodiments, the adjuvant is keyhole limpet hemocyanin (KLH), which may be conjugated to the peptide antigen or administered with the peptide. Recombinant GM-CSF is, in other embodiments, a human protein grown in a yeast ( S.cerevisiae ) vector. GM-CSF promotes the clonal expansion and differentiation of hematopoietic progenitor cells, APCs, and dendritic cells and T cells.

[0156] In other embodiments, the adjuvant is a cytokine. In other embodiments, the adjuvant is a growth factor. In other embodiments, the adjuvant is a cell population. In other embodiments, the adjuvant is QS21. In other embodiments, the adjuvant is Freund's incomplete adjuvant. In other embodiments, the adjuvant is aluminum phosphate. In other embodiments, the adjuvant is aluminum hydroxide. In other embodiments, the adjuvant is BCG. In other embodiments, the adjuvant is alum. In other embodiments, the adjuvant is an interleukin. In other embodiments, the adjuvant is a chemokine. In other embodiments, the adjuvant is any other type of adjuvant known in the art. In other embodiments, the WT1 vaccine comprises two of the above adjuvants. In other embodiments, the WT1 vaccine comprises three or more of the above adjuvants. Each possibility corresponds to a separate embodiment of the invention.

[0157] In other embodiments, the WT1 vaccine used in the method of the invention is one or more nucleic acid molecules (DNA or RNA) encoding one or more WT1 peptides of the invention. In the practice of this embodiment, a vaccine (nucleic acid vaccine) comprising a nucleic acid molecule encoding one or more WT1 peptides is administered and one or more checkpoint inhibitors are administered to the patient. In all other embodiments of the invention, the nucleic acid vaccine can be used in place of the peptide vaccine. The nucleic acid is introduced alone, as part of a viral carrier, or inside a cell, perhaps as a plasmid, or integrated into the nucleic acid of the cell. The cell carrier is a patient's cell removed from the patient, or a cell from a donor, or a cell line. The cell may be an antigen-presenting cell such as a dendritic cell or a monocyte / macrophage lineage cell. The cell vector is selected from the group consisting of autologous cells, allogeneic cells, cell lines, cells such as dendritic cells or antigen-presenting cells, or fusions to any of the above cells of hybrid cells.

[0158] The WT1 peptide, or a nucleic acid encoding the same, or its carrier in any of the forms described herein, is exposed to CTL ex vivo or in vivo. In the case of in vitro or ex vivo, cells can be grown or proliferated and then introduced into the patient.

[0159] As used interchangeably herein, the terms "nucleic acid", "nucleic acid molecule", "oligonucleotide", and "polynucleotide" include RNA, DNA, or RNA / DNA hybrid sequences of two or more nucleotides in either single-stranded or double-stranded form. The terms include "modified nucleotides" containing at least one modification, including, by way of non-limiting example: (a) alternative linking groups, (b) purine analog formation, (c) pyrimidine analog formation, or (d) analog sugars. For examples of similar linking groups, purines, pyrimidines, and sugars, see, for example, PCT Publication No. WO95 / 04064. The nucleic acid sequences of the present invention are prepared by any known method, including synthesis, recombination, ex vivo production, or combinations thereof, and utilizing any purification methods known in the art. As used herein, the term "nucleic acid vaccine" includes DNA vaccines and RNA vaccines, as well as vaccines containing viral or non-viral vectors.

[0160] In other embodiments, the use of the present invention provides a vector containing a nucleic acid molecule (DNA or RNA). In other embodiments, the compositions or vaccines used in the practice of the present invention can include any of the embodiments of the WT1 peptides of the present invention and combinations thereof. Each possibility corresponds to a separate embodiment of the present invention.

[0161] In other embodiments, the vaccine or composition of the invention used in the practice of the invention comprises two peptides derived from the same WT1 fragment, each containing a different HLA class I heteroclitic peptide. In other embodiments, the two HLA class I heteroclitic peptides contain mutations in different HLA class I molecule anchor residues. In other embodiments, the two HLA class I heteroclitic peptides contain different mutations in the same anchor residue. Each possibility corresponds to a distinct embodiment of the invention.

[0162] In other embodiments, the peptides in the composition used in the invention bind to two different HLA class II molecules. In other embodiments, the peptides bind to three different HLA class II molecules. In other embodiments, the peptides bind to four different HLA class II molecules. In other embodiments, the peptides bind to five different HLA class II molecules. In other embodiments, the peptides bind to more than five distinct HLA class II molecules. In other embodiments, the peptides in the composition bind to the same HLA class II molecule.

[0163] In other embodiments, each of the peptides in the composition or method of use of the invention binds to a set of HLA class II molecules. In other embodiments, each of the peptides binds to a distinct set of HLA class II molecules. In other embodiments, the peptides in the composition bind to the same set of HLA class II molecules. In other embodiments, two of the peptides bind to different overlapping sets of HLA class II molecules. In other embodiments, two or more peptides bind to the same set of HLA class II molecules, while another peptide binds to a different set. In other embodiments, two or more peptides bind to overlapping sets of HLA class II molecules, while another peptide binds to a distinct set.

[0164] In other embodiments, the peptides for use in practicing the invention or in the compositions of the invention bind to two different HLA class I molecules. In other embodiments, the peptides bind to three different HLA class I molecules. In other embodiments, the peptides bind to four different HLA class I molecules. In other embodiments, the peptides bind to five different HLA class I molecules. In other embodiments, the peptides bind to more than five distinct HLA class I molecules. In other embodiments, the peptides in the composition bind to the same HLA class I molecule.

[0165] In other embodiments, each of the peptides for use in practicing the invention or in the compositions of the invention binds to a set of HLA class I molecules. In other embodiments, each of the peptides binds to a distinct set of HLA class I molecules. In other embodiments, the peptides in the composition bind to the same set of HLA class I molecules. In other embodiments, two of the peptides bind to different but overlapping sets of HLA class I molecules. In other embodiments, two or more peptides bind to the same set of HLA class I molecules while another peptide binds to a different set. In other embodiments, two or more peptides bind to overlapping sets of HLA class I molecules while another peptide binds to a distinct set.

[0166] In other embodiments, a "set of HLA class II molecules" or a "set of HLA class I molecules" refers to HLA molecules encoded by different alleles at a particular locus. In other embodiments, the term refers to HLA molecules having a particular binding specificity. In other embodiments, the term refers to HLA molecules having a particular peptide consensus sequence. In other embodiments, the term refers to the superfamily of HLA class II molecules. Each possibility corresponds to a distinct embodiment of the invention.

[0167] Each of the above compositions and types of compositions represents a distinct embodiment of the invention.

[0168] Any embodiment described herein with respect to the peptides, nucleic acids, compositions, and vaccines of the invention is used in any method of the invention. Each combination of a peptide, nucleic acid, composition, or vaccine and a method represents a separate embodiment thereof.

[0169] In other embodiments, the invention provides a method of treating a subject having a WT1-expressing cancer, the method comprising administering to the subject a WT1 immunotherapeutic composition as described herein, and optionally a checkpoint inhibitor, thereby treating the subject having a WT1-expressing cancer. In other embodiments, the invention provides a method of treating a subject having a WT1-expressing cancer, the method comprising administering to the subject a composition of the invention comprising at least a WT1 delivery agent for delivering a combination of at least 7 WT1 peptides, or CTLs induced by at least 7 peptides, and optionally at least 1 checkpoint inhibitor, thereby treating the subject having a WT1-expressing cancer. In other embodiments, the invention provides a method of treating a subject having a WT1-expressing cancer, the method comprising administering to the subject an immunogenic composition such as a vaccine, and optionally a checkpoint inhibitor, thereby treating the subject having a WT1-expressing cancer.

[0170] In other embodiments, the present invention provides a method of suppressing or arresting the progression of WT1-expressing cancer in a subject, the method comprising administering to the subject one or more WT1 delivery agents for delivering a combination of at least seven WT1 peptides, or CTLs induced by at least seven WT1 peptides, and optionally, at least one checkpoint inhibitor, thereby suppressing or arresting the progression of WT1-expressing cancer. In other embodiments, the present invention provides a method of suppressing or arresting the progression of WT1-expressing cancer in a subject, the method comprising administering to the subject a composition comprising a combination of at least seven WT1 peptides and optionally, at least one checkpoint inhibitor, thereby suppressing or arresting the progression of WT1-expressing cancer. In other embodiments, the present invention provides a method of suppressing or arresting the progression of WT1-expressing cancer in a subject, the method comprising administering to the subject an immunogenic composition such as the immunotherapeutic composition of the present invention, the composition comprising a combination of at least seven WT1 peptides and optionally, at least one checkpoint inhibitor, thereby suppressing or arresting the progression of WT1-expressing cancer.

