Methods and compositions for treating cancer

Abstract

The present invention provides compositions and kits used to treat WT1-expressing cancer, reduce the incidence of WT1-expressing cancer, or induce an immune response against WT1-expressing cancer. [Solution] The composition comprises (A) a combination of at least four WT1 peptides, a nucleic acid encoding the combination of the at least four WT1 peptides, a vector containing the nucleic acid, or immune cells containing or presenting the combination of at least four WT1 peptides, or containing nucleic acids encoding the combination of the at least four WT1 peptides, and (B) at least one checkpoint inhibitor.

Description

[Technical Field]

[0001] The present invention provides methods for treating WT1-expressing cancer, reducing the incidence of WT1-expressing cancer, or inducing an immune response against WT1-expressing cancer, and compositions useful for these purposes. [Overview of the project] [Means for solving the problem]

[0002] The present invention provides methods for treating WT1-expressing cancer, reducing the incidence of WT1-expressing cancer, or inducing an immune response against WT1-expressing cancer, and compositions comprising immunogenic compositions useful for these purposes. In one embodiment, the present invention includes the step of administering to a subject in need of treatment, etc., (a) one or more WT1 peptides or cytotoxic T cells (CTLs) against WT1-expressing cancer, and (b) one or more checkpoint inhibitors. One or more WT1 peptides are administered to a subject by administering one or more agents (WT1 delivery agents) to the subject that deliver one or more WT1 peptides to induce an immune response against WT1-expressing cancer. Examples of WT1 delivery agents that can be used include (i) isolated WT1 peptides, (ii) nucleic acids encoding at least one WT1 peptide, or (iii) immune cells containing or presenting at least one WT1 peptide or nucleic acids encoding said peptide.

[0003] One or more WT1 peptides may be native peptides that are fragments of the WT1 protein, or peptides having one or more modifications that enhance their immunogenicity. These modifications may be amino acid changes (e.g., heterocritical peptides) or any other modifications. CTLs may be WT1-specific CTLs produced in vitro or ex vivo, or may be obtained from a donor. WT1 delivery agents or CTLs may be administered in the form of a composition, together with a carrier, excipient, diluent, or adjuvant. A non-limiting selection of peptide components used in the methods and compositions embodied by the present invention will be described later.

[0004] One or more checkpoint inhibitors (also known as immune checkpoint inhibitors) are compositions or drugs that block or inhibit immune checkpoint proteins. Non-limiting examples of compositions or drugs that are checkpoint inhibitors include small molecules, peptides, or antibodies. Non-limiting examples of antibodies include nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011), MEDI-0680 (AMP-514), AMP-224, AUNP-12, BMS-936559, atezolizumab (MPDL-3280A), durvalumab (MEDI-4736), avelumab ( This includes MSB-0010718C), BMS-935559 (MDX-1105), rHIgM12B7, BMS-986016, GSK-2831781, IMP-321, lirilumab (BMS-986015), IPH-2101 (1-7F9), indoximod (NLG-9189), NLG-919, INCB-024360, PF-05082566, urelumab (BMS-663513), and MEDI-6469.

[0005] In one embodiment, a method is embodied in which one or more WT1 delivery agents or CTLs and one or more checkpoint inhibitors are administered to a subject according to a schedule that yields the greatest benefit to the subject. Therefore, the one or more WT1 delivery agents or CTLs and the one or more checkpoint inhibitors do not necessarily have to be administered simultaneously, in the same composition, for the same duration, or via the same route. Each WT1 peptide may be administered according to a specific schedule. The same applies to each checkpoint inhibitor. In one embodiment, the administration schedules of at least one WT1 peptide and at least one checkpoint inhibitor are simultaneous. In one embodiment, the administration 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 contained in the same composition. In one embodiment, the method of the present invention improves or enhances the effect of treating WT1-expressing cancer, reducing the incidence of WT1-expressing cancer, or inducing an immune response against WT1-expressing cancer compared to the administration of a WT1 delivery agent or CTL alone, or the administration of a checkpoint inhibitor alone. In one embodiment, the effects of the method of the present invention on treating WT1-expressing cancer, reducing the incidence of WT1-expressing cancer, or inducing an immune response against WT1-expressing cancer are greater than the combined effects of administering a WT1 delivery agent or CTL alone and administering a checkpoint inhibitor alone.

[0006] The dosage and administration schedule, route of administration, and other aspects of administration for WT1 delivery agents or CTLs and checkpoint inhibitors are optimized to provide the greatest benefit to the target. Embodiments of the present invention provide improved methods for treating WT1-expressing cancers, reducing the incidence of WT1-expressing cancers, or inducing an immune response against WT1-expressing cancers, and improved compositions useful for these purposes.

[0007] The cancer to which the method of the present invention is applicable is any cancer that expresses the WT1 protein or a fragment thereof. In one embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is mesothelioma. In yet another embodiment, the cancer is leukemia. In yet another embodiment, the cancer is Wilms' tumor, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), melanoma, gastric cancer, prostate cancer, biliary tract cancer, urinary tract cancer, glioblastoma, soft tissue sarcoma, osteosarcoma, or non-small cell lung cancer (NSCLC). [Modes for carrying out the invention]

[0008] The present invention provides methods for treating WT1-expressing cancer, reducing the incidence of WT1-expressing cancer, or inducing an immune response against WT1-expressing cancer, and compositions comprising immunogenic compositions useful for these purposes. In one embodiment, the present invention includes the step of administering to a subject in need of treatment, etc., (a) one or more WT1 peptides or cytotoxic T cells (CTLs) against WT1-expressing cancer, and (b) one or more checkpoint inhibitors. One or more WT1 peptides are administered to a subject by administering one or more agents (WT1 delivery agents) to the subject that deliver one or more WT1 peptides to induce an immune response against WT1-expressing cancer. Examples of WT1 delivery agents that can be used include (i) isolated WT1 peptides, (ii) nucleic acids encoding at least one WT1 peptide, or (iii) immune cells containing or presenting at least one WT1 peptide or nucleic acids encoding said peptide.

[0009] Ovarian cancer is one of the most common gynecologic 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 disease at the time of diagnosis [2]. The 5-year survival rate for advanced disease remains less than 30% [1]. Although a complete clinical remission can be expected in many patients after initial chemotherapy, evaluation by second-look laparotomy, which is often performed as part of routine care, indicates that less than 50% of patients are free of recurrence [3]. Furthermore, nearly half of patients with a negative evaluation by second-look laparotomy relapse and require additional treatment [4]. Many patients will achieve a second complete clinical response with additional chemotherapy. However, almost all patients will relapse after a short remission period of 9-11 months [5]. Until chemoresistance develops extensively, subsequent remission periods will gradually shorten, and there is a need for effective strategies to prolong remission or prevent recurrence [2].

[0010] Both antibodies and T cell effectors have been shown to be beneficial in ovarian cancer models. Antibodies have been shown to inhibit initial tissue infiltration [6]. Preclinical models have also demonstrated the elimination of circulating tumor cells and systemic micrometastases by the use of both passively administered antibodies and antibodies induced by vaccines. With regard to T cell effectors, an overall activated immune response has been shown to be associated with improved clinical outcomes in patients with advanced ovarian cancer. Zhang et al. showed that the presence of tumor infiltrating T cells within tumor cell islands was associated with improved both progression-free survival and overall survival [7]. Conversely, infiltration of regulatory T cells worsens the prognosis [8].

[0011] Data from subsequent remissions in patients with ovarian cancer support the expectation of relapses in predictable forms.[9] In recent years, ovarian cancer has become a target for a variety of novel immune-based approaches. Antibody therapies include oregovomab, a monoclonal antibody therapy targeting the CA125 antigen

[10] , avagovomab, an anti-idiotype antibody targeting the CA-125 antigen

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

[12] . Other strategies include, for example, interferon-gamma therapy[13, 14] and cytokine therapies such as IL-2

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

[16] , MUC1

[17] , the HLA restriction peptide NY-ESO-1b

[18] , and KH-1-KLH conjugates has also been evaluated. Because conventional strategies have been ineffective, there is a need for new therapies to enhance the effectiveness of treatments for ovarian cancer and various other cancers that cannot be effectively treated with current therapies.

[0012] WT1 refers to the gene product of the Wilms tumor 1 or WT1 gene. The Wilms tumor suppressor gene WT1 was first identified in pediatric renal tumors, but is also highly expressed in a number of other hematological malignancies and solid tumors, including mesothelioma [19, 20]. WT1 was originally identified by cDNA mapping to the region of chromosome 11p13. The WT1 cDNA encodes a protein containing four Kruppel zinc fingers and includes a complex pattern of alternative splicing that generates four distinct transcription factors. Each WT1 isoform has different DNA-binding and transcriptional activities

[20] 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 (e.g., kidney, gonad, heart, mesothelial cells, spleen) during embryogenesis

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

[23] . WT1 shows a high degree of homology in mice and humans (96% at the amino acid level) and has similar tissue distribution and functions [24, 25]. Although originally considered a tumor suppressor gene, the WT1 protein appears to be involved in tumor formation.

[0013] The potent expression of the WT1 protein in ovarian cancer, coupled with its proposed mechanism of action, makes it a reasonable target for immunotherapy in a variety of other cancers that express the WT1 protein, including, but not limited to, mesothelioma, leukemia, Wilms' tumor, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), melanoma, gastric cancer, prostate cancer, biliary tract cancer, urinary tract cancer, glioblastoma, soft tissue sarcoma, osteosarcoma, or non-small cell lung cancer (NSCLC). In ovarian cancer, expression is so frequent that pathologists typically use immunohistochemical staining for WT1 (using standardized methods to show expression and determine "positive" or "negative" to distinguish epithelial ovarian cancer from other tumors). WT1 is a particularly sensitive and specific marker for serous ovarian cancer

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

[0014] One or more WT1 peptides useful for the purposes of the present invention may be native peptides that are fragments of the WT1 protein. In one embodiment, the WT1 peptide is RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), LVRHHNMHQRNMTKL (SEQ ID NO: 3), or NKRYFKLSHLQMHSR (SEQ ID NO: 4). In another embodiment, the WT1 peptide is SGQARMFPNAPYLPSCLES (SEQ ID NO: 5), or QARMFPNAPYLPSCL (SEQ ID NO: 6). In another embodiment, the WT1 peptide is 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).

[0015] In another embodiment, the WT1 peptide is NQMNLGATL (SEQ ID NO: 20), NLMNLGATL (SEQ ID NO: 21), NYMNLGATL (SEQ ID NO: 22), CMTWNQMNLGATLKG (SEQ ID NO: 23), CMTWNLMNLGATLKG (SEQ ID NO: 24), WNQMNLGATLKGVAA (SEQ ID NO: 25), WNLMNLGATLKGVAA (SEQ ID NO: 26), 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), RRLPHPGAL(SEQ ID NO:54), RIRPHPGAL(SEQ ID NO:55), GALRNPTAC(SEQ IDNO:56), GALRNPTAL(SEQ ID NO:57), RQRPHPGAL(SEQ ID NO:58), RRLPHPGAL(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), FMCAYPGCN(SEQ ID NO:69), FMCAYPGCY(SEQ ID NO:70) or FMCAYPGCK(SEQ ID NO:71)

[0016] In another embodiment, the 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:95), 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 IDSelect from 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), YPGCNKRYF (SEQ ID NO:122), or APVLDFAPPGASAYG (SEQ ID NO:123).

[0017] In another embodiment, the WT1 peptide is any natural peptide disclosed in WO2005053618, WO2007047763, WO2007047764, WO2007120673, US20060084609, WO2014113490, and WO2013106834. The above-mentioned patent documents are incorporated herein in their entirety by this reference.

[0018] In another embodiment, the WT1 peptide is any natural peptide disclosed in US20110070251A1, US7063854B1, US7063854, US7901693, US7662386, US7063854, US7115272, US7368119, US7329410, US7144581, US7323181, US7655249, US7553494, US7608685, US7380871, US7030212, US7807792, US7517950, US2010 / 0166738, US2011 / 0070251, US2009 / 0143291, and WO2003037060. The above-mentioned patent documents are incorporated herein in their entirety by this reference.

[0019] In another embodiment, the WT1 peptide is any natural peptide disclosed in US7666985B2, US20080070835A1, US20070128207A1, US7915393B2, US20110136141A1, US7598221B2, US20100111986A1, US20100092522A1, US20030082194A1, and WO2001025273A2. The above-mentioned patent documents are incorporated in their entirety herein by this reference.

[0020] One or more WT1 peptides may be modified WT1 peptide fragments containing one or more heterocritical modifications, for example, to enhance immunogenicity to the native peptide sequence. In one embodiment, the WT1 peptide is YMFPNAPYL (SEQ ID NO: 124). In another embodiment, the WT1 peptide is SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125). In yet another embodiment, the WT1 peptide is QAYMFPNAPYLPSCL (SEQ ID NO: 126). In another embodiment, the WT1 peptides are 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: Selected from 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).

[0021] In another embodiment, the WT1 peptide is NQMNLGATL (SEQ ID NO: 147), NLMNLGATL (SEQ ID NO: 148), NYMNLGATL (SEQ ID NO: 149), CMTWNQMNLGATLKG (SEQ ID NO: 150), CMTWNLMNLGATLKG (SEQ ID NO: 151), WNQMNLGATLKGVAA (SEQ ID NO: 152), WNLMNLGATLKGVAA (SEQ ID NO: 153), 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 IDNO: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:194), SEKRPFMCAYPGCNK(SEQ ID NO:194), KRPFMCAYPGCNK(SEQ ID It is any modified WT1 peptide selected from NO:195), FMCAYPGCN (SEQ ID NO:196), FMCAYPGCY (SEQ ID NO:197), or FMCAYPGCK (SEQ ID NO:198).

[0022] In another embodiment, the WT1 peptide is any natural peptide disclosed in WO2005053618, WO2007047763, WO2007047764, WO2007120673, US20060084609, WO2014113490, and WO2013106834. The above-mentioned patent documents are incorporated herein in their entirety by this reference.

[0023] In another embodiment, the WT1 peptide is any natural peptide disclosed in US20110070251A1, US7063854B1, US7063854, US7901693, US7662386, US7063854, US7115272, US7368119, US7329410, US7144581, US7323181, US7655249, US7553494, US7608685, US7380871, US7030212, US7807792, US7517950, US2010 / 0166738, US2011 / 0070251, US2009 / 0143291, and WO2003037060. The above-mentioned patent documents are incorporated herein in their entirety by this reference.

[0024] In another embodiment, the WT1 peptide is any natural peptide disclosed in US7666985B2, US20080070835A1, US20070128207A1, US7915393B2, US20110136141A1, US7598221B2, US20100111986A1, US20100092522A1, US20030082194A1, and WO2001025273A2. The above-mentioned patent documents are incorporated in their entirety herein by this reference.

[0025] One or more WT1 peptides useful for the purposes described herein may be a single peptide or a combination of several peptides. Each peptide may be a natural WT1 peptide or a modified WT1 peptide. When two or more peptides are used, each peptide may be administered individually (in separate formulations) or in combination with one or more other peptides (in the same formulation). One or more peptides may be administered in combination with a carrier, diluent, or excipient. In one embodiment, the peptides are administered in combination with an adjuvant. Each peptide may be administered with different adjuvants, or with a combination of different adjuvants. Alternatively, a combination of two or more peptides may be administered with different adjuvants, or with a combination of different adjuvants. In this specification, an immunogen or composition containing one or more peptides is referred to as a vaccine, peptide vaccine, WT1 vaccine, etc.