[0171] In other embodiments, the present invention provides a method of reducing the incidence of WT1-expressing cancer in a subject, the method comprising administering to the subject one or more WT1 delivery agents to deliver a combination of at least seven WT1 peptides, or CTLs induced by at least seven peptides, and optionally, at least one checkpoint inhibitor, thereby reducing the incidence of WT1-expressing cancer in the subject. In other embodiments, the present invention provides a method of reducing the incidence of WT1-expressing cancer in a subject, the method comprising administering to the subject a composition of the present invention comprising one or more WT1 delivery agents to deliver a combination of at least seven WT1 peptides, or CTLs induced by at least seven WT1 peptides, and optionally, at least one checkpoint inhibitor, thereby reducing the incidence of WT1-expressing cancer in the subject. In other embodiments, the present invention provides a method of reducing the incidence of WT1-expressing cancer in a subject, the method comprising administering to the subject a composition of the present invention comprising a combination of at least seven WT1 peptides and optionally at least one checkpoint inhibitor, thereby reducing the incidence of WT1-expressing cancer in the subject.

[0172] In other embodiments, the present invention provides a method of reducing the incidence of recurrence of WT1-expressing cancer in a subject, the method comprising administering to the subject a composition comprising one or more WT1 delivery agents to deliver a combination of at least seven WT1 peptides, or CTLs induced by at least seven WT1 peptides, and optionally at least one checkpoint inhibitor, thereby reducing the incidence of recurrence of WT1-expressing cancer in the subject. In other embodiments, the present invention provides a method of reducing the incidence of recurrence of WT1-expressing cancer in a subject, the method comprising administering to the subject a composition of the present invention comprising a combination of at least seven WT1 peptides and optionally at least one checkpoint inhibitor, thereby reducing the incidence of recurrence of WT1-expressing cancer in the subject.

[0173] In other embodiments, the present invention provides a method for overcoming T cell tolerance of a subject to WT1-expressing cancer, the method comprising administering to the subject one or more WT1 delivery agents for delivering a combination of at least seven WT1 peptides, or CTLs induced by at least seven WT1 peptides, and optionally at least one checkpoint inhibitor, thereby overcoming T cell tolerance to WT1-expressing cancer. In other embodiments, the present invention provides a method for overcoming T cell tolerance of a subject to WT1-expressing cancer, the method comprising administering to the subject a composition of the present invention, the composition comprising a combination of at least seven WT1 peptides and optionally at least one checkpoint inhibitor, thereby overcoming T cell tolerance to WT1-expressing cancer. In other embodiments, the present invention provides a method for overcoming T cell tolerance of a subject to WT1-expressing cancer, the method comprising administering to the subject an immunogenic composition such as an immunotherapeutic composition of the present invention, comprising a combination of at least seven WT1 peptides and optionally at least one checkpoint inhibitor, thereby overcoming T cell tolerance to WT1-expressing cancer.

[0174] In other embodiments, the present invention provides a method of treating a subject having a WT1-expressing cancer, comprising: (a) inducing the formation and proliferation of donor human cytotoxic T lymphocytes (CTLs) that recognize malignant cells of cancer by the method of the present invention; and (b) injecting the human CTLs into the subject, thereby treating the subject having cancer. In one embodiment, a combination of at least 7 WT1 peptides is administered to the donor, the CTLs from the donor are injected into the subject, and optionally, a checkpoint inhibitor is administered to the subject, thereby treating the subject having cancer. In one embodiment, a combination of at least 7 WT1 peptides and optionally at least 1 checkpoint inhibitor is administered to the donor, the CTLs from the donor are injected into the subject, thereby treating the subject having cancer. In one embodiment, a combination of at least 7 WT1 peptides and optionally at least 1 checkpoint inhibitor is administered to the donor, the CTLs from the donor are injected into the subject, and optionally, a checkpoint inhibitor is administered to the subject, thereby treating the subject having cancer.

[0175] In other embodiments, the present invention provides a method of treating a subject having a WT1-expressing cancer, comprising: (a) inducing the ex vivo formation and proliferation of human CTLs that recognize malignant cells of cancer by the method of the present invention, wherein the human immune cells are obtained from a donor; and (b) injecting the human CTLs into the subject, thereby treating the subject having cancer. In one embodiment, a checkpoint inhibitor is included in the ex vivo process. In other embodiments, a checkpoint inhibitor is administered to the subject. In other embodiments, both the ex vivo processes include a checkpoint inhibitor and a checkpoint inhibitor is also administered to the subject.

[0176] Methods of ex vivo immunotherapy are known in the art and are described, for example, in Davis ID et al. (Blood dendritic cells efficiently generated from cancer patients by Flt3 ligand and CD40 ligand prime CD8+ T cells, J Immuno September - October 2006 29(5):499 - 511) and Mitchell MS et al. (Cytometric T cell responses to peptide analogs of HLA - A*0201 - restricted MUC1 signal sequence epitopes, M1.2. Cancer Immunol Immunother. July 28, 2006). Each method represents a separate embodiment of the invention.

[0177] In other embodiments, the invention provides a method of inducing the formation and proliferation of WT1 - protein - specific CTLs, the method comprising contacting a lymphocyte population with an immunogenic composition (e.g., the immunotherapy composition of the invention) and optionally at least one checkpoint inhibitor, thereby inducing the formation and proliferation of WT1 - protein - specific CTLs. In other embodiments, the immunogenic composition comprises antigen - presenting cells (APCs) associated with the peptides of the invention and a checkpoint inhibitor. In other embodiments, the invention provides a method of inducing the formation and proliferation of WT1 - protein - specific CTLs, the method comprising contacting a lymphocyte population with a peptide or composition of the invention and at least one checkpoint inhibitor, thereby inducing the formation and proliferation of WT1 - protein - specific CTLs. In other embodiments, the invention provides a method of inducing the formation and proliferation of WT1 - protein - specific CTLs, the method comprising contacting a lymphocyte population with the vaccine of the invention together with at least one checkpoint inhibitor, thereby inducing the formation and proliferation of WT1 - protein - specific CTLs. In other embodiments, the CTLs are specific for WT1 - expressing cells. In other embodiments, the target cells are cells of WT1 - expressing cancer. Each possibility corresponds to a separate embodiment of the invention.

[0178] In other embodiments, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs in a subject, the method comprising contacting the subject with an immunogenic composition such as the immunotherapeutic composition of the present invention, optionally together with at least one checkpoint inhibitor, thereby inducing the formation and proliferation of WT1 protein-specific CTLs in the subject. In other embodiments, the immunogenic composition comprises APCs associated with a mixture of at least seven WT1 peptides of the present invention, which are administered together with at least one checkpoint inhibitor. In other embodiments, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs in a subject, the method comprising contacting the subject with a combination of at least seven WT1 peptides together with at least one checkpoint inhibitor or composition of the present invention, thereby inducing the formation and proliferation of WT1 protein-specific CTLs in the subject. In other embodiments, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs in a subject, the method comprising contacting the subject with the vaccine of the present invention together with at least one checkpoint inhibitor, thereby inducing the formation and proliferation of WT1 protein-specific CTLs in the subject. In other embodiments, the target cells are cells of a WT1-expressing cancer. In other embodiments, the subject has a WT1-expressing cancer. In other embodiments, the CTLs are specific for WT1-expressing cells.

[0179] In other embodiments, the present invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against WT1-expressing cancer, the method comprising administering to the subject a combination of at least 7 WT1 peptides, optionally together with at least one checkpoint inhibitor or a composition of the present invention, thereby generating a heteroclitic immune response. In other embodiments, the present invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against WT1-expressing cancer, the method comprising administering to the subject an immunogenic composition such as a vaccine of the present invention together with at least one checkpoint inhibitor, thereby generating a heteroclitic immune response. In other embodiments, the present invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against WT1-expressing cancer, the method comprising administering to the subject a vaccine of the present invention together with at least one checkpoint inhibitor, thereby generating a heteroclitic immune response.

[0180] Each method represents a separate embodiment of the present invention.

[0181] In other embodiments, the WT1-expressing cancer is acute myeloid leukemia (AML). In other embodiments, the WT1-expressing cancer is chronic myeloid leukemia (CML). In other embodiments, the WT1-expressing cancer is associated with myelodysplastic syndrome (MDS). In other embodiments, the WT1-expressing cancer is MDS. In other embodiments, the WT1-expressing cancer is non-small cell lung cancer (NSCLC). In other embodiments, the WT1-expressing cancer is esophageal squamous cell carcinoma. In other embodiments, the WT1-expressing cancer is acute lymphoblastic leukemia (ALL). In other embodiments, the WT1-expressing cancer is bone or soft tissue sarcoma. In other embodiments, the WT1-expressing cancer is Wilms tumor. In other embodiments, the WT1-expressing cancer is leukemia. In other embodiments, the WT1-expressing cancer is blood cancer. In other embodiments, the WT1-expressing cancer is lymphoma. In other embodiments, the WT1-expressing cancer is desmoplastic small round cell tumor. In other embodiments, the WT1-expressing cancer is mesothelioma. In other embodiments, the WT1-expressing cancer is malignant mesothelioma. In other embodiments, the WT1-expressing cancer is gastric cancer. In other embodiments, the WT1-expressing cancer is colon cancer. In other embodiments, the WT1-expressing cancer is lung cancer. In other embodiments, the WT1-expressing cancer is breast cancer. In other embodiments, the WT1-expressing cancer is germ cell tumor. In other embodiments, the WT1-expressing cancer is malignant pleural mesothelioma. In other embodiments, the WT1-expressing cancer is multiple myeloma. In other embodiments, the WT1-expressing cancer is myeloid leukemia. In other embodiments, the WT1-expressing cancer is astrocytoma. In other embodiments, the WT1-expressing cancer is glioblastoma (e.g., glioblastoma multiforme). In other embodiments, the WT1-expressing cancer is colorectal adenocarcinoma. In other embodiments, the WT1-expressing cancer is ovarian cancer (e.g., serous, epithelial, or endometrial). In other embodiments, the WT1-expressing cancer is breast cancer. In other embodiments, the WT1-expressing cancer is melanoma. In other embodiments, the WT1-expressing cancer is head and neck squamous cell carcinoma. In other embodiments, the WT1-expressing cancer is pancreatic ductal adenocarcinoma. In other embodiments, the WT1-expressing cancer is neuroblastoma. In other embodiments, the WT1-expressing cancer is uterine cancer. In other embodiments, the WT1-expressing cancer is thyroid cancer.In other embodiments, the WT1-expressing cancer is hepatocellular carcinoma. In other embodiments, the WT1-expressing cancer is thyroid cancer. In other embodiments, the WT1-expressing cancer is liver cancer. In other embodiments, the WT1-expressing cancer is renal cancer (e.g., renal cell carcinoma). In other embodiments, the WT1-expressing cancer is Kaposi's sarcoma. In other embodiments, the WT1-expressing cancer is sarcoma. In other embodiments, the WT1-expressing cancer is any other carcinoma or sarcoma.