[0026] The adjuvant may be any class of adjuvants, such as alum salts or other mineral adjuvants, bacterial products or bacterial-derived adjuvants, surfactants (e.g., saponins), oil-in-water (o / w) or water-in-oil (w) emulsions, liposomal adjuvants, cytokines (e.g., IL-2, GM-CSF, IL-12, IFN-gamma), and α-galactosylceramide analogs. Non-limiting examples of adjuvants include montanide emulsion, QS21, Freund's complete or incomplete adjuvants, aluminum phosphate, aluminum hydroxide, Bacillus calmette-Guélain (BCG), and alum. In one embodiment, the adjuvant is an agent that enhances the immune system's CTL response to WT1 peptides, such as a surfactant mannide monooleate containing vegetable-grade (VG) oleic acid derived from olive oil (Montanide ISA 51 VG w / o emulsion). The adjuvant may be 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 it may be contained in a composition separate from one or more WT1 peptides and one or more checkpoint inhibitors.

[0027] In one embodiment, one or more WT1 peptides useful for the purposes of the present invention are a combination of any two peptides selected from YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125). In another embodiment, one or more WT1 peptides useful for the purposes of the present invention are a combination of any three peptides selected from YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125). In one embodiment, one or more WT1 peptides useful for the purposes of the present invention are a combination of any four peptides selected from YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125). In one embodiment, one or more peptides may be used in combination with any of the above-described combinations for the purposes of the present invention.

[0028] In one embodiment, the WT1 peptide comprises the amino acid sequence:SGQAYMFPNAPYLPSCLES(SEQ ID NO:125), and this peptide has one or more point mutations located in the major or auxiliary anchor residues of the binding motif of an HLA class I or HLA class II molecule. In one embodiment, the WT1 peptide has at least 83% sequence identity to the amino acid sequence:SGQAYMFPNAPYLPSCLES(SEQ ID NO:125). In one embodiment, the WT1 peptide has an amino acid length of 20-26 and comprises the amino acid sequence:SGQAYMFPNAPYLPSCLES(SEQ ID NO:125). In another embodiment, the WT1 peptide has an amino acid length of 17 or 18 and comprises a fragment of the amino acid sequence:SGQAYMFPNAPYLPSCLES(SEQ ID NO:125). In another embodiment, the WT1 peptide has at least 93% sequence identity to the amino acid sequence:SGQAYMFPNAPYLPSCLES(SEQ ID NO:125). In another embodiment, any of the above-described peptides have one or more point mutations located at the primary or auxiliary anchor residue of the HLA class I binding motif. In one embodiment, the peptide has a point mutation at position 2 or 9 of the HLA class I binding motif, or at position 1, 3, 4, 5, 6, 7, or 8 of the auxiliary anchor residue of the HLA class I binding motif. In one embodiment, the position of the HLA class I binding motif of the peptide 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.

[0029] In one embodiment, the WT1 peptides useful for the purposes of the present invention are combinations of two, three, or four peptides selected from YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 125), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 2), and NQMNLGATL (SEQ ID NO: 147), NLMNLGATL (SEQ ID NO: 148), NYMNLGATL (SEQ ID NO: 149), CMTWNQMNLGATLKG (SEQ ID NO: 150), CMTWNLMNLGATLKG (SEQ ID NO: 151), WNQMNLGATLKGVAA (SEQ ID NO: 150) as disclosed in WO2014113490. NO:152), WNLMNLGATLKGVAA(SEQ ID NO:153), 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 IDNO: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 This is a combination with one or more natural or modified WT1 peptides selected from NO:189), HSFKHEDPM (SEQ ID NO:190), 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).

[0030] Each peptide in the combination may be administered individually as a separate formulation, or two, three, four, five or more peptides in the combination may be administered simultaneously in the same formulation.

[0031] The dosage of each peptide, the frequency of administration of each peptide or combination of peptides, the duration of administration, and other aspects of immunization by the WT1 peptide may be optimized according to the patient's clinical symptoms, duration or course of the disease, comorbidities, and other aspects of clinical care. Therefore, the present invention is not limited to specific embodiments of the immunizing components of the methods embodied herein.

[0032] In one embodiment, the WT1 vaccine contains 280 mcg each of the four peptides described above (YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125)), combined to a total volume of 0.7 mL (each peptide is 0.4 mg / mL). In one embodiment, 200 mcg of each peptide is administered in each dose (0.5 mL). In another embodiment, 100-2000 mcg of each peptide is administered in each dose. In another embodiment, the above doses are administered every other week for 10 weeks (i.e., 6 doses). In another embodiment, administration is subcutaneous. In another embodiment, the adjuvant is mixed (emulsified) with the vaccine before administration. In one embodiment, 0.5 mL of vaccine (i.e., 200 mcg of each peptide) is emulsified with 1.0 mL of adjuvant before administration. In another embodiment, the adjuvant is injected at the same site as the vaccine, either before or after the vaccine injection. In one embodiment, the adjuvant is an emulsion. In one embodiment, the emulsion is a montanide emulsion. In one embodiment, the montanide emulsion is the immunoadjuvant montanide ISA 51 VG. When the present invention is implemented, a checkpoint inhibitor is also administered to the subject along with the WT1 vaccine (details will be described later).

[0033] As described above, one or more WT1 peptides may be administered as an immunogenic composition to induce an immune response against WT1-expressing cancer. In another embodiment, one or more WT1 peptides are used to produce WT1-specific CTLs that induce an immune response against WT1-expressing cancer when administered to a patient, either in vitro or ex vivo. In one embodiment, one or more WT1 peptides are used to induce in vitro production of CTLs using cells, for example, from a cell line. In another embodiment, one or more WT1 peptides are used to induce CTL production in a cell sample obtained from a patient. The CTLs produced ex vivo are injected back into the same patient who needs them. In another embodiment, one or more WT1 peptides are used to induce CTL production in a cell sample obtained from a donor, and the CTLs produced ex vivo are injected into a patient who is not the donor. In another embodiment, one or more WT1 peptides described herein are administered to a subject who is not a patient in need of treatment to induce CTL production. The produced CTLs are transferred from the donor to the patient. Each of these embodiments represents another aspect of the present invention and is also a source of WT1-specific cells useful for treating cancer or reducing the incidence or recurrence rate of cancer, as described herein.

[0034] In all of the methods described above, whether involving patient vaccination or acquisition of WT1-specific CTLs from a donor to induce a CTL response against WT1-expressing cancer, or using immune cells from a cell line in an in vitro or ex vivo manner, or whether involving a patient or a non-patient donor, and regardless of whether the method of treating WT1-expressing cancer or reducing its incidence or recurrence rate is by immunization of a target requiring it with one or more WT1 peptides, or by the production of CTLs in vitro or ex vivo in a donor, the use of checkpoint inhibitors in combination is embodied herein. In all of these methods, the use of one or more checkpoint inhibitors in combination is embodied herein. One or more checkpoint inhibitors may be administered to a patient immunized with one or more WT1 peptides. Checkpoint inhibitors may be used in vitro or ex vivo to enhance the formation of WT1-specific CTLs that are subsequently injected into the patient. One or more checkpoint inhibitors may be used in a donor to enhance the formation of WT1-specific CTLs. WT1-specific CTLs can then be transferred to the patient. Checkpoint inhibitors can be used in patients receiving CTLs produced in vitro, ex vivo, or in the donor, regardless of whether the checkpoint inhibitor was administered in vitro, ex vivo, or to the donor. In the latter embodiment, one or more identical or different checkpoint inhibitors can be used in vitro, ex vivo, or in the donor or in the patient.

[0035] Immune checkpoints regulate T cell function in the immune system. T cells play a central role in cellular immunity. Checkpoint proteins interact with specific ligands that signal T cells, essentially switching off or inhibiting T cell function. Cancer cells utilize this system by expressing high levels of checkpoint proteins on their surface, controlling T cells that express checkpoint proteins on the surface of T cells entering the tumor microenvironment, thereby suppressing the anti-cancer immune response. Therefore, inhibition of checkpoint proteins may lead to the restoration of T cell function and an immune response against cancer cells. Immune checkpoint inhibitors (checkpoint inhibitors) are compositions or drugs that block or inhibit immune checkpoint proteins (i.e., block or inhibit checkpoint receptors or checkpoint receptor ligands). Examples of checkpoint proteins include, but are not limited to, CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, and 2B4 (belonging to the CD2 molecular family, all NK cells and memory CD8). +These include T cells (expressed on T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. PD-1 (Programmed Death-1) is a member of the immunoglobulin superfamily (IGSF) of molecules involved in regulating T cell activation. PD-1 was named "programmed death" when it was identified in 1992 as an upregulated gene in T cell hybridomas undergoing 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 systems [39, 40]. It is found in T cells, B cells, macrophages, NK cells, DCs, and mast cells, as well as in peripheral tissues [41, 42]. The involvement of PD-1 indicates one way in which tumors evade immune surveillance and clearance

[43] . Blockade of the PD-1 pathway has been demonstrated by nivolumab, which is active in immune-responsive mouse cancer models

[44] .

[0036] Non-exclusive examples of checkpoint inhibitors include small molecules, peptides, or antibodies. Non-exclusive examples of antibodies include nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011), MEDI-0680 (AMP-514), AMP-224, AUNP-12, BMS-936559, atezolizumab (MPDL-3280A), durvalumab (MEDI-4736), and avelumab. This includes MSB-0010718C), BMS-935559 (MDX-1105), rHIgM12B7, BMS-986016, GSK-2831781, IMP-321, lirilumab (BMS-986015), IPH-2101 (1-7F9), indoximod (NLG-9189), NLG-919, INCB-024360, PF-05082566, urelumab (BMS-663513), and MEDI-6469.

[0037] Nivolumab (OPDIVO) is a fully human IgG4 monoclonal antibody that targets PD-1 receptors on activated T lymphocytes and B lymphocytes

[47] . Pembrolizumab (KEYTRUDA) is another non-limiting example of an antibody that targets PD-1. Other compositions and drugs that block, inhibit, or target checkpoint proteins include experimental compositions that are not yet commercially available. The present invention is not limited by any particular checkpoint inhibitor. A non-limiting list of checkpoint inhibitors that may be used is given in Table 1.

[0038] [Table 1]

[0039] In one embodiment, a combination of two or more checkpoint inhibitors is administered to the subject. In one embodiment, the combination of checkpoint inhibitors is selected from the examples listed in Table 1. The two or more checkpoint inhibitors can be administered simultaneously or sequentially with respect to each other and to one or more WT1 peptides. In a further embodiment, a combination of two or more checkpoint inhibitors targeting two or more distinct checkpoint proteins, such as PD-1 (e.g., nivolumab or other PD-1 inhibitors) and CTLA-4 (e.g., ipilumumab or other CTLA-4 inhibitors), is administered simultaneously or sequentially with respect to each other and to one or more WT1 peptides. In one embodiment, a combination of two or more checkpoint inhibitors targeting two or more distinct checkpoint proteins is selected from the group consisting 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, a combination of two or more checkpoint inhibitors targeting two or more distinct checkpoint proteins is selected from the examples listed in Table 1.

[0040] The dosage, frequency, timing of administration, and other aspects of administration of checkpoint inhibitors may be optimized according to the patient's clinical symptoms, duration or course of the disease, comorbidities, and other aspects of clinical care. Therefore, the present invention is not limited to specific embodiments of the checkpoint inhibitor components of the methods embodied herein.

[0041] In one embodiment, nivolumab is administered at a dose of 3 mg / kg every two weeks for 12 weeks. In one embodiment, administration is intravenous. In one embodiment, the checkpoint inhibitor is administered simultaneously with the WT1 vaccine. In one embodiment, the checkpoint inhibitor is administered concurrently with the WT1 vaccine. In one embodiment, the checkpoint inhibitor is administered approximately simultaneously with the WT1 vaccine.

[0042] In one embodiment, the WT1 vaccine contained 200 mcg each of the following peptides: YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125). This WT1 vaccine was combined to a total volume of 0.5 ml, emulsified with 1.0 ml of montanide ISA 51 VG, and administered subcutaneously every two weeks for six weeks. In addition, nivolumab 3 mg / kg was started simultaneously with the administration of the WT1 vaccine and administered intravenously for 60 minutes every two weeks (seven doses in total).

[0043] In one embodiment, a method is provided herein in which one or more WT1 peptides and one or more checkpoint inhibitors are administered to a subject according to a schedule that yields the greatest benefit to the subject. Therefore, the one or more WT1 peptides and one or more checkpoint inhibitors do not necessarily have to be administered simultaneously, in the same composition, or for the same duration. Each WT1 peptide, like each checkpoint inhibitor, may be administered according to a specific schedule. In one non-limiting embodiment, one or more WT1 peptides and one or more checkpoint inhibitors are contained in the same composition.

[0044] As described herein, the dosage and administration schedule (including the frequency and route of administration), route of administration, and other aspects of administration for one or more WT1 peptides (administered individually or concurrently) and one or more checkpoint inhibitors (administered individually or concurrently) are optimized to provide the greatest benefit to the subject. Similar aspects are considered when the donor is a recipient of one or more WT1 peptides and checkpoint inhibitors for the purpose of generating WT1-specific CTLs for administration to a patient.

[0045] In one embodiment, a composition is provided comprising at least one WT1 peptide and at least one checkpoint inhibitor. In one embodiment, one or more WT1 peptides in the composition are selected from those disclosed herein. In one embodiment, the checkpoint inhibitor is selected from those disclosed herein. In one embodiment, the composition of the present invention comprises one, two, or three peptides selected from the WT1 peptides YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125). In one embodiment, the composition of the present invention comprises YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125). In one embodiment, the composition of the present invention comprises nivolumab, pembrolizumab, or a combination thereof as a checkpoint inhibitor. The composition of the present invention may further comprise excipients, diluents, or carriers. The composition of the present invention may further comprise one or more adjuvants.

[0046] The embodiments described above provide improved methods for treating WT1-expressing cancers, reducing the incidence of WT1-expressing cancers, and inducing immune responses against WT1-expressing cancers, as well as improved compositions useful for these purposes. Other aspects of the present invention are further described below.

[0047] In one embodiment, the modified WT1 peptide has one or more modified amino acids, which are referred to herein as the mutant WT1 peptide. In one embodiment, the mutant WT1 peptide includes (a) a binding motif for a human leukocyte antigen (HLA) class II molecule and (b) a binding motif for an HLA class I molecule having a point mutation present on one or more anchor residues of the binding motif for an HLA class I molecule. In another embodiment, the peptide has an amino acid length of 11 or more. In some embodiments, the peptide has an amino acid length of 11-22, 11-30, 16-22, or 16-30. In another embodiment, the point mutation is present within the 1-3 anchor residues of the HLA class I molecule binding motif. In another embodiment, the point mutation is present within the 1 anchor residue of the HLA class I molecule binding motif. In another embodiment, the point mutation is present within the 2 anchor residues of the HLA class I molecule binding motif. In another embodiment, the point mutation is present within the 1-2 anchor residues of the HLA class I molecule binding motif. In another embodiment, the point mutation is located within the 2-3 anchor residues of the HLA class I molecule binding motif. In yet another embodiment, the point mutation is located within the 1-4 anchor residues of the HLA class I molecule binding motif. Each possible form represents a separate embodiment of the present invention.

[0048] In another embodiment, the present invention provides a method for treating a subject having WT1-expressing cancer, comprising the step of administering to the subject at least one WT1 peptide and at least one checkpoint inhibitor, thereby providing a method for treating a subject having WT1-expressing cancer.

[0049] In another embodiment, the present invention provides a method for reducing the incidence or recurrence rate of WT1-expressing cancer in a subject, comprising the step of administering to the subject at least one WT1 peptide and at least one checkpoint inhibitor, thereby reducing the incidence or recurrence rate of WT1-expressing cancer in the subject.

[0050] In another embodiment, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs, comprising the step of contacting a lymphocyte population with at least one WT1 peptide and at least one checkpoint inhibitor, thereby inducing the formation and proliferation of WT1 protein-specific CTLs.

[0051] In another embodiment, the present invention relates to (a) WT1 protein-specific CD8 + CD4 specific to lymphocytes or (b)WT1 protein + A method for inducing the formation and proliferation of lymphocytes, comprising the step of contacting a lymphocyte population with at least one WT1 peptide and at least one checkpoint inhibitor, thereby (a) WT1 protein-specific CD8 + CD4 specific to lymphocytes or (b)WT1 protein + This invention provides a method for inducing the formation and proliferation of lymphocytes.

[0052] In one embodiment, the method for treating WT1-expressing cancer described above reduces the incidence or recurrence rate of WT1-expressing cancer, or induces the formation and proliferation of WT1 protein-specific CTLs, and is more effective than the administration of WT1 peptide alone or checkpoint inhibitors alone. In one embodiment, the administration of the WT1 vaccine and the administration of one or more checkpoint inhibitors are performed simultaneously, overlapping, or concurrently with each other, so that the biological response to the WT1 vaccine is enhanced by the administration of one or more checkpoint inhibitors. Concurrent administration includes the administration of the WT1 vaccine to induce WT1-specific CTLs and the administration of one or more checkpoint inhibitors to enhance the activity of the CTLs against cancer. In one embodiment, the administration of the WT1 vaccine may be terminated before the initiation of checkpoint inhibitor therapy, insofar as the effect of the CTLs induced by the WT1 vaccine administration is enhanced by checkpoint inhibitor therapy. In one embodiment, the first checkpoint inhibitor therapy is performed on the same day as the previous WT1 vaccine administration. In one embodiment, the termination of WT1 vaccination and the initiation of checkpoint inhibitor therapy are spaced 1-7 days or 1-4 weeks apart.

[0053] As described herein, the WT1 peptide may be a native fragment or a continuous amino acid sequence of the WT1 protein. Alternatively, the WT1 peptide may have one or more modifications to its amino acid sequence to enhance the immunogenicity or any other beneficial properties of the peptide to express immunity against WT1-expressing cancer. In some embodiments, one or more amino acids are modified to enhance immunogenicity. In one embodiment, the use of the present invention involves using an isolated mutant WT1 peptide comprising (a) a binding motif for a human leukocyte antigen (HLA) class II molecule and (b) a binding motif for an HLA class I molecule having a point mutation present in one or more anchor residues of the binding motif for an HLA class I molecule. In another embodiment, the peptide has a length of 11 or more. Each possible form represents a separate embodiment of the present invention.

[0054] In another embodiment, a "point mutation" indicates that the fragment is mutated relative to the native sequence of the protein, thereby generating an HLA class I molecule-binding motif. In yet another embodiment, the "point mutation" enhances the binding ability of the HLA class I molecule-binding motif present in the native sequence. Each possible form corresponds to a separate embodiment of the present invention.

[0055] In another embodiment, the point mutation is located within anchor residues 1-3 of the HLA class I molecule binding motif. In another embodiment, the point mutation is located within anchor residue 1 of the HLA class I molecule binding motif. In another embodiment, the point mutation is located within anchor residue 2 of the HLA class I molecule binding motif. In another embodiment, the point mutation is located within anchor residues 1-2 of the HLA class I molecule binding motif. In another embodiment, the point mutation is located within anchor residues 2-3 of the HLA class I molecule binding motif. In another embodiment, the point mutation is located within anchor residues 1-4 of the HLA class I molecule binding motif. Each possible form represents a separate embodiment of the present invention.

[0056] In another embodiment, the peptide of the present invention has an amino acid (AA) length of 11-453. In another embodiment, the amino acid length is 12-453AA. In another embodiment, the amino acid length is 13-453AA. In another embodiment, the amino acid length is 14-453AA. In another embodiment, the amino acid length is 15-453AA. In another embodiment, the amino acid length is 16-453AA. In another embodiment, the amino acid length is 17-453AA. In another embodiment, the amino acid length is 18-453AA. In another embodiment, the amino acid length is 19-453AA. In another embodiment, the amino acid length is 20-453AA.

[0057] In another embodiment, the amino acid length is 11-449AA. In another embodiment, the amino acid length is 12-449AA. In another embodiment, the amino acid length is 13-449AA. In another embodiment, the amino acid length is 14-449AA. In another embodiment, the amino acid length is 15-449AA. In another embodiment, the amino acid length is 16-449AA. In another embodiment, the amino acid length is 17-449AA. In another embodiment, the amino acid length is 18-449AA. In another embodiment, the amino acid length is 19-449AA. In another embodiment, the amino acid length is 20-449AA.

[0058] In another embodiment, the amino acid length is 11-30AA. In another embodiment, the amino acid length is 16-22AA. In another embodiment, the amino acid length is 19AA. In another embodiment, the amino acid length is 15-23AA. In another embodiment, the amino acid length is 15-24AA. In another embodiment, the amino acid length is 15-25AA. In another embodiment, the amino acid length is 15-26AA. In another embodiment, the amino acid length is 15-27AA. In another embodiment, the amino acid length is 15-28AA. In another embodiment, the amino acid length is 14-30AA. In another embodiment, the amino acid length is 14-29AA. In another embodiment, the amino acid length is 14-28AA. In another embodiment, the amino acid length is 14-26AA. In another embodiment, the amino acid length is 14-24AA. In another embodiment, the amino acid length is 14-22AA. In another embodiment, the amino acid length is 14-20AA. In another embodiment, the amino acid length is 16-30AA. In another embodiment, the amino acid length is 16-28AA. In another embodiment, the amino acid length is 16-26AA. In another embodiment, the amino acid length is 16-24AA. In another embodiment, the amino acid length is 16-22AA. In another embodiment, the amino acid length is 18-30AA. In another embodiment, the amino acid length is 18-28AA. In another embodiment, the amino acid length is 18-26AA. In another embodiment, the amino acid length is 18-24AA. In another embodiment, the amino acid length is 18-22AA. In another embodiment, the amino acid length is 18-20AA. In another embodiment, the amino acid length is 20-30AA. In another embodiment, the amino acid length is 20-28AA. In another embodiment, the amino acid length is 20-26AA. In another embodiment, the amino acid length is 20-24AA. In another embodiment, the amino acid length is 22-30AA. In another embodiment, the amino acid length is 22-28AA. In yet another embodiment, the amino acid length is 22-26AA. In yet another embodiment, the amino acid length is 24-30AA.In another embodiment, the amino acid length is 24-28AA. In yet another embodiment, the amino acid length is 24-26AA.

[0059] In another embodiment, the peptide useful for the methods and compositions of the present invention is longer than the minimum length required to bind to an HLA class II molecule, and in another embodiment, is about 12AA. In another embodiment, extending the length of the HLA class II binding peptide allows binding to two or more HLA class II molecules. In another embodiment, extending the length of the HLA class II binding peptide allows binding to an HLA class II molecule whose binding motif is unknown. In another embodiment, extending the length of the HLA class II binding peptide allows binding to an HLA class I molecule. In another embodiment, the binding motif of the HLA class I molecule is known. In another embodiment, the binding motif of the HLA class I molecule is unknown. Each possible form represents a separate embodiment of the present invention.

[0060] Each of the peptide lengths described above represents a separate embodiment of the present invention.

[0061] HLA molecules (also referred to as major histocompatibility complex (MHC) molecules in another embodiment) bind to peptides and present immune cells to the peptides. Therefore, in another embodiment, the immunogenicity of a peptide is partially determined by its affinity for HLA molecules. HLA class I molecules interact with CD8 molecules, which are normally present on cytotoxic T lymphocytes (CTLs). HLA class II molecules interact with CD4 molecules, which are normally present on helper T lymphocytes.

[0062] In another embodiment, the peptide of the present invention is immunogenic. In another embodiment, the term “immunogenicity” refers to the ability to promote or induce an immune response, or the ability to participate in an immune response. In another embodiment, the immune response induced is a cellular immune response. In another embodiment, the immune response is a combination of a cellular immune response and a humoral immune response.

[0063] In another embodiment, T cells are activated upon binding to an MHC molecule-peptide complex and are induced to proliferate and lyse cells expressing a protein containing the peptide. T cells are typically initially activated by “professional” antigen-presenting cells (“APCs,” e.g., dendritic cells, monocytes, and macrophages). APCs present co-stimulatory molecules that promote T cell activation, as opposed to anergy or apoptosis. In another embodiment, the response is heterocritic as described herein, and the CTLs lyse tumor cells expressing a protein having an AA sequence homologous to the peptide of the present invention, or a peptide different from the peptide used to initially stimulate the T cells.

[0064] In another embodiment, contact of T cells with the peptide of the present invention induces differentiation into effector and / or memory T cells. Subsequently, contact of the effector or memory T cells with the same peptide, or in another embodiment, with the heterocritical peptide of the present invention, induces a faster and more potent immune response. Such a response is evaluated in another embodiment by measuring the degree of proliferation of the T cell population exposed to the peptide. In another embodiment, such a response is evaluated by one of the methods listed below.

[0065] In another embodiment, as described herein, when a subject is exposed to a peptide or a composition / cell population containing a peptide of this specification that is different from the expressed native protein, a host immune response with cross-reactivity to the native protein / antigen is subsequently expressed.

[0066] In another embodiment, the peptides, compositions, and vaccines of the present invention promote an immune response that lyses tumor cells. In all of the above embodiments, the immune response against tumors can be enhanced by using them in combination with checkpoint inhibitors.

[0067] In another embodiment, the HLA class I molecule binding motif of the peptide of the present invention is included in the HLA class II molecule binding motif of the peptide. In another embodiment, the HLA class I molecule binding motif overlaps with the HLA class II molecule binding motif. In another embodiment, the HLA class I molecule binding motif does not overlap with the HLA class II molecule binding motif. Each possible form represents a separate embodiment of the present invention.

[0068] In another embodiment, the HLA class II molecule whose binding motif is contained in the peptide of the present invention is an HLA-DR molecule. In another embodiment, the HLA class II molecule is an HLA-DP molecule. In another embodiment, the HLA class II molecule is an HLA-DQ molecule.

[0069] In another embodiment, the HLA class II molecule is an HLA-DRB molecule. In another embodiment, the HLA class II molecule is DRB101. In another embodiment, the HLA class II molecule is DRB301. In another embodiment, the HLA class II molecule is DRB401. In another embodiment, the HLA class II molecule is DRB701. In another embodiment, the HLA class II molecule is DRB1101. In another embodiment, the HLA class II molecule is DRB1501. In another embodiment, the HLA class II molecule is any other HLA-DR molecule known in the art. In another embodiment, the HLA class II molecule is an HLA-DRA molecule. In another embodiment, the HLA class II molecule is an HLA-DQA1 molecule. In another embodiment, the HLA class II molecule is an HLA-DQB1 molecule. In another embodiment, the HLA class II molecule is an HLA-DPA1 molecule. In another embodiment, the HLA class II molecule is an HLA-DPB1 molecule. In another embodiment, the HLA class II molecule is an HLA-DMA molecule. In another embodiment, the HLA class II molecule is an HLA-DMB molecule. In another embodiment, the HLA class II molecule is an HLA-DOA molecule. In another embodiment, the HLA class II molecule is an HLA-DOB molecule. In another embodiment, the HLA class II molecule is any other HLA class II molecule known in the art.

[0070] In another embodiment, the peptide of the present invention binds to two distinct HLA class II molecules. In another embodiment, the peptide of the present invention binds to three distinct HLA class II molecules. In another embodiment, the peptide of the present invention binds to four distinct HLA class II molecules. In another embodiment, the peptide of the present invention binds to five distinct HLA class II molecules. In another embodiment, the peptide of the present invention binds to six distinct HLA class II molecules. In another embodiment, the peptide of the present invention binds to seven or more distinct HLA class II molecules.

[0071] In another embodiment, the HLA class II molecule that binds to the peptide of the present invention is encoded by two or more distinct alleles located at a given HLA class II locus. In another embodiment, the HLA class II molecule is encoded by three distinct alleles located at one locus. In another embodiment, the HLA class II molecule is encoded by four distinct alleles located at one locus. In another embodiment, the HLA class II molecule is encoded by five distinct alleles located at one locus. In another embodiment, the HLA class II molecule is encoded by six distinct alleles located at one locus. In another embodiment, the HLA class II molecule is encoded by seven or more distinct alleles located at one locus.

[0072] In another embodiment, the HLA class II molecule that binds to the peptide of the present invention is encoded by HLA class II genes located at two distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at two or more distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at three distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at three or more distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at four distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at four or more distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at five distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at five or more distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at six distinct loci. In yet another embodiment, the HLA class II molecule is encoded by HLA class II genes located at six or more distinct loci. In yet another embodiment, the HLA class II molecule is encoded by HLA class II genes located at seven or more distinct loci. Each possible form represents a separate embodiment of the present invention.

[0073] In another embodiment, the peptide of the present invention binds to two distinct HLA-DRB molecules. In another embodiment, the peptide of the present invention binds to three distinct HLA-DRB molecules. In another embodiment, the peptide of the present invention binds to four distinct HLA-DRB molecules. In another embodiment, the peptide of the present invention binds to five distinct HLA-DRB molecules. In another embodiment, the peptide of the present invention binds to six distinct HLA-DRB molecules. In another embodiment, the peptide of the present invention binds to seven or more distinct HLA-DRB molecules.

[0074] In another embodiment, the HLA class II molecule that binds to the WT1 peptide is encoded by HLA class II genes located at two distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at two or more distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at three distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at three or more distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at four distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at four or more distinct loci. In another embodiment, the HLA class II molecule is encoded by HLA class II genes located at five or more distinct loci. In yet another embodiment, the loci are selected from the HLA-DRB locus. In yet another embodiment, the HLA class II molecule is an HLA-DRA binding peptide. In another embodiment, the peptide is an HLA-DQA1-binding peptide. In another embodiment, the peptide is an HLA-DQB1-binding peptide. In another embodiment, the peptide is an HLA-DPA1-binding peptide. In another embodiment, the peptide is an HLA-DPB1-binding peptide. In another embodiment, the peptide is an HLA-DMA-binding peptide. In another embodiment, the peptide is an HLA-DMB-binding peptide. In another embodiment, the peptide is an HLA-DOA-binding peptide. In another embodiment, the peptide is an HLA-DOB-binding peptide. In another embodiment, the peptide binds to any other HLA class II molecule known in the art. Each possible form represents a separate embodiment of the present invention.

[0075] In another embodiment, the peptide of the present invention binds to an HLA-DRB molecule encoded by two distinct HLA-DRB alleles selected from DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. In yet another embodiment, the peptide of the present invention binds to an HLA-DRB molecule encoded by three distinct HLA-DRB alleles selected from DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. In yet another embodiment, the peptide of the present invention binds to an HLA-DRB molecule encoded by four distinct HLA-DRB alleles selected from DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. In another embodiment, the peptide of the present invention binds to an HLA-DRB molecule encoded by five distinct HLA-DRB alleles selected from DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. In yet another embodiment, the peptide of the present invention binds to an HLA-DRB molecule encoded by each of the HLA-DRB alleles DRB101, DRB301, DRB401, DRB701, DRB1101, and DRB1501. Each possible form corresponds to a distinct embodiment of the present invention.

[0076] Each of the above-mentioned HLA class II molecules, types, classes, and combinations thereof corresponds to a separate embodiment of the present invention.

[0077] In another embodiment, the HLA class I molecule whose binding motif is contained in the peptide of the present invention is an HLA-A molecule. In another embodiment, the HLA class I molecule is an HLA-B molecule. In another embodiment, the HLA class I molecule is an HLA-C molecule. In another embodiment, the HLA class I molecule is an HLA-A0201 molecule. In another embodiment, the HLA class I molecule is an HLA-A1 molecule. In another embodiment, the HLA class I molecule is an HLA-A2 molecule. In another embodiment, the HLA class I molecule is an HLA-A2.1 molecule. In another embodiment, the HLA class I molecule is an HLA-A3 molecule. In another embodiment, the HLA class I molecule is an HLA-A3.2 molecule. In another embodiment, the HLA class I molecule is an HLA-A11 molecule. In another embodiment, the HLA class I molecule is an HLA-A24 molecule. In another embodiment, the HLA class I molecule is an HLA-B7 molecule. In another embodiment, the HLA class I molecule is HLA-B27. In yet another embodiment, the HLA class I molecule is HLA-B8. Each possible form represents a separate embodiment of the present invention.