[0182] In other embodiments, the WT1-expressing cancer is a solid tumor. In other embodiments, the solid tumor is associated with the WT1-expressing cancer. In other embodiments, the solid tumor is associated with myelodysplastic syndrome (MDS). In other embodiments, the solid tumor is associated with non-small cell lung cancer (NSCLC). In other embodiments, the solid tumor is associated with lung cancer. In other embodiments, the solid tumor is associated with breast cancer. In other embodiments, the solid tumor is associated with colorectal cancer. In other embodiments, the solid tumor is associated with prostate cancer. In other embodiments, the solid tumor is associated with ovarian cancer. In other embodiments, the solid tumor is associated with renal cancer. In other embodiments, the solid tumor is associated with pancreatic cancer. In other embodiments, the solid tumor is associated with brain cancer. In other embodiments, the solid tumor is associated with gastrointestinal cancer. In other embodiments, the solid tumor is associated with skin cancer. In other embodiments, the solid tumor is associated with melanoma.

[0183] In other embodiments, the cancer or tumor treated by the method of the present invention is suspected of expressing WT1. In other embodiments, the WT1 expression has not been verified by testing of actual tumor samples. In other embodiments, the cancer or tumor is often of a type known to express WT1. In other embodiments, this type expresses WT1 in the majority of cases.

[0184] Each type of WT1-expressing cancer or tumor and cancer or tumor suspected of expressing WT1 represents a separate embodiment of the present invention.

[0185] A non-exhaustive list of cancer types treated using the compositions and methods of the present invention is provided in Table 2.

[0186] [Table 3] [Table 4] [Table 5]

[0187] In other embodiments, the plurality of peptides of the present invention are used to stimulate an immune response in the methods of the present invention, together with at least one checkpoint inhibitor.

[0188] The heteroclitic peptides provided herein that induce an antigen-specific CD8 + T cell response are made using the methods of the present invention. CD4 + WT1 peptides that induce a T cell response against multiple HLA class II molecules can be identified. CD4 + T cells recognize peptides bound to HLA class II molecules on APCs. In other embodiments, the antigen-specific CD4 + T cell response aids in the induction and maintenance of a CD8 + cytotoxic T lymphocyte (CTL) response.

[0189] In other embodiments, the peptides of the present invention administered with at least one checkpoint inhibitor exhibit an enhanced ability to induce a CTL response due to their ability to bind to both HLA class I and HLA class II molecules. In other embodiments, the peptides of the present invention administered with at least one checkpoint inhibitor exhibit an enhanced ability to induce a CTL response due to the ability of the checkpoint inhibitor to increase the survival and proliferation of WT1-specific CTLs. In other embodiments, the vaccines of the present invention administered with at least one checkpoint inhibitor are CD4 +and CD8 + has the advantage of activating or inducing both CD4 + and CD8 + T cells. In other embodiments, the activation or induction of both CD4

[0190] and CD8 + T cells provides a synergistic anti-WT1 immune response compared to the activation of either population alone. In other embodiments, the enhanced immunogenicity of the peptides of the invention is demonstrated in individual ones of a plurality of HLA class II subtypes due to the ability of the peptides of the invention to bind to a plurality of HLA class II subtypes. Each possibility corresponds to a separate embodiment of the invention. + T cells enhance immunity by licensing dendritic cells, thereby maintaining the activation and survival of cytotoxic T cells. In other embodiments, activated CD4

[0191] T cells induce tumor cell death by direct contact with tumor cells or by activation of the apoptotic pathway. For example, mesothelioma tumor cells can process and present antigens in the context of HLA class I and class II molecules.

[0192] It will be understood by those skilled in the art that the methods disclosed herein enable the design of other WT1-derived peptides that can bind to both HLA class I and HLA class II molecules. The method further enables the design of immunogenic compositions and vaccines that combine the WT1-derived peptides of the invention. Each possibility corresponds to a separate embodiment of the invention. + T cells. In other embodiments, the vaccine activates or induces WT1-specific CD4 +It has the advantage of activating or inducing T cells. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 10% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 15% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 20% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 25% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 30% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 35% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 40% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 45% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 50% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 55% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 60% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 70% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 75% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 80% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 85% of the population. In other embodiments, the peptide activates WT1-specific CD4 + T cells in 90% of the population. In other embodiments, the peptide activates WT1-specific CD4 +Activates T cells. In other embodiments, the peptide activates WT1-specific CD4 + T cells in more than 95% of the population. In other embodiments, the vaccine activates WT1-specific CD4 + T cells or induces them in a significant proportion of a particular population (e.g., white Americans). Each possibility corresponds to a separate embodiment of the invention.

[0193] In other embodiments, the methods of the invention provide for enhancing an immune response already initiated by a subject. In other embodiments, the methods of the invention comprise administering a peptide, composition, or vaccine one or more or two or more times in combination with at least one checkpoint inhibitor. In other embodiments, the peptides vary in their composition, concentration, or combinations thereof. In other embodiments, a peptide administered in combination with at least one checkpoint inhibitor provides for initiation of an immune response against an antigen of a subject in a subject in which the immune response against the antigen of the subject has not yet been initiated. In other embodiments, the induced CTLs proliferate in response to presentation of the peptide on APCs or cancer cells. In other embodiments, modulation of the immune response includes either or both of the humoral and cell-mediated arms of the immune system, which are accompanied by the presence of Th2 and Th1 T helper cells, respectively, or, in other embodiments, each arm is accompanied individually.

[0194] In other embodiments, methods of affecting tumor growth result in (1) direct inhibition of tumor cell division, or (2) immune cell-mediated tumor cell lysis, or both, which results in suppression of net tumor cell growth. Each possibility corresponds to a separate embodiment of the invention. The use of a peptide or vaccine administered in combination with at least one checkpoint inhibitor increases direct inhibition of tumor cell division, immune cell-mediated cell lysis, or both, as compared to not using a checkpoint inhibitor.

[0195] Inhibition of tumor growth by either of these two mechanisms can be readily determined by one of ordinary skill in the art based on any of a number of well-known methods. In other embodiments, tumor inhibition is determined by measuring the actual tumor size over a period of time. In other embodiments, tumor inhibition is determined by estimating the size of the tumor over a period of time using methods well known to one of ordinary skill in the art. More specifically, the size of the tumor can be estimated using various radiological imaging methods (see, e.g., single photon and positron emission computed tomography, “Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.), Springer-Verilog, New York, 1986). Such methods can also utilize various imaging agents, including, for example, conventional imaging agents (e.g., gallium-67 citrate), as well as specialized reagents for metabolic imaging, receptor imaging, or immunological imaging (e.g., radiolabeled monoclonal antibody tumor markers). Additionally, non-radiological 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 the tumor.

[0196] In addition to the in vivo methods for determining tumor inhibition described above, various in vitro methods can be utilized to determine in vivo tumor inhibition. Representative examples include, for example, 51Lymphocyte-mediated antitumor cytolytic activity determined by Cr release assay, 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 cell growth inhibition in vitro (Gazit et al., Cancer Immunol 35:135-144, 1992), and determination of cell precursor frequency for any of these assays (Vose, Int. J. Cancer 30:135-142(1982)), etc. are included.

[0197] In other embodiments, a method of suppressing tumor growth exhibits a reduced growth state as compared to growth that does not contact at least one checkpoint inhibitor of the present invention or is not exposed to a peptide administered therewith. Tumor cell growth can be measured by measuring the size of the tumor, determining whether tumor cells are proliferating using an H-thymidine incorporation assay, or counting the tumor cells, and can be evaluated by any means known in the art, including but not limited to these. 3 "Suppressing" tumor cell growth means, in other embodiments, retarding, delaying, or halting tumor growth or causing tumor shrinkage. Each possibility corresponds to a separate embodiment of the present invention.

[0198] In other embodiments of the methods and compositions of the present invention, WT1 expression is measured before administration of the treatment, after administration of the treatment, or both before and after administration of the treatment. In other embodiments, WT1 transcript expression is measured. In other embodiments, the WT1 protein level in tumor cells or cancer cells is measured. In other embodiments, WT1 protein or peptide released from cancer cells or tumor cells into other body fluids such as circulating fluid or urine is measured, including but not limited to these. Each possibility corresponds to a separate embodiment of the present invention.

[0199] In other embodiments of the methods and compositions of the present invention, the expression of checkpoint proteins targeted by one or more checkpoint inhibitors administered to a subject is measured (at the transcriptional or protein level) in tumors or cancer cells, or in whole blood, serum, or plasma, before administration of the treatment (baseline), after administration of the treatment, or both before and after administration of the treatment. In one embodiment of the methods and compositions of the present invention, one or more checkpoint proteins are selected from CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1 kinase, CHK2 kinase, A2aR, and B-7 family ligands. In one embodiment of the methods and compositions of the present invention, the expression of PD1, PD2, CTLA4, or a combination of two or more of the above is measured before administration of the treatment, after administration of the treatment, or both before and after administration of the treatment. In one embodiment, checkpoint protein expression is measured at the primary tumor site. In other embodiments the cancer is metastatic and checkpoint protein expression is measured at the metastatic site, or at the primary tumor site, or both.

[0200] In other embodiments of the methods and compositions of the present invention, one or more of the following markers: monocytic myeloid-derived suppressor cells (m-MDSC), C-reactive protein (CRP), absolute lymphocytes, absolute lymphocytes, and lactate dehydrogenase (LDH) are measured before administration of the treatment (baseline), after administration of the treatment, or both before and after administration of the treatment. In other embodiments, the use of one or more markers to predict or identify responsiveness to checkpoint modulation is encompassed herein.

[0201] Methods for determining the presence and magnitude of an immune response are well known in the art. In other embodiments, in a lymphocyte proliferation assay, T cell uptake of a radioactive substance (e.g., 3 3H-thymidine) is measured as a function of cell proliferation. In other embodiments, detection of T cell proliferation is by interleukin-2 (IL-2) production, Ca 2+This is achieved by measuring an increase in the uptake of dyes such as a flux or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Each possibility corresponds to a separate embodiment of the present invention.

[0202] In other embodiments, CTL stimulation is determined by means known to those skilled in the art, including cell proliferation, cytokine production, and other assays. Analysis of the type and amount of cytokines secreted by T cells upon contact with the ligand pulse target is a measure of functional activity. Cytokines are measured by ELISA, ELISPOT assay, or fluorescence-activated cell sorting (FACS) to determine cytokine production rates and total amounts. (Fujihashi K et al. (1993) J. Immunol. Meth. 160:181; Tanguay S and Killion J.J. (1994) Lymphokine Cytokine Res. 13:259).

[0203] In other embodiments, CTL activity is 51 determined by a Cr release lysis assay. Lysis of the peptide-pulsed 51 Cr-labeled target can be compared to target cells pulsed with a control peptide. In other embodiments, T cells are stimulated with the peptides of the present invention, and lysis of target cells expressing native peptides in the context of MHC is determined. In other embodiments, the kinetics of lysis and overall target lysis at a fixed time point (e.g., 4 hours) are used to evaluate ligand performance. (Ware C.F. et al. (1983) J Immunol 131: 1312).

[0204] Methods for determining the affinity of a peptide for an HLA molecule are well known in the art. In other embodiments, the affinity is determined by a TAP stabilization assay.

[0205] In other embodiments, the affinity is determined by competitive radioimmunoassay. In other embodiments, the following protocol is utilized. Target cells are washed twice in PBS containing 1% bovine serum albumin (BSA; Fisher Chemicals, Fairlawn, NJ). The cells are resuspended on ice at 10 7 / ml and stripped of native cell surface-bound peptides at 0 °C for 2 minutes using citrate-phosphate buffer in the presence of 3 mg / ml beta-2 microglobulin. In the presence of 3 mg / ml beta-2 microglobulin and 30 mg / ml deoxyribonuclease, resuspend the pellet at 5×10 6 cells / ml in PBS / 1% BSA and aliquot 200 ml and incubate at 20 °C for 10 minutes in the presence or absence of HLA-specific peptides and then 125 incubate at 20 °C for 30 minutes with I-labeled peptide. Total bound 125 I is measured after washing twice with PBS / 2% BSA and once with PBS. Relative affinity is determined by comparison of the increasing concentrations of the test peptide to a known binding peptide.

[0206] In other embodiments, the specificity of peptide binding to HLA on the surface of live cells (e.g., SKLY-16 cells) is analyzed to confirm that the binding is to the appropriate HLA molecule and to identify its restriction. This includes, in other embodiments, competition with an excess of unlabeled peptides known to bind to the same or different HLA molecules, and the use of target cells expressing the same or different HLA types. In other embodiments, this assay is performed on live fresh or 0.25% paraformaldehyde-fixed human PBMCs, leukemia cell lines, and EBV-transformed T cell lines of specific HLA types. The relative binding activity of peptides found to bind to MHC molecules on specific cells is assayed by a competitive assay as described above for known high-affinity 125 I-labeled peptides against related HLA molecules, e.g., tyrosinase or HBV peptide sequences.

[0207] In other embodiments, any WT1 peptide used in the methods and compositions of the invention comprises one or more non-classical amino acids 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. Takeda Res. 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(1):68-77) ((1992) Acta. Cryst., Crystal Struc. Comm. 48(IV):1239-124). Such non-classical amino acids are embodied in the modified peptides of the invention.

[0208] In other embodiments, any peptide used in the methods and compositions of the invention comprises one or more AA analogs or is a peptidomimetic, which in other embodiments induces or is advantageous for a particular secondary structure. Such peptides, in other embodiments, include the following. LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), β-turn-inducing dipeptide analogs (Kemp et al. (1985) J. Org. Chem. 50:5834-5838), β-sheet-inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5081-5082), β-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, Gly-Ala turn analogs (Kahn et al. (1989) Tetrahedron Lett. 30:2317), amide bond isosteres (Jones et al. (1988) Tetrahedron Left. 29(31):3853-3856), tetrazole (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880), DTC (Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501:509) and Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 and Garvey et al. (1990) J. Org. Chem. 55(3):936-940. Stereochemically restricted mimetics of beta turns and beta bulges, and peptides containing them, are described in U.S. Patent No. 5,440,013, issued to Kahn on August 8, 1995.

[0209] In other embodiments, any peptide used in the methods of the invention is conjugated to one of a variety of other molecules via covalent or non-covalent (complexation) bonds, and its properties vary according to a particular purpose in other embodiments. In other embodiments, 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 other embodiments, the peptides of the invention are linked to a substrate. In other embodiments, the peptide is conjugated to a fatty acid for introduction into liposomes (U.S. Patent No. 5,837,249). In other embodiments, the peptides of the invention are complexed to a solid support, either covalently or non-covalently, and various such are known in the art. In other embodiments, the attachment of the peptide to the carrier, substrate, fatty acid, or solid support serves to increase the induced immune response.

[0210] In other embodiments, the carrier is thyroglobulin, albumin (e.g., human serum albumin), tetanus toxoid, a polyamino acid such as poly(lysine:glutamic acid), influenza protein, hepatitis B virus core protein, keyhole limpet hemocyanin, albumin, or another carrier protein or carrier peptide, hepatitis B virus recombinant vaccine, or an APC. Each possibility corresponds to a separate embodiment of the invention.

[0211] In other embodiments, the term "amino acid" refers to natural, or in other embodiments non-natural or synthetic AAs, and in other embodiments can include glycine, D- or L optical isomers, AA analogs, peptidomimetics, or combinations thereof.

[0212] In other embodiments, the terms “cancer,” “neoplasm,” “neoplastic,” or “tumor” are used interchangeably and refer to cells that have undergone malignant transformation rendering them pathogenic to the host organism. Cancer is any stage within a numbered staging system (e.g., stage 0, 1, 2, 3, or 4), and any stage within the TNM staging system. Primary cancer cells (i.e., 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 cancer cells as used herein includes not only primary cancer cells, but also any cells derived from cancer cell progenitors. This includes metastatic cancer cells, and in vitro cultures and cell lines derived from cancer cells. In other embodiments, a tumor is detectable based on a tumor mass, for example, by procedures such as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation, and in other embodiments, identified by biochemical or immunological findings, the latter of which are also used in other embodiments to identify cancerous cells. A tumor is a solid tumor or a non-solid tumor.

[0213] Methods for synthesizing peptides are well known in the art. In other embodiments, the peptides of the invention are synthesized using suitable solid-phase synthesis procedures (see, e.g., Steward and Young, Solid Phase Peptide Synthesis , Freemantle, San Francisco, CA (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.

[0214] In other embodiments, the peptides of the invention are purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. In other embodiments, immunoaffinity chromatography is used, whereby the epitope is isolated by binding to an affinity column containing an antibody raised against the peptide or a related peptide of the invention and is attached to a solid support.