[0078] In another embodiment, the HLA class I molecule-binding WT1 peptide of the method and composition of the present invention binds to a superfamily of HLA class I molecules. In another embodiment, the superfamily is the A2 superfamily. In another embodiment, the superfamily is the A3 superfamily. In another embodiment, the superfamily is the A24 superfamily. In another embodiment, the superfamily is the B7 superfamily. In another embodiment, the superfamily is the B27 superfamily. In another embodiment, the superfamily is the B44 superfamily. In another embodiment, the superfamily is the C1 superfamily. In another embodiment, the superfamily is the C4 superfamily. In another embodiment, the superfamily is any other superfamily known in the art. Each possible form represents a separate embodiment of the present invention.

[0079] In another embodiment, the HLA class I molecule-binding motif of the peptide of the present invention exhibits increased affinity for HLA class I molecules compared to the unmutated counterpart of the peptide. In another embodiment, the point mutation increases the affinity of the isolated mutant WT1 peptide for HLA class I molecules. In yet another embodiment, the increase in affinity is related to the affinity (for the same HLA class I molecules) of the isolated unmutated WT1 peptide from which the isolated mutant WT1 peptide is derived. Each possible form represents a separate embodiment of the present invention.

[0080] In another embodiment, the HLA class I molecule-binding WT1 peptide of the method and composition of the present invention has a length of 9–13AA. In another embodiment, the length is 8–13AA. In yet another embodiment, the peptide has any of the lengths of the peptides of the present invention listed herein.

[0081] In another embodiment, the HLA class I molecule-binding WT1 peptide has a length of 9AA. In another embodiment, the peptide has a length of 9AA. In another embodiment, the peptide has a length of 10AA. Natural and heterocritical peptides having a length of 9-10AA substantially bind to HLA class I molecules and induce cytokine secretion and CTL-mediated cell lysis.

[0082] In another embodiment, the HLA class I molecule-binding WT1 peptide contained within the WT1 peptide of the present invention has any of the lengths described above. Each possible form represents a separate embodiment of the present invention. In one embodiment, the WT1 peptide of the present invention is a peptide having a longer length than the HLA class I molecule-binding WT1 peptide. This longer peptide is broken down by the cell to an appropriate length presented by the HLA class I molecule.

[0083] In another embodiment, the HLA class I molecule that binds to the HLA class I molecule-binding WT1 peptide is an HLA-A molecule. In another embodiment, the HLA class I molecule is an HLA-A2 molecule. In another embodiment, the HLA class I molecule is an HLA-A3 molecule. In another embodiment, the HLA class I molecule is an HLA-A11 molecule. In another embodiment, the HLA class I molecule is an HLA-B8 molecule. In another embodiment, the HLA class I molecule is an HLA-0201 molecule. In another embodiment, the HLA class I molecule binds to any other HLA class I molecule known in the art. Each possible form represents a separate embodiment of the present invention.

[0084] In another embodiment, the peptide of the present invention has the ability to bind to multiple HLA class II molecules, as demonstrated by the isolated WT1 peptide from which the peptide of the present invention is derived.

[0085] In all embodiments herein, the selection of one or more WT1 peptides useful in the vaccine herein, or peptides or peptide sequences that match the patient or donor's HLA type(s) for generating CTLs in vitro, ex vivo, or in a donor, whether natural or modified, is materialized herein.

[0086] In another embodiment, the WT1 molecule derived from the peptide of the present invention has the sequence:MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCT It has GSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO:199; GenBank access number AY245105).

[0087] In another embodiment, the WT1 molecule has the sequence: AAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSV It has PPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO:200; GenBank access number NM000378).

[0088] In another embodiment, the WT1 molecule has the sequence: MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDP It has MGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO:201; GenBank access number NP077742).

[0089] In another embodiment, the WT1 molecule contains the sequence:MGHHHHHHHHHHSSGHIEGRHMRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFFRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO: 202).

[0090] In other embodiments, the WT1 protein comprises one of the following GenBank sequence entries, namely NM024426, NM024425, NM024424, NM000378, S95530, D13624, D12496, D12497, AH003034, or X77549. In other embodiments, the WT1 protein has one of the sequences described in one of the above GenBank sequence entries. In another embodiment, the WT1 protein is any WT1 protein known in the art. In another embodiment, the WT1 protein has any other WT1 sequence known in the art.

[0091] In another embodiment, the peptide useful for the purposes of the present invention is derived from a fragment of the WT1 protein. In another embodiment, the induction process involves introducing a point mutation to the anchor residue of the HLA class I molecule binding motif. In yet another embodiment, the induction process consists of introducing a point mutation to the anchor residue of the HLA class I molecule binding motif. In yet another embodiment, the peptide of the present invention differs from the corresponding fragment of the WT1 protein solely by a point mutation in the anchor residue of the HLA class I molecule binding motif. In yet another embodiment, the HLA class I molecule binding motif of the peptide of the present invention differs from the corresponding WT1 sequence solely by a point mutation in the anchor residue. Each possible form represents a separate embodiment of the present invention.

[0092] In another embodiment, the peptide induction process of the present invention further comprises one or more amino acid (AA) modifications to an AA analog. In another embodiment, the induction process further comprises one or more peptide bond modifications linking two or more AAs. In another embodiment, the AA analog or peptide bond modification is one of the AA analogs or peptide bond modifications listed below. Each possible form represents a separate embodiment of the present invention.

[0093] In other embodiments, the unmutated fragment of the WT1 protein into which the peptide of the present invention (the "correspondent" of the wild-type sequence) is induced has the sequence SGQARMFPNAPYLPSCLES (SEQ ID NO: 5). In another embodiment, the unmutated WT1 fragment has the sequence QARMFPNAPYLPSCL (SEQ ID NO: 6). In another embodiment, the unmutated WT1 fragment has the sequence LVRHHNMHQRNMTKL (SEQ ID NO: 3). In another embodiment, the unmutated WT1 fragment has the sequence RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1). In another embodiment, the unmutated WT1 fragment has the sequence NKRYFKLSHLQMHSR (SEQ ID NO: 4). In another embodiment, the unmutated WT1 fragment has the sequence PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 2). In another embodiment, the unmutated WT1 fragment is any other WT1 fragment containing an HLA class II molecule-binding motif. In another embodiment, the unmutated WT1 fragment is any other WT1 fragment containing an HLA-DR molecule binding motif. In another embodiment, the unmutated WT1 fragment contains multiple HLA-DR molecule binding motifs. In another embodiment, the unmutated WT1 fragment is any other WT1 fragment containing an HLA-DRB molecule binding motif. In another embodiment, the unmutated WT1 fragment contains multiple HLA-DRB molecule binding motifs. In another embodiment, the peptide of the present invention differs from its counterpart only in point mutations contained therein. In another embodiment, the peptide of the present invention differs from its counterpart only in mutations of HLA class I anchor residues. Each possible form represents a separate embodiment of the present invention.

[0094] In another embodiment, the peptide of the present invention retains the ability to bind to HLA class II molecules, as indicated by the unamutated WT1 fragment from which the peptide is derived. In yet another embodiment, the peptide of the present invention retains the ability to bind to multiple HLA class II molecules, as indicated by the unamutated WT1 fragment. Each possible form represents a separate embodiment of the present invention.

[0095] In another embodiment, the present invention provides isolated peptides comprising the AA sequences GATLKGVAAGSSSSVKWT (SEQ ID NO: 203) and LKGVAAGSSSSVKWT (SEQ ID NO: 204).

[0096] In other embodiments of the methods and compositions of the present invention, “peptide” refers to a compound of subunits AA linked by peptide bonds. In other embodiments, the peptide includes AA analogs. In other embodiments, the peptide is a peptide mimetic. In other embodiments, the peptide of the present invention includes one of the AA analogs listed below. In other embodiments, the subunits are linked by peptide bonds. In other embodiments, the subunits are linked by a different type of bond, such as an ester or ether. In other embodiments, the peptide of the present invention is one of the types of peptide mimetic listed below. Each possible form represents a separate embodiment of the present invention.

[0097] In another embodiment, the peptide of the method and composition of the present invention binds with high affinity to an HLA class I molecule whose binding motif is contained therein. In yet another embodiment, the HLA class I molecule is any HLA class I molecule listed herein. In yet another embodiment, the peptide binds to the HLA class I molecule with moderate affinity. In yet another embodiment, the peptide binds to the HLA class I molecule with significant affinity. In yet another embodiment, the peptide binds to the HLA class I molecule with measurable affinity. In yet another embodiment, the peptide exhibits stable binding to the HLA class I molecule. Each possible form represents a separate embodiment of the present invention.

[0098] In another embodiment, the peptide of the method and composition of the present invention binds with high affinity to an HLA class II molecule containing a binding motif. In another embodiment, the HLA class II molecule is any HLA class II molecule listed herein. In another embodiment, the peptide binds with high affinity to two or more HLA class II molecules. In another embodiment, the peptide binds to an HLA class II molecule with moderate affinity. In another embodiment, the peptide binds to two or more HLA class II molecules with moderate affinity. In another embodiment, the peptide binds to an HLA class II molecule with significant affinity. In another embodiment, the peptide binds to two or more HLA class II molecules with significant affinity. In another embodiment, the peptide binds to an HLA class II molecule with measurable affinity. In another embodiment, the peptide binds to two or more HLA class II molecules with measurable affinity. In another embodiment, the peptide exhibits stable binding to an HLA class II molecule. In another embodiment, the peptide exhibits stable binding to two or more HLA class II molecules. Each possible form represents a separate embodiment of the present invention.

[0099] In another embodiment, the peptide of the method and composition of the present invention binds to both HLA class I and HLA class II molecules with significant affinity. In another embodiment, the peptide binds to both HLA class I and HLA class II molecules with high affinity. In another embodiment, the peptide binds to both HLA class I and HLA class II molecules with moderate affinity. In another embodiment, the peptide binds to both HLA class I and HLA class II molecules with measurable affinity. Each possible form represents a separate embodiment of the present invention.

[0100] In another embodiment, “fragment” refers to a peptide with 11 or more AAs in length. In another embodiment, the peptide fragment of the present invention has a length of 16 or more AAs. In another embodiment, the fragment has a length of 12 or more AAs. In another embodiment, the fragment has 13 or more AAs. In another embodiment, the fragment has 14 or more AAs. In another embodiment, the fragment has 15 or more AAs. In another embodiment, the fragment has 17 or more AAs. In another embodiment, the fragment has 18 or more AAs. In another embodiment, the fragment has 19 or more AAs. In another embodiment, the fragment has 22 or more AAs. In another embodiment, the fragment is 8-12 AAs. In another embodiment, the fragment is approximately 8-12 AAs. In another embodiment, the fragment is 16-19 AAs. In another embodiment, the fragment is approximately 16-19 AAs. In another embodiment, the fragment is 10-25 AAs. In another embodiment, the fragment is approximately 10-25 AAs. In another embodiment, the fragment has any other length. Each possible form represents a separate embodiment of the present invention.

[0101] In another embodiment, the present invention provides a composition comprising an isolated peptide of the present invention in combination with at least one further WT1 peptide. In a particular embodiment, a composition comprising at least two different isolated peptides of the present invention is provided. In a particular embodiment, a composition comprising at least three or at least four different isolated peptides of the present invention is provided. Each possible form represents a separate embodiment of the present invention. In a particular embodiment, the composition of the present invention is a vaccine.

[0102] In another embodiment, the peptides of the method and composition of the present invention bind to HLA class II molecules with significant affinity, while the peptides derived from the original peptide bind to HLA class I molecules with significant affinity.

[0103] In another embodiment, "affinity" refers to the concentration of the peptide required to inhibit the binding of the standard peptide to the indicated MHC molecule by 50%. In another embodiment, "high affinity" refers to the affinity requiring a peptide concentration of approximately 500 nanomoles (nM) or less to inhibit the binding of the standard peptide by 50%. In another embodiment, a peptide concentration of approximately 400 nM or less is required. In another embodiment, the binding affinity is 300 nM. In another embodiment, the binding affinity is 200 nM. In another embodiment, the binding affinity is 150 nM. In another embodiment, the binding affinity is 100 nM. In another embodiment, the binding affinity is 80 nM. In another embodiment, the binding affinity is 60 nM. In another embodiment, the binding affinity is 40 nM. In another embodiment, the binding affinity is 30 nM. In another embodiment, the binding affinity is 20 nM. In another embodiment, the binding affinity is 15 nM. In another embodiment, the binding affinity is 10 nM. In another embodiment, the binding affinity is 8 nM. In another embodiment, the binding affinity is 6 nM. In another embodiment, the binding affinity is 4 nM. In another embodiment, the binding affinity is 3 nM. In another embodiment, the binding affinity is 2 nM. In another embodiment, the binding affinity is 1.5 nM. In another embodiment, the binding affinity is 1 nM. In another embodiment, the binding affinity is 0.8 nM. In another embodiment, the binding affinity is 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. In another embodiment, the binding affinity is 0.4 nM. In another embodiment, the binding affinity is 0.3 nM. In another embodiment, the binding affinity is less than 0.3 nM.

[0104] In another embodiment, "affinity" refers to a measure of binding strength to an MHC molecule. In another embodiment, affinity is measured using methods known in the art to measure competitive binding affinity. In another embodiment, affinity is measured using methods known in the art to measure relative binding affinity. In another embodiment, the method is a competitive binding assay. In another embodiment, the method is a radioimmunoassay, i.e., RIA. In another embodiment, the method is a BiaCore analysis. In another embodiment, the method is any other method known in the art. In another embodiment, the method produces an IC50 in relation to the IC50 of a reference peptide having known affinity.

[0105] Each type of affinity and the method for measuring affinity represent a separate embodiment of the present invention.

[0106] In another embodiment, "high affinity" refers to an IC50 of 0.5-100 nM. In another embodiment, the IC50 is 1-100 nM. In another embodiment, the IC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. In another embodiment, the IC50 is 3-100 nM. In another embodiment, the IC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. In another embodiment, the IC50 is 10-100 nM. In another embodiment, the IC50 is 30-100 nM. In another embodiment, the IC50 is 3-80 nM. In another embodiment, the IC50 is 4-60 nM. In another embodiment, the IC50 is 5-50 nM. In another embodiment, the IC50 is 6-50 nM. In another embodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is 10-50 nM. In another embodiment, IC50 is 20-50 nM. In another embodiment, IC50 is 6-40 nM. In another embodiment, IC50 is 8-30 nM. In another embodiment, IC50 is 10-25 nM. In another embodiment, IC50 is 15-25 nM. Each affinity and affinity range represents a distinct embodiment of the present invention.

[0107] In another embodiment, “moderate affinity” refers to an IC50 of 100–500 nM. In another embodiment, the IC50 is 100–300 nM. In another embodiment, the IC50 is 100–200 nM. In another embodiment, the IC50 is 50–100 nM. In another embodiment, the IC50 is 50–80 nM. In another embodiment, the IC50 is 50–60 nM. Each affinity and affinity range represents a distinct embodiment of the present invention.

[0108] In another embodiment, “significant affinity” refers to sufficient affinity to mediate the recognition of target cells by T cells having a T cell receptor (TCR) that recognizes MHC molecule-peptide complexes. In another embodiment, this term refers to sufficient affinity to mediate the recognition of cancer cells by T cells having a TCR that recognizes MHC molecule-peptide complexes. In another embodiment, this term refers to sufficient affinity to mediate the activation of naive T cells by peptide-presenting dendritic cells. In another embodiment, this term refers to sufficient affinity to mediate the activation of naive T cells by peptide-presenting APCs. In another embodiment, this term refers to sufficient affinity to mediate the reactivation of memory T cells by peptide-presenting dendritic cells. In another embodiment, this term refers to sufficient affinity to mediate the reactivation of memory T cells by peptide-presenting APCs. In another embodiment, this term refers to sufficient affinity to mediate the reactivation of memory T cells by peptide-presenting somatic cells. Each possible form represents a separate embodiment of the present invention.

[0109] In another embodiment, “measurable affinity” refers to sufficient affinity that can be measured by an immunological assay. In another embodiment, the immunological assay is any assay listed herein. Each possible form represents a separate embodiment of the present invention.

[0110] In another embodiment, the peptides of the methods and compositions of the present invention bind to a superfamily of HLA molecules. The superfamily of HLA molecules shares very similar or identical binding motifs. In another embodiment, the superfamily is the HLA class I superfamily. In yet another embodiment, the superfamily is the HLA class II superfamily. Each possible form represents a separate embodiment of the present invention.

[0111] 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 terms refer to peptides that bind to HLA molecules with high affinity. In other embodiments, the terms refer to peptides that bind to HLA molecules with sufficient affinity to activate T cell precursors. In other embodiments, the terms refer to peptides that bind to HLA molecules with sufficient affinity to mediate recognition by T cells. In other embodiments, the HLA molecule is any HLA molecule listed herein. Each possible form represents a separate embodiment of the present invention.

[0112] In another embodiment, the peptides of the methods and compositions of the present invention are heterocritic. In another embodiment, "heterocritic" refers to a peptide that generates an immune response that recognizes the original peptide from which the heterocritic peptide is derived (e.g., a peptide that does not contain anchor residue mutations). In another embodiment, "original peptide" refers to a fragment of the WT1 protein. For example, a peptide called "WT1 122A1" having the sequence SGQAYMFPNAPYLPSCLES (SEQ ID NO: 124) was generated from the wild-type WT1 peptide SGQARMFPNAPYLPSCLES (SEQ ID NO: 5) by a mutation to arginine at residue 5. The heterocritic mutation was introduced into the CD8+WT1 peptide RMFPNAPYL (SEQ ID NO: 7) peptide, which generated the WT1A1 peptide YMFPNAPYL (SEQ ID NO: 124). In another embodiment, "heterocritic" refers to a peptide that generates an immune response that recognizes the original peptide from which the heterocritic peptide is derived, and the immune response generated by vaccination with the heterocritic peptide is greater than the immune response generated by vaccination with the original peptide. In another embodiment, the “heterocritical” immune response refers to an immune response that recognizes the original peptide from which the improved peptide originates (e.g., a peptide that does not contain anchor residue mutations). In yet another embodiment, the “heterocritical” immune response refers to an immune response that recognizes the original peptide from which the heterocritical peptide originates, and the immune response produced by vaccination with the heterocritical peptide is greater than the immune response produced by vaccination with the original peptide. In yet another embodiment, the magnitude of the immune response produced by vaccination with the heterocritical peptide is greater than an immune response substantially equal to the response to vaccination with the original peptide. In yet another embodiment, the magnitude of the immune response produced by vaccination with the heterocritical peptide is greater than an immune response smaller than the response to vaccination with the original peptide. Each possible form represents a separate embodiment of the present invention.

[0113] In another embodiment, the heterocritic peptide of the present invention induces an immune response that is at least twice as strong as that of the WT1 peptide ("natural peptide") from which the heterocritic peptide is derived. In another embodiment, the increase is three times that of the natural peptide. In another embodiment, the increase is five times that of the natural peptide. In another embodiment, the increase is seven times that of the natural peptide. In another embodiment, the increase is ten times that of the natural peptide. In another embodiment, the increase is fifteen times that of the natural peptide. In another embodiment, the increase is twenty times that of the natural peptide. In another embodiment, the increase is thirty times that of the natural peptide. In another embodiment, the increase is fifty times that of the natural peptide. In another embodiment, the increase is one hundred times that of the natural peptide. In another embodiment, the increase is one fifty times that of the natural peptide. In another embodiment, the increase is one hundred times that of the natural peptide. In another embodiment, the increase is one fifty times that of the natural peptide. In another embodiment, the increase is one hundred times that of the natural peptide. In another embodiment, the increase is one hundred times that of the natural peptide. In another embodiment, the increase is one hundred times that of the natural peptide. In another embodiment, the increase is one hundred thousand times that of the natural peptide. In another embodiment, the increase exceeds 1000 times compared to the natural peptide. Each possible form represents a separate embodiment of the present invention.

[0114] In another embodiment, the heterocritic peptide of the present invention is an HLA class I heterocritic peptide. In another embodiment, the heterocritic peptide of the present invention is an HLA class II heterocritic peptide. In another embodiment, the heterocritic class II peptide of the present invention is mutated at the class II binding residue. In another embodiment, as illustrated herein, the heterocritic class II peptide of the present invention is identified and tested in the same manner as the identification and testing of the HLA class I heterocritic peptide. Each possible form represents a separate embodiment of the present invention.

[0115] In other embodiments, an "anchor motif" or "anchor residue" refers to one or a set of preferred residues at specific positions in an HLA-binding sequence. For example, 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 play a crucial role in binding to 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 possible form represents a separate embodiment of the present invention.

[0116] In another embodiment, “anchor residue” refers to the residues at positions 1, 3, 6, and 9 of the HLA class I binding motif. In another embodiment, this term refers to positions 1, 2, 6, and 9 of the HLA class I binding motif. In another embodiment, this term refers to positions 1, 6, and 9 of the HLA class I binding motif. In another embodiment, this term refers to positions 1, 2, and 9 of the HLA class I binding motif. In another embodiment, this term refers to positions 1, 3, and 9 of the HLA class I binding motif. In another embodiment, this term refers to positions 2 and 9 of the HLA class I binding motif. In another embodiment, this term refers to positions 6 and 9 of the HLA class I binding motif. Each possible form represents a separate embodiment of the present invention.

[0117] Methods for identifying MHC class II epitopes are known in the art. In another embodiment, MHC class II epitopes are predicted using TEPITOPE (Meister GE, Roberts CG et al, Vaccine 1995 13: 581-91). In another embodiment, MHC class II epitopes are identified using EpiMatrix (De Groot AS, Jesdale BM et al, AIDS Res Hum Retroviruses 1997 13: 529-31). In another embodiment, MHC class II epitopes are identified using Predict Method (Yu K, Petrovsky N et al, Mol Med. 2002 8: 137-48). In another embodiment, MHC class II epitopes are identified using the SYFPEITHI epitope prediction algorithm. SYFPEITHI is a database containing more than 4500 peptide sequences known to bind to class I and class II MHC molecules. SYFPEITHI provides a score based on the presence of specific amino acids at specific positions along the MHC binding groove. An ideal amino acid anchor is worth 10 points, an abnormal anchor is worth 6-8 points, a co-anchor is worth 4-6 points, and a preferred residue is worth 1-4 points. The negative amino acid effect on the binding score is between -1 and -3. The maximum score for HLA-A*0201 is 36.

[0118] In another embodiment, MHC class II epitopes are identified using Rankpep. Rankpep uses a position-specific scoring matrix (PSSM) or profile from a set of aligned peptides known to bind to a given MHC molecule as a predictor of MHC-peptide bonding. Using selected profiles, Rankpep includes information on the peptide score relative to the consensus sequence that yields the highest score, and the optimal percentile score of the predicted peptide. Rankpep includes selections of 102 and 80 PSSMs for predicting peptide bonding to MHCI and MHCII molecules, respectively. Several PSSMs for predicting peptide binding agents of different sizes are typically available for each MHCI molecule.

[0119] In another embodiment, 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 yet another embodiment, MHC class II epitopes are identified using any other method known in the art. In yet another embodiment, the above method is utilized to confirm that MHC class II binding is disrupted by introducing MHC class I anchor residue mutations into the WT1 sequence. Each possible form represents a separate embodiment of the present invention.

[0120] Methods for identifying MHC class I epitopes are known in this art. In another embodiment, 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-1 / β2-microglobulin / peptide complex, which is a measure of peptide binding affinity. This program uses information on HLA-1 peptides with a length of 8-10 amino acids. The higher the binding affinity of the peptide to the MHC, the more likely it is that this peptide represents 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 a coefficient significantly higher than 1 in the table. If an amino acid is not known to make a favorable or unfavorable contribution to binding, it is assigned a value of 1. All the values ​​assigned to the amino acids are multiplied, and the resulting running score is multiplied by a constant to obtain an estimate of the dissociation half-life.

[0121] In another embodiment, MHFP class I epitopes are identified using SYFPEITHI. In another embodiment, 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 another embodiment, MHC class I epitopes are identified using NetMHC-2.0 (Sensitive quantitative predictions 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 another embodiment, MHC class I epitopes are identified using any other method known in the art. In another embodiment, the method described above is used to identify MHC class I epitopes that can be generated by introducing anchor residue mutations into the WT1 sequence. Each possible form represents a separate embodiment of the present invention.

[0122] In another embodiment, the mutation that enhances MHC binding is located at the residue at position 1 of the HLA class I binding motif. In another embodiment, the residue is changed to tyrosine. In another embodiment, the residue is changed to glycine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to phenylalanine. In another embodiment, the residue is changed to another residue known in the art. In another embodiment, the substitution at position 1 (e.g., substitution to tyrosine) stabilizes the binding of the anchor residue at position 2.

[0123] In another embodiment, the mutation is located at position 2 of the HLA class I binding motif. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to methionine. In another embodiment, the residue is changed to another residue known in the art.

[0124] In another embodiment, the mutation is located at position 6 of the HLA class I binding motif. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to glutamine. In another embodiment, the residue is changed to histidine. In another embodiment, the residue is changed to another residue known in the art.

[0125] In another embodiment, the mutation is located at position 9 of the HLA class I binding motif. In another embodiment, the mutation alters the residue at its C-terminal position. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to alanine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to another residue known in the art.

[0126] In another embodiment, the point mutation is located in a primary anchor residue. In another embodiment, the primary anchor residues for HLA class I are at positions 2 and 9. In another embodiment, the point mutation is located in a secondary anchor residue. In another embodiment, the secondary anchor residues for HLA class I are at positions 1 and 8. In another embodiment, the secondary anchor residues for HLA class I are at positions 1, 3, 6, 7, and 8. In another embodiment, the point mutation is at a position selected from positions 4, 5, and 8. Each possible form represents a separate embodiment of the present invention.

[0127] In another embodiment, the point mutation is located at one or more residues at positions 1, 2, 8, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is located at one or more residues at positions 1, 3, 6, and 9. In another embodiment, the point mutation is located at one or more residues at positions 1, 2, 6, and 9. In another embodiment, the point mutation is located at one or more residues at positions 1, 6, and 9. In another embodiment, the point mutation is located at one or more residues at positions 1, 2, and 9. In another embodiment, the point mutation is located at one or more residues at positions 1, 3, and 9. In another embodiment, the point mutation is located at one or more residues at positions 2 and 9. In another embodiment, the point mutation is located at one or more residues at positions 6 and 9. Each possible form represents a separate embodiment of the present invention.

[0128] In another embodiment, the mutation is located at position 4 of the HLA class I binding motif. In another embodiment, the mutation is located at position 5 of the HLA class I binding motif. In another embodiment, the mutation is located at position 7 of the HLA class I binding motif. In yet another embodiment, the mutation is located at position 8 of the HLA class I binding motif. Each possible form represents a separate embodiment of the present invention.

[0129] Each of the above anchor residues and substitutions represents a separate embodiment of the present invention.

[0130] In another embodiment, the HLA class II binding site in the peptide of the present invention is generated or improved by mutation of an HLA class II motif anchor residue. In another embodiment, the modified anchor residue is located at position P1. In another embodiment, the anchor residue is located at position P2. In another embodiment, the anchor residue is located at position P6. In another embodiment, the anchor residue is located at position P9. In another embodiment, the anchor residue is selected from positions P1, P2, P6, and P9. In another embodiment, the anchor residue is located at position P3. In another embodiment, the anchor residue is located at position P4. In another embodiment, the anchor residue is located at position P5. In another embodiment, the anchor residue is located at position P6. In another embodiment, the anchor residue is located at position P8. In another embodiment, the anchor residue is located at position P10. In another embodiment, the anchor residue is located at position P11. In another embodiment, the anchor residue is located at position P12. In another embodiment, the anchor residue is located at position P13. In another embodiment, the anchor residue is located at any other anchor residue of an HLA class II molecule known in the art. In another embodiment, residues other than P1, P2, P6, and P9 serve as secondary anchor residues. Therefore, by mutating them, HLA class II binding can be improved. In another embodiment, any combination of the above residues is mutated. Each possible form represents a separate embodiment of the present invention.

[0131] In another embodiment, the present invention provides a method for inducing an anti-mesothelioma immune response in a subject, the method comprising the steps of contacting the subject with an immunogenic composition comprising (a) WT1 protein; (b) a fragment of the WT protein; (c) a nucleotide molecule encoding the WT1 protein; or (d) a nucleotide molecule encoding a fragment of the WT1 protein, and at least one checkpoint inhibitor, thereby inducing an anti-mesothelioma immune response in the subject.

[0132] In another embodiment, the present invention provides a method for treating a subject with mesothelioma, the method comprising the step of administering an immunogenic composition to a subject comprising (a) WT1 protein; (b) a fragment of WT protein; (c) a nucleotide molecule encoding WT1 protein; or (d) a nucleotide molecule encoding a fragment of WT1 protein, and at least one checkpoint inhibitor, thereby treating the subject with mesothelioma.

[0133] In another embodiment, the present invention provides a method for reducing the incidence or recurrence of mesothelioma in a subject, the method comprising the step of administering an immunogenic composition to a subject comprising (a) WT1 protein; (b) a fragment of WT protein; (c) a nucleotide molecule encoding WT1 protein; or (d) a nucleotide molecule encoding a fragment of WT1 protein; and at least one checkpoint inhibitor, thereby reducing the incidence or recurrence of mesothelioma in the subject.

[0134] When referring to any protein or peptide, terms such as “homologous” refer, in another embodiment, to the ratio of AA residues in a candidate sequence that are identical to the residues of the corresponding native polypeptide after aligning the sequences as necessary and introducing gaps, without considering conserved substitutions as part of sequence homology, in order to achieve maximum ratio homology. Methods and computer programs for alignment are known in the art.

[0135] In another embodiment, when the term “homology” refers to any nucleic acid sequence, it similarly indicates the ratio of nucleotides in the candidate sequence that are identical to the nucleotides in the corresponding native nucleic acid sequence.

[0136] Homology is determined in another embodiment by a computer algorithm for sequence alignment using methods well described in the Art. In other embodiments, computer algorithmic analysis of nucleic acid sequence homology involves the use of any number of available software packages, such as BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT, and TREMBL packages.

[0137] The identity ratio between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., identity % = number of identical positions / total number of positions × 100). Sequence comparison and determination of the identity ratio between two sequences can be achieved using mathematical algorithms in sequence analysis software. Protein analysis software obtains similar sequences using similar measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions.

[0138] The identity ratio between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm, which is incorporated into the GAP program of the GCG software package (available at www.gcg.com), for example, using either a BLOSUM62 matrix or a 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 can also be compared using FASTA with default or recommended parameters applied. The programs in GCG Version 6.1, FASTA (e.g., FASTA2 and FASTA3) provide alignment and sequence identity ratios for the region with the most overlap between the query and the search sequence (Pearson, Methods Enzymol. 1990; 183:63-98; Pearson, Methods Mol. Biol. 2000; 132:185-219). The identity ratio between two amino acid sequences can also be determined using the E. Meyers and W. Miller algorithm (Comput. Appl. Biosci., 1988; 11-17), incorporated into the ALIGN program (version 2.0), using the PAM120 residue weight table, gap length penalty 12, and gap penalty 4.