[0215] In other embodiments, affinity tags such as hexahistidine (Invitrogen), maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Meth. Enzymol. 194:508 - 509), glutathione - S - transferase, etc. can be attached to the peptides of the invention and easily purified by passing through an appropriate affinity column. The isolated peptides can also be physically characterized in other embodiments using techniques such as proteolysis, nuclear magnetic resonance, and x - ray crystallography.

[0216] In other embodiments, the peptides of the invention are generated by in vitro translation via known techniques as will be apparent to those skilled in the art. In other embodiments, the peptides are differentially modified during or after translation, for example, by phosphorylation, glycosylation, cross - linking, acylation, proteolytic cleavage, binding to antibody molecules, membrane molecules, or other ligands (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285 - 320).

[0217] In other embodiments, the peptides of the invention further comprise a detectable label, which in other embodiments is fluorescent, or in other embodiments is luminescent, or in other embodiments is radioactive, or in other embodiments has a high electron density. In other embodiments, the detectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase, 32 P, 125 I, 3 H and 14 C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin, or any number of other such labels known to those of skill in the art. The particular label used depends on the type of immunoassay being used.

[0218] In other embodiments, the peptides of the invention are linked to a substrate, which in other embodiments serves as a carrier. In other embodiments, the linking of the peptide to the substrate helps to increase the induced immune response.

[0219] In other embodiments, the peptides of the invention are linked to other molecules as described herein using conventional cross-linking agents such as carbodiimide. Examples of carbodiimide include 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.

[0220] In other embodiments, the crosslinking agent includes cyanogen bromide, glutaraldehyde, and succinic anhydride. Generally, any of a number of homobifunctional agents can be used, including homobifunctional aldehydes, homobifunctional epoxides, homobifunctional imido esters, homobifunctional N-hydroxysuccinimide esters, homobifunctional maleimides, homobifunctional alkyl halides, homobifunctional pyridyl disulfides, homobifunctional aryl halides, homobifunctional hydrazides, homobifunctional diazonium derivatives, and homobifunctional photoreactive compounds. Also, in other embodiments, heterobifunctional compounds are envisioned, such as compounds having an amine-reactive group and a sulfhydryl-reactive group, compounds having an amine-reactive group and a photoreactive group, and compounds having a carbonyl-reactive group and a sulfhydryl-reactive group.

[0221] In other embodiments, the homobifunctional crosslinking agent includes bifunctional N-hydroxysuccinimide esters dithiobis(succinimidyl propionate), disuccinimidyl suberate, disuccinimidyl tartrate, bifunctional imide esters dimethyl adipimidate, dimethyl pimelimidate, dimethyl suberimidate, bifunctional sulfhydryl-reactive crosslinking agents 1,4-di-[3'-(2'-pyridylthio)propionamide]butane, bismaleimide hexane, bis-N-maleimide-1,8-octane, bifunctional aryl halides 1,5-difluoro-2,4-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone, bifunctional photoreactive agents such as bis-[b-(4-azidosalicylamide)ethyl] disulfide, bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, adipaldehyde, bifunctional epoxides such as 1,4-butanediol diglycidyl ether, bifunctional hydrazides adipic acid dihydrazide, carbohydrazide, and succinic acid dihydrazide, bifunctional diazonium o-tolidine, diazotized and bis-diazotized benzidine, bifunctional alkyl halides N1N'-ethylene-bis(iodoacetamide), N1N'-hexamethylene-bis(iodoacetamide) N1N'-undecamethylene-bis(iodoacetamide), and benzyl halides and halomustards such as a1a'-diiodo-p-xylene sulfonic acid and tri(2-chloroethyl)amine.

[0222] In other embodiments, as described herein, hetero-bifunctional crosslinking agents used to link peptides to other molecules include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl (4-iodoacetyl) aminobenzoic acid), SMPB (succinimidyl-4-(p-maleimidophenyl) butyrate), GMBS (N-(gamma-maleimidobutyryloxy) succinimide ester), MPBH (4-(4-N-maleimidophenyl) 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).

[0223] In other embodiments, the peptides of the invention are formulated as non-covalent adducts of monomers by ionic, adsorptive, or biospecific interactions. Complexes of peptides with highly positively or negatively charged molecules can, in other embodiments, be obtained by salt bridge formation in low ionic strength environments such as in deionized water. In other embodiments, charged polymers such as poly-(L-glutamic acid) or poly-(L-lysine), each containing a large number of negative and positive charges respectively, can be used to generate large complexes. In other embodiments, the peptide is adsorbed to the surface of microparticle latex beads or other hydrophobic polymers, and in other embodiments, non-covalent peptide-superantigen complexes are formed that effectively mimic cross-linked or chemically polymerized proteins. In other embodiments, the peptide is non-covalently linked through the use of biospecific interactions between other molecules. For example, the strong affinity of biotin for proteins such as avidin or streptavidin or their derivatives can be utilized to form peptide complexes. According to this aspect, in other embodiments, the peptide is modified to have a biotin group using common biotinylation reagents such as the N-hydroxysuccinimidyl ester of D-biotin (NHS-biotin) that react with available amine groups.

[0224] In other embodiments, the peptides of the invention are linked to a carrier. In other embodiments, the carrier is KLH. In other embodiments, the carrier is any other carrier known in the art, including, for example, albumin such as thyroglobulin, human serum albumin, polyamino acids such as poly(lysine:glutamic acid), tetanus toxoid, influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine, and the like. Each possibility corresponds to a separate embodiment of the invention.

[0225] In other embodiments, the peptides of the invention are conjugated to a lipid (e.g., P3 CSS). In other embodiments, the peptides of the invention are conjugated to beads.

[0226] In any of the foregoing embodiments, a peptide, a cross-linked peptide, a binding peptide, or any other form of peptide is used in the method of the invention together with at least one checkpoint inhibitor.

[0227] In other embodiments, in addition to the use of at least one checkpoint inhibitor, the methods and compositions of the invention further comprise an immunomodulatory compound. In other embodiments, the immunomodulatory compound is a cytokine, chemokine, or complement component that enhances the expression of immune system accessory or adhesion molecules, their receptors, or combinations thereof. In some embodiments, the immunomodulatory compound comprises an interleukin, such as interleukin 1-15, interferon alpha, beta or gamma, tumor 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 chemotactic and activating factor (MCAF), RANTES, macrophage inflammatory peptide MIP-1a and MIP-1b, complement components, or combinations thereof. In other embodiments, the immunomodulatory compound stimulates or enhances the expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or combinations thereof.

[0228] In other embodiments, the immunomodulatory compound induces or enhances the expression of co-stimulatory molecules involved in the immune response in some embodiments.

[0229] In one embodiment, patients administered the WT1 vaccine and a checkpoint inhibitor according to the invention are also administered GM-CSF either before or on the day of the first vaccination, or in combination therewith. In one embodiment, the patient is administered 70 mcg of GM-CSF subcutaneously two days before and on the day of the first vaccine administration.

[0230] In other embodiments, the composition comprises a solvent comprising water, a dispersion medium, a cell culture medium, an isotonic agent, and the like. In other embodiments, the solvent is an aqueous isotonic buffer solution having a pH of about 7.0. In other embodiments, the composition comprises a diluent such as water, phosphate buffered saline, or saline. In other embodiments, the composition comprises a non-aqueous solvent such as propylene glycol, polyethylene glycol, and vegetable oil.

[0231] In other embodiments, the composition is formulated for administration by any of a number of techniques known to those of skill in the art. For example, the present invention provides for parenteral, intravenous, subcutaneous, intradermal, intramucosal, topical, oral, or inhaled administration of the pharmaceutical composition.

[0232] In other embodiments, in the use of a vaccine comprising one or more WT1 delivery agents for delivering a combination of at least seven WT1 peptides, or CTLs induced by at least seven WT1 peptides, of the present invention, the vaccine, in other embodiments, comprises lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells, or combinations thereof, and in other embodiments, further comprises cell populations that are autologous, syngeneic, or allogeneic to each other. In other embodiments, the cell population comprises the peptide of the present invention. In other embodiments, the cell population takes up the peptide. In one embodiment, the cell is an antigen presenting cell (APC). In a further embodiment, the APC is a professional APC. Each possibility corresponds to a separate embodiment of the present invention.

[0233] In other embodiments, the cell population of the present invention is obtained from an in vivo source, such as peripheral blood, leukapheresis blood products, apheresis blood products, peripheral lymph nodes, gut-associated lymphoid tissue, spleen, thymus, cord blood, mesenteric lymph nodes, liver, immune lesion sites (e.g., synovial fluid, pancreas, cerebrospinal fluid, tumor samples, granuloma tissue), or any other source from which such cells can be obtained. In other embodiments, the cell population is obtained from a human source, which, in other embodiments, is a human fetal, neonatal, pediatric, or adult source. In other embodiments, the cell population of the present invention is obtained from an animal source (e.g., pig or monkey, or any other animal of interest). In other embodiments, the cell population of the present invention is obtained from a subject that is normal, or in other embodiments, is diseased, or in other embodiments, is susceptible to the disease in question.