[0139] Another algorithm for comparing a sequence with other sequences in a database is the computer program BLAST, in particular blastp using 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 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 (see above). To obtain amino acid sequences homologous to the WT1 peptide of the present invention, a BLAST protein search can be performed using the XBLAST program, score=50, word length=3. To obtain gapped alignments for comparative purposes, Gapped BLAST can be used, as described in Altschul et al., 1997 (see above). When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0140] In another embodiment, “homology” with respect to homologous sequences refers to an identity ratio 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% with respect to sequences disclosed herein. Each possible form represents a separate embodiment of the present invention.

[0141] In another embodiment, the present invention provides a composition comprising a peptide and at least one checkpoint inhibitor. In another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the composition further comprises an adjuvant. In another embodiment, the composition comprises two or more peptides of the present invention. In another embodiment, the composition further comprises any of the additives, compounds, or excipients listed below. In another embodiment, the adjuvant is an alum salt or other mineral adjuvant, a bacterial product or bacterial-derived adjuvant, a surfactant (e.g., saponin), an oil-in-water (o / w) or water-in-oil (w) emulsion, a liposome adjuvant, a cytokine (e.g., IL-2, GM-CSF, IL-12, IFN-gamma), and an α-galactosylceramide analog. In another embodiment, the adjuvant is QS21, a Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG, or alum. In yet another embodiment, the carrier is any carrier listed herein. In other embodiments, the adjuvant is any adjuvant listed herein. Each possible form represents a separate embodiment of the present invention.

[0142] In another embodiment, the present invention provides a vaccine comprising the peptide of the present invention and at least one checkpoint inhibitor. In another embodiment, the vaccine further comprises a carrier. In another embodiment, the vaccine further comprises an adjuvant. In another embodiment, the vaccine further comprises a combination of the carrier and the adjuvant. In another embodiment, the vaccine further comprises an APC. In another embodiment, the vaccine further comprises a combination of the APC and the carrier or adjuvant. In another embodiment, the vaccine is a cell-based composition. Each possible form represents a separate embodiment of the present invention.

[0143] In another embodiment, the present invention provides an immunogenic composition comprising the peptide of the present invention and at least one checkpoint inhibitor. In another embodiment, the immunogenic composition further comprises a carrier. In another embodiment, the immunogenic composition further comprises an adjuvant. In another embodiment, the immunogenic composition further comprises a combination of the carrier and the adjuvant. Each possible form represents a separate embodiment of the present invention.

[0144] In another embodiment, the term “vaccine” refers to a material or composition that, when introduced to a subject, provides a preventive or therapeutic response to that particular disease, condition, or symptom. In another embodiment, the present invention includes a peptide-based vaccine in which the peptide comprises any embodiment listed herein and optionally further comprises immunomodulatory compounds such as cytokines and adjuvants.

[0145] In other embodiments, the composition or vaccine of the method and composition of the present invention further comprises an adjuvant. In another embodiment, the adjuvant is Montanide ISA 51. Montanide ISA 51 comprises a naturally metabolizable oil and a refined emulsifier. In another embodiment, the adjuvant is GM-CSF. In another embodiment, the adjuvant is keyhole limpet hemocyanin (KLH) that can be conjugated to a peptide antigen or administered together with a peptide. Recombinant GM-CSF is, in another embodiment, a human protein grown in a yeast (S. cerevisiae) vector. GM-CSF promotes the clonal proliferation and differentiation of hematopoietic progenitor cells, APCs, and dendritic and T cells.

[0146] In another embodiment, the adjuvant is a cytokine. In another embodiment, the adjuvant is a growth factor. In another embodiment, the adjuvant is a cell population. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is a Freund's incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. In another embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is BCG. In another embodiment, the adjuvant is alum. In another embodiment, the adjuvant is an interleukin. In another embodiment, the adjuvant is a chemokine. In another embodiment, the adjuvant is any other type of adjuvant known in the art. In another embodiment, the WT1 vaccine comprises two of the above adjuvants. In another embodiment, the WT1 vaccine comprises three or more of the above adjuvants. Each possible form represents a separate embodiment of the present invention.

[0147] In another embodiment, the WT1 vaccine used in the method of the present invention may be one or more nucleic acid molecules (DNA or RNA) encoding one or more WT1 peptides of the present invention. In the implementation of this embodiment, a vaccine comprising nucleic acid molecules encoding one or more WT1 peptides (nucleic acid vaccines) is administered to the subject, and one or more checkpoint inhibitors are administered to the subject. In all other embodiments of the present invention, a nucleic acid vaccine may be used instead of a peptide vaccine. The nucleic acid may be introduced alone, as part of a viral carrier, or inside a cell, possibly as a plasmid, or incorporated into the nucleic acid of a cell. The cell carrier may be cells of the subject isolated from the subject, or cells from a donor, or a cell line. The cells may be antigen-presenting cells such as dendritic cells or monocyte / macrophage lineage cells. 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 any combination thereof.

[0148] The WT1 peptide or the nucleic acid encoding it, or its carrier in any form described herein, can be exposed to CTLs ex vivo or in vivo. In vitro or ex vivo, the cells can be grown, i.e., proliferated, before being introduced into the target.

[0149] 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. These terms include, but are not limited to, “modified nucleotides” that include at least one modification, including: (a) alternative binding groups, (b) purine-like forms, (c) pyrimidine-like forms, or (d) sugar-like forms. Examples of purine-like binding groups include purines, pyrimidines, and sugars. See, for example, PCT Publication WO95 / 04064. The nucleic acid sequences of the present invention can be prepared by any known method, including synthesis, recombination, ex vivo generation, or a combination thereof, and by any purification method 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.

[0150] In another embodiment, the use of the present invention provides a vector comprising a nucleic acid molecule (DNA or RNA). In yet another embodiment, the composition or vaccine used in the implementation of the present invention may comprise any embodiment of the WT1 peptide of the present invention and combinations thereof. Each possible form represents a separate embodiment of the present invention.

[0151] In another embodiment, the vaccine or composition of the present invention used in the implementation of the present invention comprises two peptides derived from the same WT1 fragment, each containing a different HLA class I heterocritic peptide. In another embodiment, the two HLA class I heterocritic peptides contain mutations in different HLA class I molecular anchor residues. In yet another embodiment, the two HLA class I heterocritic peptides contain different mutations in the same anchor residue(s). Each possible form represents a separate embodiment of the present invention.

[0152] In another embodiment, the peptide in the composition for use in the present invention binds to two different HLA class II molecules. In another embodiment, the peptide binds to three different HLA class II molecules. In another embodiment, the peptide binds to four different HLA class II molecules. In another embodiment, the peptide binds to five different HLA class II molecules. In another embodiment, the peptide binds to six or more different HLA class II molecules. In another embodiment, the peptide in the composition binds to the same HLA class II molecule.

[0153] In another embodiment, each peptide in the composition or method of use of the present invention binds to a set of HLA class II molecules. In another embodiment, each peptide binds to a different set of HLA class II molecules. In another embodiment, the peptides in the composition bind to the same set of HLA class II molecules. In another embodiment, two peptides bind to different but overlapping sets of HLA class II molecules. In another embodiment, two or more peptides bind to the same set of HLA class II molecules, but another peptide binds to a different set. In another embodiment, two or more peptides bind to overlapping sets of HLA class II molecules, but another peptide binds to a different set.

[0154] In another embodiment, the peptide for use in an implementation of the present invention or in a composition of the present invention binds to two different HLA class I molecules. In another embodiment, the peptide binds to three different HLA class I molecules. In another embodiment, the peptide binds to four different HLA class I molecules. In another embodiment, the peptide binds to five different HLA class I molecules. In another embodiment, the peptide binds to six or more different HLA class I molecules. In another embodiment, the peptide in the composition binds to the same HLA class I molecule.

[0155] In another embodiment, each peptide for use in an embodiment of the present invention or in a composition of the present invention binds to a set of HLA class I molecules. In another embodiment, each peptide binds to a different set of HLA class I molecules. In another embodiment, peptides in a composition bind to the same set of HLA class I molecules. In another embodiment, two peptides bind to different but overlapping sets of HLA class I molecules. In another embodiment, two or more peptides bind to the same set of HLA class I molecules, but another peptide binds to a different set. In another embodiment, two or more peptides bind to overlapping sets of HLA class I molecules, but another peptide binds to a different set.

[0156] In another embodiment, “a set of HLA class II molecules” or “a set of HLA class I molecules” refers to HLA molecules encoded by different alleles at specific locations. In another embodiment, this term refers to HLA molecules having a specific binding specificity. In another embodiment, this term refers to HLA molecules having a specific peptide consensus sequence. In another embodiment, this term refers to a superfamily of HLA class II molecules. Each possible form represents a separate embodiment of the present invention.

[0157] Each of the above compositions and types of compositions represents a separate embodiment of the present invention.

[0158] Any embodiment described herein with respect to the peptides, nucleic acids, compositions, and vaccines of the present invention may be used in any of the methods of the present invention. Each combination of peptides, nucleic acids, compositions, or vaccines by a method represents a separate embodiment thereof.

[0159] In another embodiment, the present invention provides a method for treating a subject having WT1-expressing cancer, the method comprising the step of treating the subject by administering a WT1 vaccine and a checkpoint inhibitor as described herein to the subject. In another embodiment, the present invention provides a method for treating a subject having WT1-expressing cancer, the method comprising the step of treating the subject by administering a composition of the present invention comprising at least one WT1 peptide and at least one checkpoint inhibitor to the subject. In another embodiment, the present invention provides a method for treating a subject having WT1-expressing cancer, the method comprising the step of treating the subject by administering an immunogenic composition such as a vaccine and a checkpoint inhibitor to the subject.

[0160] In another embodiment, the present invention provides a method for inhibiting or halting the progression of WT1-expressing cancer in a subject, the method comprising the step of inhibiting or halting the progression of WT1-expressing cancer by administering to the subject at least one WT1 peptide and at least one checkpoint inhibitor of the present invention. In another embodiment, the present invention provides a method for inhibiting or halting the progression of WT1-expressing cancer in a subject, the method comprising the step of inhibiting or halting the progression of WT1-expressing cancer by administering to the subject a composition comprising at least one WT1 peptide and at least one checkpoint inhibitor of the present invention. In another embodiment, the present invention provides a method for inhibiting or halting the progression of WT1-expressing cancer in a subject, the method comprising the step of inhibiting or halting the progression of WT1-expressing cancer by administering to the subject an immunogenic composition, such as a vaccine of the present invention, comprising at least one WT1 peptide and at least one checkpoint inhibitor of the present invention.

[0161] In another embodiment, the present invention provides a method for reducing the incidence of WT1-expressing cancer in a subject, the method comprising the step of reducing the incidence of WT1-expressing cancer by administering at least one WT1 peptide and at least one checkpoint inhibitor to the subject. In another embodiment, the present invention provides a method for reducing the incidence of WT1-expressing cancer in a subject, the method comprising the step of reducing the incidence of WT1-expressing cancer by administering at least one WT1 peptide and at least one checkpoint inhibitor to the subject of the composition of the present invention. In another embodiment, the present invention provides a method for reducing the incidence of WT1-expressing cancer in a subject, the method comprising the step of reducing the incidence of WT1-expressing cancer by administering to the subject an immunogenic composition such as the vaccine of the present invention comprising at least one WT1 peptide and at least one checkpoint inhibitor to the subject.

[0162] In another embodiment, the present invention provides a method for reducing the recurrence rate of WT1-expressing cancer in a subject, the method comprising the step of reducing the recurrence rate of WT1-expressing cancer by administering to the subject a composition comprising at least one WT1 peptide and at least one checkpoint inhibitor. In another embodiment, the present invention provides a method for reducing the recurrence rate of WT1-expressing cancer in a subject, the method comprising the step of reducing the recurrence rate of WT1-expressing cancer by administering to the subject a composition of the present invention comprising at least one WT1 peptide and at least one checkpoint inhibitor. In another embodiment, the present invention provides a method for reducing the recurrence rate of WT1-expressing cancer in a subject, the method comprising the step of reducing the recurrence rate of WT1-expressing cancer by administering to the subject an immunogenic composition such as a vaccine of the present invention comprising at least one WT1 peptide and at least one checkpoint inhibitor.

[0163] In another embodiment, the present invention provides a method for disrupting T cell tolerance to a target WT1-expressing cancer, the method comprising the step of disrupting T cell tolerance to WT1-expressing cancer by administering to a target at least one WT1 peptide and at least one checkpoint inhibitor. In another embodiment, the present invention provides a method for disrupting T cell tolerance to a target WT1-expressing cancer, the method comprising the step of disrupting T cell tolerance to WT1-expressing cancer by administering to a target a composition of the present invention comprising at least one WT1 peptide and at least one checkpoint inhibitor. In another embodiment, the present invention provides a method for disrupting T cell tolerance to a target WT1-expressing cancer, the method comprising the step of disrupting T cell tolerance to WT1-expressing cancer by administering to a target an immunogenic composition, such as a vaccine of the present invention comprising at least one WT1 peptide and at least one checkpoint inhibitor.

[0164] In another embodiment, the present invention provides a method for treating a subject having WT1-expressing cancer, the method comprising the steps of (a) inducing the formation and proliferation of human CTLs that recognize cancer malignant cells in a donor by the method of the present invention, and (b) treating the subject having cancer by injecting the human CTLs into the subject. In one embodiment, the subject having cancer is treated by administering at least one WT1 peptide to a donor, injecting CTLs obtained from the donor into the subject, and administering a checkpoint inhibitor to the subject. In one embodiment, the subject having cancer is treated by administering at least one WT1 peptide and at least one checkpoint inhibitor to a donor, injecting CTLs obtained from the donor into the subject. In one embodiment, the subject having cancer is treated by administering at least one WT1 peptide and at least one checkpoint inhibitor to a donor, injecting CTLs obtained from the donor into the subject, and administering a checkpoint inhibitor to the subject.

[0165] In another embodiment, the present invention provides a method for treating a subject having WT1-expressing cancer, the method comprising: (a) ex vivo induction of the formation and proliferation of human CTLs that recognize cancerous malignant cells obtained from a donor by the method of the present invention; and (b) treatment of the subject having cancer by injecting the human CTLs into the subject. In one embodiment, the checkpoint inhibitor is included in the ex vivo step. In another embodiment, the checkpoint inhibitor is administered to the subject. In yet another embodiment, the checkpoint inhibitor is included in both ex vivo steps, and the subject is also administered the checkpoint inhibitor.

[0166] Methods of ex vivo immunotherapy are publicly known in this field and are described, for example, in Davis ID et al (Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients. J Immunother. 2006 Sep-Oct;29(5):499-511) and Mitchell MS et al (The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope, M1.2. Cancer Immunol Immunother. 2006 Jul 28). Each method represents a distinct embodiment of the present invention.

[0167] In another embodiment, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs, the method comprising the step of inducing the formation and proliferation of WT1 protein-specific CTLs by contacting a lymphocyte population with an immunogenic composition, such as the vaccine of the present invention, which comprises at least one checkpoint inhibitor. In another embodiment, the immunogenic composition comprises antigen-presenting cells (APCs) associated with the peptide of the present invention and a checkpoint inhibitor. In another embodiment, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs, the method comprising the step of inducing the formation and proliferation of WT1 protein-specific CTLs by contacting a lymphocyte population with the peptide or composition of the present invention together with at least one checkpoint inhibitor. In another embodiment, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs, the method comprising the step of inducing the formation and proliferation of WT1 protein-specific CTLs by contacting a lymphocyte population with the vaccine of the present invention together with at least one checkpoint inhibitor. In another embodiment, the CTLs are specific to WT1-expressing cells. In another embodiment, the target cells are cells of a WT1-expressing cancer. Each possible form represents a separate embodiment of the present invention.

[0168] In another embodiment, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs in a subject, the method comprising the step of inducing the formation and proliferation of WT1 protein-specific CTLs in a subject by contacting the subject with an immunogenic composition such as the vaccine of the present invention together with at least one checkpoint inhibitor. In another embodiment, the immunogenic composition comprises an APC associated with a mixture of peptides of the present invention administered together with at least one checkpoint inhibitor. In another embodiment, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs in a subject, the method comprising the step of inducing the formation and proliferation of WT1 protein-specific CTLs in a subject by contacting the subject with a peptide together with at least one checkpoint inhibitor or the composition of the present invention. In another embodiment, the present invention provides a method for inducing the formation and proliferation of WT1 protein-specific CTLs in a subject, the method comprising the step of inducing the formation and proliferation of WT1 protein-specific CTLs in a subject by contacting the subject with the vaccine of the present invention together with at least one checkpoint inhibitor. In another embodiment, the target cells are cells of a WT1-expressing cancer. In another embodiment, the subject has a WT1-expressing cancer. In another embodiment, the CTLs are specific to WT1-expressing cells.

[0169] In another embodiment, the present invention provides a method for generating a heterocritical immune response directed to WT1-expressing cancer in a subject, the method comprising the step of generating a heterocritical immune response by administering to a subject at least one heterocritical WT1 peptide together with at least one checkpoint inhibitor or composition of the present invention. In another embodiment, the present invention provides a method for generating a heterocritical immune response directed to WT1-expressing cancer in a subject, the method comprising the step of generating a heterocritical immune response by administering to a subject an immunogenic composition such as the vaccine of the present invention together with at least one checkpoint inhibitor. In another embodiment, the present invention provides a method for generating a heterocritical immune response directed to WT1-expressing cancer in a subject, the method comprising the step of generating a heterocritical immune response by administering to a subject the vaccine of the present invention together with at least one checkpoint inhibitor.

[0170] Each method represents a distinct embodiment of the present invention.

[0171] In another embodiment, the WT1-expressing cancer is acute myeloid leukemia (AML). In another embodiment, the WT1-expressing cancer is chronic myeloid leukemia (CML). In another embodiment, the WT1-expressing cancer is associated with myelodysplastic syndrome (MDS). In another embodiment, the WT1-expressing cancer is MDS. In another embodiment, the WT1-expressing cancer is non-small cell lung cancer (NSCLC). In another embodiment, the WT1-expressing cancer is esophageal squamous cell carcinoma. In another embodiment, the WT1-expressing cancer is acute lymphoblastic leukemia (ALL). In another embodiment, the WT1-expressing cancer is bone or soft tissue sarcoma. In another embodiment, the WT1-expressing cancer is Wilms' tumor. In another embodiment, the WT1-expressing cancer is leukemia. In another embodiment, the WT1-expressing cancer is hematological cancer. In another embodiment, the WT1-expressing cancer is lymphoma. In another embodiment, the WT1-expressing cancer is fibrous round cell tumor. In another embodiment, the WT1-expressing cancer is mesothelioma. In another embodiment, the WT1-expressing cancer is malignant mesothelioma. In another embodiment, the WT1-expressing cancer is gastric cancer. In another embodiment, the WT1-expressing cancer is colon cancer. In another embodiment, the WT1-expressing cancer is lung cancer. In another embodiment, the WT1-expressing cancer is breast cancer. In another embodiment, the WT1-expressing cancer is a germ cell tumor. In another embodiment, the WT1-expressing cancer is malignant pleural mesothelioma. In another embodiment, the WT1-expressing cancer is multiple myeloma. In another embodiment, the WT1-expressing cancer is myeloid leukemia. In another embodiment, the WT1-expressing cancer is astrocytoma. In another embodiment, the WT1-expressing cancer is gliablastoma (e.g., pleomorphic gliablastoma). In another embodiment, the WT1-expressing cancer is colorectal cancer. In another embodiment, the WT1-expressing cancer is ovarian cancer (e.g., serous, epithelial, or endometrial). In another embodiment, the WT1-expressing cancer is breast cancer. In another embodiment, the WT1-expressing cancer is melanoma. In another embodiment, the WT1-expressing cancer is head and neck squamous cell carcinoma. In another embodiment, the WT1-expressing cancer is pancreatic ductal cell carcinoma. In another embodiment, the WT1-expressing cancer is neuroblastoma. In another embodiment, the WT1-expressing cancer is uterine cancer. In another embodiment, the WT1-expressing cancer is thyroid cancer. In another embodiment, the WT1-expressing cancer is hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is thyroid cancer.In another embodiment, the WT1-expressing cancer is liver cancer. In another embodiment, the WT1-expressing cancer is kidney cancer (e.g., renal cell carcinoma). In another embodiment, the WT1-expressing cancer is Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma.

[0172] In another embodiment, WT1-expressing cancer is a solid tumor. In another embodiment, solid tumors are associated with WT1-expressing cancer. In another embodiment, solid tumors are associated with myelodysplastic syndrome (MDS). In another embodiment, solid tumors are associated with non-small cell lung cancer (NSCLC). In another embodiment, solid tumors are associated with lung cancer. In another embodiment, solid tumors are associated with breast cancer. In another embodiment, solid tumors are associated with colorectal cancer. In another embodiment, solid tumors are associated with prostate cancer. In another embodiment, solid tumors are associated with ovarian cancer. In another embodiment, solid tumors are associated with kidney cancer. In another embodiment, solid tumors are associated with pancreatic cancer. In another embodiment, solid tumors are associated with brain cancer. In another embodiment, solid tumors are associated with gastrointestinal cancer. In another embodiment, solid tumors are associated with skin cancer. In another embodiment, solid tumors are associated with melanoma.

[0173] In another embodiment, the cancer or tumor treated by the method of the present invention is suspected to express WT1. In another embodiment, WT1 expression has not been confirmed by testing of actual tumor samples. In another embodiment, the cancer or tumor is of a type known to express WT1 in many cases. In another embodiment, the type expresses WT1 in the majority of cases.

[0174] Each type of cancer or tumor expressing WT1, and each type of cancer or tumor suspected of expressing WT1, represents a separate embodiment of the present invention.

[0175] Table 2 shows a non-exclusive list of cancer types that can be treated using the compositions and methods of the present invention.

[0176]

Table 2

[0177] In another embodiment, multiple peptides of the invention are used to stimulate an immune response in the methods of the invention together with at least one checkpoint inhibitor.

[0178] As provided herein, heteroclitic peptides that induce antigen-specific CD8 + T cell responses can be generated using the methods of the invention. CD4 + WT1 peptides that induce T cell responses against multiple HLA class II molecules can be identified. CD4 + T cells recognize peptides bound to HLA class II molecules on APCs. In another embodiment, antigen-specific CD4 + T cell responses assist in the induction and maintenance of CD8 + cytotoxic T lymphocyte (CTL) responses.

[0179] In another embodiment, the peptides of the invention administered together with at least one checkpoint inhibitor exhibit a high ability to induce a CTL response due to their ability to bind to both HLA class I molecules and HLA class II molecules. In another embodiment, the peptides of the invention administered together with at least one checkpoint inhibitor exhibit a high 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 another embodiment, the vaccines of the invention administered together with at least one checkpoint inhibitor have the advantage of activating or inducing both CD4 + and CD8 + T cells that recognize the WT1 antigen. In another embodiment, CD4 + and CD8 +T cell activation or induction provides a synergistic anti-WT1 immune response compared to activation of any population alone. In another embodiment, the high immunogenicity of the peptide of the present invention is demonstrated in multiple, individual HLA class II subtypes due to the peptide's ability to bind to multiple HLA class II subtypes. Each possible form represents a separate embodiment of the present invention.

[0180] In another embodiment, activated CD4 + The cells enhance immunity by licensing dendritic cells, thereby maintaining the activation and survival of cytotoxic T cells. In another embodiment, activated CD4 + T cells induce tumor cell death through direct contact with tumor cells or activation of apoptotic pathways. For example, mesothelioma tumor cells can process and present antigens in HLA class I and class II molecules.

[0181] Those skilled in the art will understand 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. These methods further enable the design of immunogenic compositions and vaccines combining the WT1-derived peptides of the present invention. Each possible form represents a separate embodiment of the present invention.

[0182] In another embodiment, a method, peptide, vaccine and / or immunogenic composition of the present invention, administered together with at least one checkpoint inhibitor, comprises a WT1-specific CD4 containing multiple different HLA class II alleles. + It has the advantage of activating or inducing T cells. In another embodiment, the vaccine has the advantage of activating or inducing WT1-specific CD4 in a significant proportion of the population. + It has the advantage of activating or inducing T cells. In another embodiment, the peptide is WT1-specific CD4 in 15% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in 20% of the population. +It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 cells in 25% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 cells in 30% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in 35% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 cells in 40% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in 45% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in 50% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 cells in 55% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in 60% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in 70% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 cells in 75% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 cells in 80% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in 85% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in 90% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in 95% of the population. + It activates T cells. In another embodiment, the peptide activates WT1-specific CD4 in over 95% of the population. + It activates T cells. In another embodiment, the vaccine has a significant proportion of WT1-specific CD4 in a particular population (e.g., the Caucasian race in the United States). + Activates or induces T cells. Each possible form represents a separate embodiment of the present invention.

[0183] In another embodiment, the method of the present invention results in an enhancement of an immune response already initiated by a subject. In another embodiment, the method of the present invention comprises the step of administering a peptide, composition, or vaccine two or more times together with at least one checkpoint inhibitor. In another embodiment, the peptides vary in their composition, concentration, or combination thereof. In another embodiment, the peptide administered together with at least one checkpoint inhibitor results in the initiation of an immune response to the target antigen in a subject that has not yet initiated an immune response to the target antigen. In another embodiment, the induced CTLs proliferate in response to the presentation of the peptide to APCs or cancer cells. In other embodiments, references to modification of the immune response include one or both of humoral immunity and cellular immunity, with the presence of Th2 helper T cells and Th1 helper T cells, respectively, or, in another embodiment, each immunity is included individually.

[0184] In other embodiments, methods of influencing tumor growth result in (1) direct inhibition of tumor cell division, or (2) immune cell-mediated lysis of tumor cells, or both, thereby suppressing net tumor cell proliferation. Each possible form represents a separate embodiment of the present invention. The use of peptides or vaccines administered with at least one checkpoint inhibitor increases the direct inhibition of tumor cell division, immune cell-mediated lysis, or both, compared to the absence of checkpoint inhibitors.

[0185] The inhibition of tumor growth by either of these two mechanisms can be readily determined by those skilled in the art based on several well-known methods. In another embodiment, tumor inhibition is determined by measuring the actual tumor size over a period of time. In yet another embodiment, tumor inhibition can be determined by estimating the tumor size (over a period of time) using methods well-known to those skilled in the art. More specifically, tumor size can be estimated using various radiological imaging techniques (e.g., single-photon emission computed tomography; generally, see "Nuclear Medicine in Clinical Oncology," Winkler, C. (ed.) Springer-Verlag, New York, 1986). Such methods can also utilize various imaging agents, including, for example, conventional imaging agents (e.g., gallium-67 citrate) and reagents specialized for metabolite imaging, receptor imaging, or immunoimaging (e.g., tumor markers specific to radiolabeled monoclonal antibodies). Furthermore, non-radioactive methods such as ultrasound (see "Ultrasonic Differential Diagnosis of Tumors", Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984) can also be used to estimate tumor size.

[0186] In addition to the in vivo methods described above for determining tumor inhibition, various in vitro methods can be used to predict tumor inhibition in vivo. Typical examples include lymphocyte-mediated antitumor cytolytic activity and tumor-dependent lymphocyte proliferation determined by the 51Cr release assay (example), as well as tumor-dependent lymphocyte proliferation (Ioannides, et al., J. Immunol. 146(5):1700-1707, 1991), in vitro production of tumor-specific antibodies (Herlyn, et al., J. Immunol. Meth. 73:157-167, 1984), in vitro inhibition of cellular (e.g., CTLs, helper T cells) or humoral (e.g., antibodies) cell growth (Gazit, et al., Cancer Immunol Immunother 35:135-144, 1992), and determination of the frequency of cell precursors for any of these assays (Vose, Int. J. Cancer 30:135-142 (1982)).

[0187] In another embodiment, a method for suppressing tumor growth has been shown to result in a shortened proliferative phase compared to growth without contact with or exposure to the peptide administered with at least one checkpoint inhibitor of the present invention. Tumor cell proliferation is measured by tumor size measurement, but is not limited to the following. 3 The determination of whether tumor cells are proliferating can be made using an H-thymidine uptake assay, or by any means known in the art, including tumor cell counting. In other embodiments, “suppression” of tumor cell proliferation refers to slowing, delaying, or stopping tumor growth, or shrinking the tumor. Each possible form represents a separate embodiment of the present invention.

[0188] In another embodiment of the method and composition of the present invention, WT1 expression is measured after administration of the treatment, before administration of the treatment, or both before and after administration of the treatment. In another embodiment, the expression of a WT1 transcript is measured. In another embodiment, WT1 protein levels in tumor or cancer cells are measured. In another embodiment, WT1 protein or peptides circulating from cancer or tumor cells, or excreted in other bodily fluids such as urine, are measured. Each possible form represents a separate embodiment of the present invention.

[0189] In another embodiment of the methods and compositions of the present invention, the expression of checkpoint proteins (may be multiple) targeted by one or more checkpoint inhibitors administered to a subject is measured in tumor or cancer cells (at the transcript level or protein level), or in whole blood, serum, or plasma before administration of treatment (baseline), after administration of treatment, or both before and after administration of 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 two or more combinations thereof is measured before administration of treatment, after administration of treatment, or both before and after administration of treatment. In one embodiment, checkpoint protein expression is measured at the primary tumor site. In another embodiment, the cancer is metastatic, and checkpoint protein expression is measured at the metastatic site, the primary tumor site, or both.

[0190] In another embodiment of the methods and compositions of the present invention, one or more of the following markers are measured before administration of the treatment (baseline), after administration of the treatment, or both before and after administration of the treatment: monocytic myeloid suppressor cells (m-MDSCs), C-reactive protein (CRP), absolute lymphocytes, and lactate dehydrogenase (LDH). Another embodiment involves the use of one or more markers to predict or identify responsiveness to checkpoint modulation.

[0191] Methods for determining the presence and magnitude of an immune response are known in the art. Another embodiment is a lymphocyte proliferation assay, in which radioactive material, for example, 3 The uptake of H-thymidine by T cells is measured as a function of cell proliferation. In other embodiments, T cell proliferation is detected by interleukin-2 (IL-2) production, Ca 2+ This is achieved by measuring the leaching or increased uptake of dyes such as 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium. Each possible form represents a separate embodiment of the present invention.

[0192] In another embodiment, CTL stimulation is determined by means known to those skilled in the art, including detection of cell proliferation and cytokine production. Analysis of the types and amounts of cytokines secreted by T cells after contact of a ligand with a pulsed target can serve as a measure of functional activity. The rate and total amount of cytokine production can be determined by measuring cytokines by ELISA or ELISPOT assays (Fujihashi K. et al. (1993) J. Immunol. Meth. 160: 181; Tanguay S. and Killion JJ (1994) Lymphokine Cytokine Res. 13:259).

[0193] In another embodiment, CTL activity is 51 Determined by a Cr-releasing lysis assay. Pulsed by a peptide. 51Lysis of Cr-labeled targets by antigen-specific T cells can be compared to target cells pulsed with the target peptide. In another embodiment, T cells are stimulated with the peptide of the present invention, and lysis of target cells expressing the native peptide in association with MHC can be determined. In another embodiment, the kinetics of lysis at the fixation time (e.g., 4 hours) and overall target lysis are used to evaluate ligand performance (Ware CF et al. (1983) J Immunol 131: 1312).

[0194] Methods for determining the affinity of peptides to HLA molecules are known in the art. In another embodiment, affinity is determined by a TAP stabilization assay.