[0234] In other embodiments, the cell population of the present invention is separated by an affinity-based separation method. Techniques for affinity separation include, in other embodiments, magnetic separation using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents conjugated to monoclonal antibodies, or use in combination with monoclonal antibodies (e.g., complement and cytotoxins), "panning" using antibodies attached to a solid matrix such as a plate, or any other convenient technique. In other embodiments, the separation technique includes the use of fluorescence-activated cell sorters with varying degrees of sophistication, such as multi-color channels, low-angle and obtuse-angle light scatter detection channels, impedance channels, etc. In other embodiments, any technique that enables the separation of the cell population of the present invention is used and should be considered as part of the present invention.

[0235] In other embodiments, dendritic cells are derived from a diverse population of morphologically similar cell types found in various lymphoid and non-lymphoid tissues (Steinman (1991) Ann. Rev. Immunol. 9:271-296). In other embodiments, the dendritic cells used in the present invention are isolated from bone marrow, or, in other embodiments, are derived from bone marrow progenitor cells, or, in other embodiments, are isolated from / derived from peripheral blood, or, in other embodiments, are derived from a cell line or are a cell line.

[0236] In other embodiments, the cell populations described herein are isolated from the leukocyte fraction of a mammal (e.g., mouse, monkey or human) (see, e.g., WO96 / 23060). The leukocyte fraction can, in other embodiments, be isolated from the peripheral blood of a mammal.

[0237] Methods for isolating dendritic cells are well known in the art. In other embodiments, DCs are isolated via a method comprising the following steps. (a) Providing a leukocyte fraction obtained from a mammalian source by a method known in the art such as leukapheresis, (b) Separating the leukocyte fraction of step (a) into four or more subfractions by counterflow centrifugal elutriation, (c) Stimulating the conversion of monocytes of step (b) to dendritic cells by contacting the cells with a 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°C.

[0238] In other embodiments, the dendritic cell-enriched fraction is identified by fluorescence-activated cell sorting, which, in other embodiments, identifies at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the following markers: CD3, CD14, CD16, 56, 57, and CD19, 20 are absent.

[0239] In other embodiments, the cell population comprises lymphocytes, which, in other embodiments, are T cells, or, in other embodiments, are B cells. T cells are, in other embodiments, NK cells, helper T cells, cytotoxic T lymphocytes (CTLs), TILs, naive T cells, or combinations thereof. Primary T cells, or cell lines, clones, etc. are considered to be part of the present invention. In other embodiments, the T cells are CTLs, or CTL lines, CTL clones, or CTLs isolated from tumors, inflammatory, or other infiltrates.

[0240] In other embodiments, hematopoietic stem cells or early progenitor cells are included in the cell population used in the present invention. In other embodiments, such populations are isolated or induced by leukapheresis. In other embodiments, leukapheresis occurs following cytokine administration from bone marrow, peripheral blood (PB), or neonatal umbilical cord blood. In other embodiments, the stem cells or progenitor cells are characterized by surface expression of the surface antigen marker known as CD34 + and exclusion of the expression of the surface lineage antigen marker Lin-.

[0241] In other embodiments, the subject is administered the peptide, composition, or vaccine of the present invention together with bone marrow cells. In other embodiments, the administration together with the bone marrow cell embodiment is made as part of the treatment process following prior irradiation of the subject in order to suppress, inhibit, or treat cancer in the subject.

[0242] In other embodiments, the phrase "contacting a cell" or "contacting a population" refers to a method of exposure, which is direct or indirect in other embodiments. In other embodiments, such contact includes direct injection of cells by any means well known in the art, such as microinjection. Also, in other embodiments, the supply to the cells is assumed to be indirect, for example, by supply in a culture medium surrounding the cells, or via any route well known in the art through administration to the subject and as described herein.

[0243] In other embodiments, CTL generation of the methods of the invention is achieved in vivo and is achieved by introducing into a subject antigen-presenting cells contacted in vitro with a peptide of the invention, administered together with at least one checkpoint inhibitor (see, e.g., Paglia et al. (1996) J. Exp. Med. 183:317-322).

[0244] In other embodiments, the peptides of the methods and compositions of the invention are delivered to antigen-presenting cells (APCs).

[0245] In other embodiments, the peptide is delivered to the APC in the form of cDNA encoding the peptide. In other embodiments, the term "antigen-presenting cell" refers to dendritic cells (DCs), monocytes / macrophages, B lymphocytes, or other cell types expressing the necessary MHC / costimulatory molecules that enable effective T cell recognition of the presented peptide. In other embodiments, the APC is a cancer cell. Each possibility corresponds to a separate embodiment of the invention. In each embodiment, vaccine or APC or delivery of the peptide in any form to a patient or subject is administered together with at least one checkpoint inhibitor. As described herein, administration of at least one checkpoint inhibitor need not be at the same vaccine, formulation, site of administration, or time of administration as the WT1 vaccine or an alternative form thereof. As embodied herein, administration of a checkpoint inhibitor in any of its various embodiments simultaneously with the WT1 vaccine enhances the formation of WT1-specific CTLs in subjects in need thereof.

[0246] In other embodiments, CTLs contact two or more antigen-presenting cell populations together with at least one checkpoint inhibitor. In other embodiments, the two or more antigen-presenting cell populations present different peptides. Each possibility corresponds to a separate embodiment of the invention.

[0247] In other embodiments, techniques that lead to the expression of an antigen in the cytosol of an APC (e.g., a DC) are used to deliver the peptide to the APC. Methods for expressing an antigen on an APC are well known in the art. In other embodiments, this technique includes (1) introduction of naked DNA encoding the peptide of the invention into the APC, (2) infection of the APC with a recombinant vector expressing the peptide of the invention, and (3) introduction of the peptide of the invention into the cytosol of the 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).

[0248] In other embodiments, foster antigen - presenting cells (referred to as T2, including mutations in its antigen - processing pathway that limit the association of endogenous peptides with cell - surface MHC class I molecules) derived from the human cell line 174xCEM.T2 (Zweerink et al. (1993) J. Immunol. 150:1763 - 1771) are used as exemplified herein.

[0249] In other embodiments, any of the methods described herein are used to induce CTLs induced in vitro. In other embodiments, CTLs are induced ex vivo. In other embodiments, CTLs are induced in vitro. The resulting CTLs are, in other embodiments, administered to a subject, thereby treating symptoms associated with a peptide, an expression product containing the peptide, or a homolog thereof, administered together with at least one checkpoint inhibitor. Each possibility corresponds to a separate embodiment of the invention.

[0250] In other embodiments, the method of the invention involves the introduction of a gene sequence encoding a combination of at least seven WT1 peptides of the invention. Since the nucleic acid is contained within one or more vectors, in other embodiments, the method involves administering to a subject a vector comprising a nucleotide sequence encoding a peptide of the invention (Tindle, RW. et al., Virology (1994) 200:54). In other embodiments, the method involves administering to a subject naked nucleic acid (DNA or RNA) encoding a peptide, or in other embodiments, two or more peptides of the invention (Nabel et al., PNAS-USA (1990) 90: 11307). In other embodiments, multi-epitope, analogue-based cancer vaccines are utilized (Fikes et al., Expert Opin Biol Ther., 2003, Sep;3(6):985-993). Each possibility corresponds to a distinct embodiment of the invention. In each of the foregoing embodiments, the nucleic acid can encode each WT1 peptide individually, or a combination of up to seven, or more than seven WT1 peptides. The nucleic acid encoding a WT1 peptide represents one form of a WT1 delivery agent. A combination of at least seven WT1 peptides is delivered by one form of a WT1 delivery agent, for example, a peptide, or a nucleic acid, or an immune cell, or any combination of two or three of the foregoing.

[0251] The nucleic acid may encode a single WT1 peptide out of the seven WT1 peptides, or the nucleic acid may encode a plurality (e.g., two, three, four, five, six, or all seven) of the seven WT1 peptides. Similarly, the nucleic acid, if utilized, may encode one or more additional WT1 peptides. Accordingly, the compositions and methods of the invention use a single nucleic acid or multiple nucleic acids to act as a WT1 delivery agent. The compositions and methods of the invention use a single vector to deliver at least seven WT1 peptides or multiple vectors.

[0252] Nucleic acids (DNA or RNA) are administered to a subject via any means known in the art, including parenteral or intravenous administration, or in other embodiments, by means of a gene gun. In other embodiments, the nucleic acids are administered in a composition corresponding to any of the embodiments recited herein in other embodiments. The DNA or RNA is administered to the subject as naked nucleic acid or is carried by a vector.

[0253] For use according to the method of the present invention, the vector can, in other embodiments, comprise any vector that promotes or enables the expression of the peptides of the present invention (e.g., one or more of at least seven WT1 peptides) in cells in vitro or in vivo. The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, particle) that can be used to transfer coding sequence information (e.g., a nucleic acid sequence encoding a WT1 peptide) to a cell or a subject. Nucleic acid vaccines for several cancers are in clinical trials (Wahren B et al., "DNA Vaccines: Recent Developments and Future", Vaccines, 2014, 2:785-796; Fioretti D. et al., "DNA Vaccines: Developing New Strategies Against Cancer, Journal of Biomedicine and Biotechnology, 2010(938):174378). Strategies for proliferative functional WT1-specific T cells using DNA vaccines are known. (Chaise C et al [DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance", Blood, 2008, 112(7):2956-2964). In other embodiments, the vector is a non-viral vector. In one embodiment, the non-viral vector is a nucleic acid vector such as a plasmid DNA or mRNA vector (e.g., see Weide B. et al., "Plasmid DNA- and messenger RNA-based Anti-Cancer Vaccination", Immunol Lett, 2008, 115(1):33-42), Kim H. et al., "Self-Assembled Messenger RNA Nanoparticles(mRNA-NP) for Efficient Gene Expression", Sci Rep, 2015, 5:12737), Ulmer J.B. et al., "RNA-based Vaccines", Vaccine, 2012, 30:4414-4418).In other embodiments, the "vector" includes attenuated viruses such as vaccinia and fowlpox as described in U.S. Pat. No. 4,722,848, incorporated herein by reference. In other embodiments, the vector is BCG (Bacille Calmette Guerin) as described in Stover et al. (Nature 351:456-460 (1991)). Other vectors useful for the therapeutic administration or immunization of the peptides of the present invention (e.g., Salmonella typhi vectors, etc.) will be apparent to those skilled in the art from the description herein. Examples of vectors used for in vivo and in vitro administration to a nucleic acid molecule include, but are not limited to, adenovirus, adeno-associated virus, retrovirus, lentivirus, poxvirus, herpesvirus, virus-like particles (VLPs), plasmid, cationic lipids, liposomes, and nanoparticles.

[0254] A "coding sequence" is a nucleic acid sequence that is transcribed into mRNA and / or translated into a polypeptide. The boundaries of the coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. Coding sequences include, but are not limited to, mRNA, cDNA, and recombinant polynucleotide sequences. Variants or analogs are made by deletion of a portion of the coding sequence, by insertion of a sequence, and / or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleic acid sequences, such as site-directed mutagenesis, are well known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, 1989; DNA Cloning, Vols. I and II, D. N. Glover ed., 1985). Optionally, the nucleic acid sequences of the present invention, and the compositions and methods of the present invention utilizing such polynucleotides, can include non-coding sequences.

[0255] The term "operably linked" is used herein to refer to the arrangement of a flanking control sequence such that the flanking array so described is configured or assembled to perform its normal function. Thus, a flanking control sequence operably linked to a coding sequence affects the replication, transcription, and / or translation of the coding sequence under conditions compatible with the control sequence. For example, a coding sequence is operably linked to a promoter if the promoter can direct transcription of the coding sequence. The flanking array need not be contiguous with the coding sequence so long as it functions correctly. Thus, for example, intervening non-translated but transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence is still considered to be "operably linked" to the coding sequence. Each nucleic acid sequence encoding a polypeptide (e.g., the WT1 peptide) typically has its own operably linked promoter sequence.

[0256] In other embodiments, the vector further encodes an immunomodulatory compound, as described herein. In other embodiments, an additional vector encoding the subject is administered continuously before or after administration of the vector encoding the peptide of the invention to the subject.

[0257] In other embodiments, the WT1 delivery agent, CTL, composition, and vaccine of the invention are administered to a subject or utilized in combination with other anti-cancer compounds and chemotherapeutic agents (monoclonal antibodies against alternative cancer antigens, or, in other embodiments, epitopes comprising AA sequences corresponding to or partially corresponding to those from which the peptides of the invention are derived) in the methods of the invention. This is in addition to the use of at least one checkpoint inhibitor in various embodiments of the invention.

[0258] In other embodiments, the invention provides for WT1-specific CD4 in a subject +Provided is a method for detecting a T cell response, the method comprising administering to a subject a WT1 delivery agent, a vaccine, or a composition of the present invention. In other embodiments, a delayed hypersensitivity test is used to detect a WT1-specific CD4 + T cell response. In other embodiments, the peptide of the present invention is superior to its non-mutated counterpart in inducing a CD4 + T cell response in a subject. Each possibility corresponds to a separate embodiment of the present invention.

[0259] As used herein, the terms "patient", "subject", and "individual" are used interchangeably and are intended to include human and non-human animal species. For example, the subject is a human or non-human mammal. In some embodiments, the subject is a non-human animal model or an affected animal. The subject can be of any age or gender.

[0260] In other embodiments, the immunogenic compositions of the methods and compositions of the present invention comprise one or more WT1 delivery agents of the present invention and / or APCs associated with CTLs. In other embodiments, the immunogenic composition consists of one or more WT1 delivery agents of the present invention and / or APCs associated with CTLs. In other embodiments, the immunogenic composition comprises, or consists of, APCs associated with a combination of at least seven WT1 peptides.

[0261] In other embodiments, the compositions of the methods and compositions of the present invention are immunogenic compositions. In other embodiments, the composition is a pharmaceutical composition. In other embodiments, the composition is any other type of composition known in the art. Each possibility corresponds to a separate embodiment of the present invention. Each composition further comprises at least one checkpoint inhibitor.

[0262] Various embodiments of the dosage range are contemplated by the present invention. In other embodiments, the dosage is 20 μg / peptide per day. In other embodiments, the dosage is 10 μg / peptide / day. In other embodiments, the dosage is 30 μg / peptide / day. In other embodiments, the dosage is 40 μg / peptide / day. In other embodiments, the dosage is 60 μg / peptide / day. In other embodiments, the dosage is 80 μg / peptide / day. In other embodiments, the dosage is 100 μg / peptide / day. In other embodiments, the dosage is 150 μg / peptide / day. In other embodiments, the dosage is 200 μg / peptide / day. In other embodiments, the dosage is 300 μg / peptide / day. In other embodiments, the dosage is 400 μg / peptide / day. In other embodiments, the dosage is 600 μg / peptide / day. In other embodiments, the dosage is 800 μg / peptide / day. In other embodiments, the dosage is 1000 μg / peptide / day.

[0263] In other embodiments, the dosage is 10 μg / peptide / dose amount. In other embodiments, the dosage is 30 μg / peptide / dose amount. In other embodiments, the dosage is 40 μg / peptide / dose amount. In other embodiments, the dosage is 60 μg / peptide / dose amount. In other embodiments, the dosage is 80 μg / peptide / dose amount. In other embodiments, the dosage is 100 μg / peptide / dose amount. In other embodiments, the dosage is 150 μg / peptide / dose amount. In other embodiments, the dosage is 200 μg / peptide / dose amount. In other embodiments, the dosage is 300 μg / peptide / dose amount. In other embodiments, the dosage is 400 μg / peptide / dose amount. In other embodiments, the dosage is 600 μg / peptide / dose amount. In other embodiments, the dosage is 800 μg / peptide / dose amount. In other embodiments, the dosage is 1000 μg / peptide / dose amount.

[0264] In other embodiments, the dosage is 10 - 20 μg / peptide / dose amount. In other embodiments, the dosage is 20 - 30 μg / peptide / dose amount. In other embodiments, the dosage is 20 - 40 μg / peptide / dose amount. In other embodiments, the dosage is 30 - 60 μg / peptide / dose amount. In other embodiments, the dosage is 40 - 80 μg / peptide / dose amount. In other embodiments, the dosage is 50 - 100 μg / peptide / dose amount. In other embodiments, the dosage is 50 - 150 μg / peptide / dose amount. In other embodiments, the dosage is 100 - 200 μg / peptide / dose amount. In other embodiments, the dosage is 200 - 300 μg / peptide / dose amount. In other embodiments, the dosage is 300 - 400 μg / peptide / dose amount. In other embodiments, the dosage is 400 - 600 μg / peptide / dose amount. In other embodiments, the dosage is 500 - 800 μg / peptide / dose amount. In other embodiments, the dosage is 800 - 1000 μg / peptide / dose amount.

[0265] In other embodiments, the total amount of peptide per dose amount or per day is one of the amounts described above. In other embodiments, the total peptide dose amount per dose amount is one of the amounts described above.

[0266] Each of the above dosage amounts represents a separate embodiment of the present invention.

[0267] In other embodiments, the present invention provides a kit comprising the peptide, composition or vaccine of the present invention together with at least one checkpoint inhibitor. In other embodiments, the kit further comprises a label or a packaging insert. In other embodiments, the kit is used to detect a WT1-specific CD4 response via use of a delayed hypersensitivity test. In other embodiments, the kit is used for any of the other methods listed herein. In other embodiments, the kit is used for any other method known in the art. Each possibility corresponds to a separate embodiment of the present invention.

[0268] Example Evaluation of the Efficacy of a 7-Valent WT1 Immunotherapy Composition Administered with Nivolumab in Ovarian Cancer Patients Eligible patients diagnosed with ovarian cancer start the vaccination schedule within 4 months after the end of chemotherapy. Patients first receive 6 vaccinations of WT1 peptide over 12 weeks and 7 infusions of the immune checkpoint inhibitor nivolumab over 14 weeks. Toxicity evaluation is performed 3 weeks after the end of treatment at week 15 for each dose of the vaccine. Patients are observed by the study staff for 30 minutes after treatment. Dose escalation is not planned. Routine toxicity evaluation is continued throughout the trial period.

[0269] Patients in whom disease progression is not observed at the 15-week evaluation are allowed to receive 4 additional vaccinations approximately every 8 weeks. This maintenance vaccine course starts at week 19.

[0270] Immune responses are evaluated at 6 time points: at baseline (at the time of consent and before the first dose, to confirm baseline fluctuations), 5 weeks, 6 weeks, and 3 weeks after the last nivolumab infusion, with 40 ml of heparinized blood samples. If possible, additional blood sampling is performed at the 3-month follow-up.

[0271] Using ELISA, the antibody levels generated against four WT1 peptides in the vaccine are measured. Antibodies generally persist until completion of the fourth vaccination. The T cell proliferation response assay is performed on peripheral blood lymphocytes, including: flow cytometry for phenotypic analysis by FACS, including white blood cell subset analysis; T regulatory cell assay (including CD3, CD4, CD8, FOXP3, ICOS, and PD1); and myeloid-derived suppressor cells (MDSC, CD14+HLA-DRlow cells) in peripheral blood and in tumors (if any biopsies are obtained). The WT1 T cell-specific CD4 and CD8 proliferation responses are measured using a cytotoxicity assay based on flow cytometry using the Meso Scale Discovery System with functionality measured by multifunctional intracellular cytokine staining (ICS) and IFN-gamma production. Detailed procedures for blood sample processing, T cell monitoring, antibody ELISA, and multifunctional T cell assays are described in

[29] .

[0272] The results of baseline values and T cell responses correlate with the duration of clinical remission.

[0273] If patients are excluded from the study before 15 weeks, blood for post-study immunological testing is collected. CT scans are performed at baseline and at week 15 (earlier if medically necessary), and then every 3 months up to 1 year until disease progression. Abdominal and pelvic MRI may be used instead of abdominal and pelvic CT. The reference radiologist uses immune-related response criteria

[57] to determine disease progression. CA125 is measured at baseline, at 6 weeks and 15 weeks, and then every 3 months up to 1 year until disease progression. CA125 is not used to determine disease progression because the possibility of inflammation in vaccinated patients is confounded. The trial continues until progression, the development of unacceptable toxicity, completion of the vaccination sequence, or patient withdrawal is noted.

[0274] WT1 vaccine: The vaccine used in this study contains seven different WT1 peptides: ·YMFPNAPYL (SEQ ID NO: 124, WT1-A1): An HLA class I peptide with the mutant amino acid R126Y that stimulates CD8+ responses. ·SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125, WT1-122A1 long): An HLA class II peptide containing the WT1-A1 heteroclitic sequence embedded within a longer peptide to stimulate both CD4+ and CD8+ responses according to data from preclinical and phase 1 trials. ·RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1, WT1-427 long) and PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2, WT1-331 long): HLA class II peptides that induce CD4+ responses that help sustain CD8+ T cell responses. ·NLMNLGATL (SEQ ID NO: 21, NLM short) ·WNLMNLGATLKGVAA (SEQ ID NO: 26, NLM long) ·WNYMNLGATLKGVAA (SEQ ID NO: 205, NLM long).

[0275] Drug product: Seven peptides are provided in a sterile solution containing phosphate-buffered saline for manufacturing a vaccine product (“WT1Vax”). Each vial contains 280 mcg of each peptide at a total volume of 0.7 ml (0.4 mg / ml of each peptide, 40% overfill). Vial and sterility testing were performed under GMP conditions. The vaccine emulsion is prepared individually before use. This requires a mixture of the peptide solution and the immunological adjuvant Montanide ISA51VG.

[0276] Intended dose: A dose of 200 mcg is selected for each peptide, as it is within the range of safe and active doses used otherwise. Peptide vaccines have elicited immune and clinical responses within a wide range of doses (100 - 2000 mcg injection) without clear evidence of a dose - response relationship. At higher doses, there is a theoretical possibility of stimulating low - affinity TCRs on T cells and reducing the response [30, 33, 34]. Vial size: Each single - dose vial is 0.7 ml, Route of administration: Subcutaneous. Nivolumab: Intended dose: 3 mg / kg, Vial size: 10 mL, Route of administration: Intravenous. Nivolumab is administered at 3 mg / kg and is given intravenously by a 60 - minute intravenous infusion once every two weeks. At the end of the infusion, the line is flushed with a sufficient amount of normal saline. If the subject's weight differs by >10% from the previous weight used to calculate the required dose, the required dose, the corrected dose, should be calculated. No dose escalation or de - escalation of nivolumab is recognized. There is no recommended pre - medication for the first nivolumab treatment.

[0277] Subjects are administered within 12 days or less after the scheduled dosing day and within 3 days or less after the previous nivolumab administration. Administration after the 3 - day time frame is considered a dosing delay. Treatment may be postponed for up to 6 weeks from the previous administration.

[0278] Tumor evaluation by CT or MRI should continue according to the protocol even if dosing is delayed.

[0279] Treatment / Intervention Plan · Patients are treated as outpatients. · The WT1 vaccine is administered at weeks 0, 2, 4, 6, 8, and 10. · All injections are administered subcutaneously at sites rotated between the extremities. · All patients are injected subcutaneously with 70 mcg of sargramostim (GM - CSF) on days 0 and - 2. If the patient has been properly instructed about SQ injection administration, GM - CSF may be self - administered. The patient should be informed about the expected reactions such as irritation at the injection site. The patient keeps a record book of the injection time and location. · The patient also receives an emulsion of 1.0 ml of WT1 peptide and Montanide. It is administered subcutaneously at the same anatomical site as GM-CSF by a nurse (it may not be self-administered). · Observe the patient for approximately 30 minutes after vaccination. · At weeks 0, 2, 4, 6, 8, 10, and 12, nivolumab is administered as a 60-minute infusion. For subjects, nivolumab is administered more than 12 days after the scheduled administration date and within 3 days after the scheduled administration date. Administration after the 3-day time frame is considered a delayed administration. Treatment may be delayed for up to 6 weeks from the previous administration.

[0280] The combined treatment of WT1 vaccine and nivolumab is expected to increase the WT1-specific CTL population in patients and increase the activity against WT1-expressing tumors compared to WT1 vaccine alone or nivolumab monotherapy.

Claims

1. YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26) and WNYMNLGATLKGVAA (SEQ ID NO: 205) A combination (a) of at least seven isolated peptides consisting of, A nucleic acid (b) encoding the combination of at least seven isolated peptides of (a), An immune cell (c) comprising the nucleic acid encoding the combination of at least seven isolated peptides of (a) and / or presenting or containing at least seven peptides of (a), Cytotoxic T cells (CTLs) (d) induced by the combination of at least seven isolated peptides of (a), or A combination (e) of two, three, or all four of (a), (b), (c), and (d), An immunotherapy composition.

2. A composition for use in treating WT1-expressing cancer in a subject in need thereof, YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26) and WNYMNLGATLKGVAA (SEQ ID NO: 205) A combination (a) of at least seven isolated peptides consisting of, A nucleic acid (b) encoding the combination of at least seven isolated peptides of (a), An immune cell (c) comprising the nucleic acid encoding the combination of at least seven isolated peptides of (a) and / or presenting or containing at least seven peptides of (a), Cytotoxic T cells (CTLs) (d) induced by the combination of at least seven isolated peptides of (a), or A combination (e) of two, three, or all four of (a), (b), (c), and (d), A composition.

3. A composition for use in reducing the incidence of WT1-expressing cancer in a subject in need thereof, YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26) and WNYMNLGATLKGVAA (SEQ ID NO: 205) A combination (a) of at least seven isolated peptides consisting of: A nucleic acid (b) encoding the combination of at least seven isolated peptides of (a), An immune cell (c) comprising the nucleic acid encoding the combination of at least seven isolated peptides of (a) and / or presenting at least seven peptides of (a), Cytotoxic T cells (CTLs) (d) induced by the combination of at least seven isolated peptides of (a), or A composition comprising combinations (e) of two, three, or all four of (a), (b), (c), and (d). Composition.

4. A composition for use in inducing an immune response against WT1-expressing cancer in a subject in need thereof, YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26) and WNYMNLGATLKGVAA (SEQ ID NO: 205) A combination (a) of at least seven isolated peptides consisting of: A nucleic acid (b) encoding the combination of at least seven isolated peptides of (a), An immune cell (c) comprising the nucleic acid encoding the combination of at least seven isolated peptides of (a) and / or presenting at least seven peptides of (a), Cytotoxic T cells (CTLs) (d) induced by the combination of at least seven isolated peptides of (a), or A composition comprising combinations (e) of two, three, or all four of (a), (b), (c), and (d). Composition.

5. A composition for use in inducing the formation and proliferation of T cells specific for WT1-expressing cancer in a subject, YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), NLMNLGATL (SEQ ID NO: 21), WNLMNLGATLKGVAA (SEQ ID NO: 26) and WNYMNLGATLKGVAA (SEQ ID NO: 205) a combination (a) of at least seven isolated peptides consisting of a nucleic acid (b) encoding the combination of at least seven isolated peptides of (a), an immune cell (c) comprising the nucleic acid encoding the combination of at least seven isolated peptides of (a) and / or comprising or presenting at least seven peptides of (a), or a composition comprising a combination (d) of two or three of (a), (b), and (c). Composition.

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