[0195] In another embodiment, affinity is determined by competitive radioimmunoassay. In another embodiment, the following protocol is used: Target cells are washed twice in PBS containing 1% bovine serum albumin (BSA; Fisher Chemicals, Fairlawn, New Jersey). The cells are resuspended on ice in 10⁷ / ml, and peptides originally bound to the cell surface are detached in citrate-phosphate buffer in the presence of 3 mg / ml β2-microglobulin for 2 minutes at 0°C. The pellet is refracted in PBS / 1% BSA for 5 × 10⁻⁶ times in the presence of 3 mg / ml β2-microglobulin and 30 mg / ml deoxyribonuclease. 6 Resuspend in cells / ml, incubate 200ml portions at 20°C for 10 minutes in or without HLA-specific peptide, and then... 125 Incubate with I-labeled peptide at 20°C for 30 minutes. Wash twice with PBS / 2% BSA, then wash once with PBS, and remove the bound total 125 Determine I. Relative affinity is determined by comparing the test peptide, with its concentration gradually increased, with a known binding peptide.

[0196] In another embodiment, the specificity of peptide binding to HLA on the surface of living cells (e.g., SKLY-16 cells) is analyzed to confirm that the binding is to the appropriate HLA molecule and to characterize its limitation. In another embodiment, this includes competition with excess 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 another embodiment, this assay is performed on living, 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 known to bind to MHC molecules on specific cells is known to have high affinity for the relevant HLA molecules, e.g., tyrosinase or HBV peptide sequences. 125 The assay is performed using the competitive assay described above for I-labeled peptides.

[0197] In another embodiment, the peptides used in the methods and compositions of the present invention include 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991) J. Am Chem. Soc. 113:2275-2283); (2S,3S)-methylphenylalanine, (2S,3R)-methylphenylalanine, (2R,3S)-methylphenylalanine and (2R,3R)-methylphenylalanine (Kazmierski and Hruby (1991) Tetrahedron Lett. 32(41): 5769-5772); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) doctoral dissertation, University of Arizona); hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al. (1984) J. Takeda Res. It contains one or more non-classical amino acids, such as histidine isoquinoline carboxylic acid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138) and HIC (histidine cyclic urea) (Dharanipragada et al. (1993) Int. J. Pep. Protein Res. 42(l):68-77), ((1992) Acta. Crst., Crystal Struc. Comm. 48(IV): 1239-124).

[0198] In another embodiment, the peptide of the present invention comprises one or more AA analogs or peptide mimes, which, in other embodiments, induce or select a particular secondary structure. In other embodiments, such peptides include: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), β-turn-inducible dipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838); β-sheet-inducible analog (Kemp et al. (1988) Tetrahedron Lett. 29:5081-5082); β-turn-inducible analog (Kemp et al. (1988) Tetrahedron Lett. 29:5057-5060); α-helix-inducible analog (Kemp et al. (1988) Tetrahedron Lett. 29:4935-4938); γ-turn-inducible analog (Kemp et al. (1989) J. Org. Chem. 54:109:115); Analogues provided in the following references: Nagai and Sato (1985) Tetrahedron Lett. 26:647-650; and DiMaio et al. (1989) J. Chem. Soc. Perkin Trans, p. 1687; GIy-Ala turn analogue (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide linkage isoconformity (Jones et al. (1988) Tetrahedron Lett. 29(31):3853-3856); tretrazol (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880); DTC (Samanen et al. (1990) Int. J. Protein Pep. Res. Analogues taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 and Garvey et al. (1990) J. Org. Chem. 55(3):936-940 (35:501:509).Structurally restricted β-turn mimetic and β-bulge mimetic, as well as peptides containing them, are described in U.S. Patent No. 5,440,013, published in Kahn in August 1995.

[0199] In other embodiments, the peptide of the present invention is bound to one of various other molecules, such as those described below, and the binding may be covalent or non-covalent (complex), and in other embodiments, its properties vary depending on the specific purpose. In other embodiments, the peptide is covalently or non-covalently complexed with a polymeric carrier (e.g., an immunogenic carrier), which includes but is not limited to natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinylpyrrolidone, and lipids. In other embodiments, the peptide of the present invention is linked to a substrate. In other embodiments, the peptide is linked to a fatty acid for introduction into liposomes (U.S. Patent No. 5,837,249). In other embodiments, the peptide of the present invention is covalently or non-covalently complexed with a solid phase, various solid phases known in the art. In other embodiments, the linkage of the peptide to a carrier, substrate, fatty acid, or solid phase enhances the induction of an immune response.

[0200] In other embodiments, the carrier may be thyroglobulin, albumin (e.g., human serum albumin), tetanus toxoid, polyamino acids 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 APC. Each possible form represents a separate embodiment of the present invention.

[0201] In another embodiment, the term “amino acid” may refer to natural AA, in another embodiment to unnatural or synthetic AA, and in yet another embodiment may include glycine, D-optical isomers or L-optical isomers, AA analogs, peptide mimes, or combinations thereof.

[0202] In another embodiment, the terms “cancer,” “neoplasm,” “neoplastic,” or “tumor” are used synonymously and refer to cells that have undergone malignant transformation, making them pathological to the host organism. Cancer can be any stage of a numbered staging system (e.g., stage 0, stage 1, stage 2, stage 3, or stage 4) and any stage of the TNM staging system. Primary cancer cells (i.e., cells obtained near the site of malignant transformation) can be readily distinguished from non-cancerous cells by established techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. In another embodiment, tumors are detectable based on tumor volume; for example, by methods such as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation; in another embodiment, they are identified by biochemical or immunological findings; and in yet another embodiment, immunological findings are also used to identify cancer cells. Tumors can be solid or non-solid.

[0203] Peptide synthesis methods are known in the art. In another embodiment, the peptides of the present invention are synthesized using a suitable solid-state synthesis method (see, for example, Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco, Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23: 451). In other embodiments, the activity of these peptides is tested using assays such as those described herein.

[0204] In another embodiment, the peptides of the present invention are purified by standard methods including chromatography (e.g., ion-exchange chromatography, affinity chromatography, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification. In yet another embodiment, immunoaffinity chromatography is used, in which the epitope is isolated by binding to an affinity column containing an antibody produced against the peptide or a related peptide of the present invention and immobilized on a stabilizing support.

[0205] In another embodiment, affinity tags such as hexa-His (Invitrogen), maltose-binding domain (New England Biolabs), influenza encasing sequence (Kolodziej et al. (1991) Meth. Enzymol. 194:508-509), and glutathione-S-transferase are attached to the peptide of the present invention, enabling easy purification by passage on a suitable affinity column. In yet another embodiment, the isolated peptide may also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0206] In another embodiment, the peptide of the present invention is produced by in vitro translation through known techniques, as will be apparent to those skilled in the art. In another embodiment, the peptide is otherwise modified during or after translation, for example, by phosphorylation, glycosylation, crosslinking, acylation, protein cleavage, or linkage to antibody molecules, membrane molecules, or other ligands (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).

[0207] In another embodiment, the peptide of the present invention further comprises a detectable label, which in another embodiment is a fluorescent label, or in another embodiment is an luminescent label, or in another embodiment is an radioactive label, or in another embodiment is a high electron density label. In other embodiments, the detectable label is, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), β-galactosidase, luciferase, 32 P, 125 I, 3 H and 14 This includes C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin, or many other labels known to those skilled in the art. The specific label used depends on the type of immunoassay used.

[0208] In another embodiment, the peptide of the present invention is linked to a substrate, and in another embodiment, the substrate acts as a carrier. In another embodiment, the linkage of the peptide to the substrate enhances the induction of an immune response.

[0209] In another embodiment, the peptides of the present invention are linked to other molecules as described herein using conventional crosslinking agents such as carbodiimides. Examples of carbodiimides are 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl))carbodiimide (CMC), 1-ethyl-3-(3-dimethiaminopropyl)carbodiimide (EDC), and 1-ethyl-3-(4-azonia-44-dimethylpentyl)carbodiimide.

[0210] In other embodiments, the crosslinking agent includes cyanogen bromide, glutaraldehyde, and succinic anhydride. Generally, any of the many homobifunctional substances can be used, including homobifunctional aldehydes, homobifunctional epoxides, homobifunctional imide esters, homobifunctional N-hydroxysuccinimide esters, homobifunctional maleimides, homobifunctional alkyl halides, homobifunctional pyridyl disulfide, homobifunctional aryl halides, homobifunctional hydrazides, homobifunctional diazonium derivatives, and homobifunctional photoreactive compounds. In other embodiments, heterobifunctional compounds, 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, are also conceivable.

[0211] In other embodiments, the homobifunctional crosslinking agents include the bifunctional N-hydroxysuccinimidole dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartrate; the bifunctional imido-esters dimethyl adipiimide, dimethyl pimilimidate, and dimethyl suberilimidate; the bifunctional sulfhydryl reactive crosslinking agents 1,4-di-[3'-(2'-pyridyldithio)propionamide]butane, bismaleimidehexane, and bis-N-maleimide-1,8-octane; the bifunctional aryl halides 1,5-difluoro-2,4-dibenzene and 4,4'-difluoro-3,3'-dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4-azidosalicylamide)ethyl]disulfide; and the bifunctional aldehyde formaldehyde. This includes dihydrofluoric acid dihydrides such as malondialdehyde, succinaldehyde, glutaraldehyde, and adipoaldehyde; dihydrofluoric acid dihydrazides such as 1,4-butaneodiol diglycidyl ether; dihydrofluoric acid dihydrazides such as adipic acid dihydrazide, carbohydrazide, and succinic acid dihydrazide; dihydrofluoric acid dihydrazides such as o-tolidine, benzidine diazotide, and benzidine bis-diazotide; dihydrofluoric acid dihydrides such as NlN'-ethylene-bis(iodoacetamide), NlN'-hexamethylene-bis(iodoacetamide), and NlN'-undemethylene-bis(iodoacetamide), as well as benzyl halides and halomustards such as ala'-diiodo-p-xylenesulfonic acid and tri(2-chloroethyl)amine, respectively.

[0212] In other embodiments, the heterobifunctional crosslinking agent used to link the peptide to other molecules as described herein may include, but is not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MBS This includes (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl (4-iodoacetyl) aminobenzoate), SMPB (succinimidyl-4-(p-maleimidophenyl) butyrate), GMBS (N-(γ-maleimidobutyryloxy)succinimide ester), MPBH (4-(4-N-maleimidophenyl) butyrate hydrazide), M2C2H (4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide), SMPT (succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), and SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate).

[0213] In another embodiment, the peptides of the present invention are formulated as non-covalent monomers via ionic, adsorbent, or biomolecule-specific interactions. In another embodiment, complexes of peptides with highly positively or negatively charged molecules may be obtained through salt bridge formation in a low ionic strength environment such as deionized water. In another embodiment, large complexes may be prepared using charged polymers such as poly-(L-glutamic acid) or poly-(L-lysine) containing numerous negative and positive charges, respectively. In another embodiment, peptides are adsorbed onto a surface such as microparticle latex beads or other hydrophobic polymers to form non-covalent peptide-superantigen complexes that efficiently mimic cross-linked proteins or, in another embodiment, chemically polymerized proteins. In another embodiment, peptides are non-covalently linked by utilizing biomolecule-specific interactions between other molecules. For example, the strong affinity of biotin for proteins such as avidin or streptavidin, or their derivatives, may be used to form peptide complexes. The peptides in this embodiment and in other embodiments may be modified to have a biotin group using a common biotinylating reagent such as N-hydroxysuccinimidyl ester of D-biotin (NHS-biotin) that reacts with an available amine group.

[0214] In another embodiment, the peptide of the present invention is linked to a carrier. In another embodiment, the carrier is KLH. In yet another embodiment, the carrier is any other carrier known in the art, such as thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid), influenza, hepatitis B virus core proteins, and recombinant hepatitis B virus vaccines. Each possible form represents a separate embodiment of the present invention.

[0215] In another embodiment, the peptide of the present invention is bound to a lipid such as P3CSS. In yet another embodiment, the peptide of the present invention is bound to beads.

[0216] In any of the embodiments described above, a peptide, a crosslinked peptide, a conjugated peptide, or any other form of peptide is used in the method of the present invention together with at least one checkpoint inhibitor.

[0217] In another embodiment, in addition to the use of at least one checkpoint inhibitor, the methods and compositions of the present invention further include immunomodulatory compounds. In other embodiments, the immunomodulatory compounds are cytokines, chemokines, or complement components that enhance the expression of accessory or adhesion molecules of the immune system, their receptors, or combinations thereof. In some embodiments, the immunomodulatory compounds include interleukins, e.g., interleukins 1-15, interferon α, β, or γ, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), chemokines, e.g., neutrophil-activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), Lantes, macrophage inflammatory peptides MIP-Ia and MIP-Ib, 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.

[0218] In another embodiment, the immunomodulatory compound induces or enhances the expression of co-stimulating molecules involved in the immune response.

[0219] In one embodiment, a patient who has been administered the WT1 vaccine and checkpoint inhibitor according to the present invention is administered GM-CSF before or on the day of the first vaccination, or a combination thereof. In one embodiment, 70 mcg of GM-CSF is administered subcutaneously to the patient two days before and on the day of the first vaccination.

[0220] In another embodiment, the composition comprises a solvent including water, a dispersion medium, a cell culture medium, and an isotonic agent. In another embodiment, the solvent is an isotonic buffered aqueous solution with a pH of about 7.0. In another embodiment, the composition comprises a diluent such as water, phosphate-buffered saline, or saline solution. In another embodiment, the composition comprises a non-aqueous solvent such as propyl ethylene glycol, polyethylene glycol, and vegetable oil.

[0221] In another embodiment, the composition is formulated for administration by any of many techniques known to those skilled in the art. For example, the present invention provides administration of a pharmaceutical composition by parenteral, intravenous, subcutaneous, intradermal, intramucosal, topical, oral, or inhalation.

[0222] In another embodiment, in the use of a vaccine containing the peptide of the present invention, the vaccine further comprises a population of cells, and in another embodiment, the cell population comprises lymphocytes, monocytes, macrophages, dendritic cells, epithelial cells, stem cells, or a combination thereof, and in another embodiment, these cells are autologous, homogeneous, or homogeneous with respect to one another. In another embodiment, the cell population contains the peptide of the present invention. In another embodiment, the cell population takes up the peptide. In one embodiment, the cells are antigen-presenting cells (APCs). In a further embodiment, the APCs are professional antigen-presenting cells (APCs). Each possible form represents a separate embodiment of the present invention.

[0223] In another embodiment, the cell population of the present invention is obtained from an in vivo source, such as peripheral blood, leukophage-induced blood products, apheresis-induced blood products, peripheral lymph nodes, gastrointestinal lymphoid tissue, spleen, thymus, umbilical cord blood, mesenteric lymph nodes, liver, immune lesions, such as synovial fluid, pancreas, cerebrospinal fluid, tumor samples, granulomatous tissue, or any other source from which such cells can be obtained. In another embodiment, the cell population is obtained from a human source, and in another embodiment, this human source is obtained from a human fetus, neonatal, child, or adult source. In another embodiment, the cell population of the present invention is obtained from an animal source, such as a pig or monkey, or another target animal. In another embodiment, the cell population of the present invention is obtained from a normal subject, or in another embodiment, from a subject with a disease, or in another embodiment, from a subject susceptible to the disease of the present invention.

[0224] In another embodiment, the cell populations of the present invention are separated by affinity-based separation methods. In other embodiments, techniques for affinity separation include magnetic separation using antibody-coated magnetic beads, affinity chromatography, cytotoxic drugs (e.g., complement and cytotoxic agents) conjugated to or used in conjunction with monoclonal antibodies, and "panning" using antibodies attached to a solid matrix such as a plate, or other convenient techniques. In other embodiments, separation techniques include the use of fluorescently labeled cell separators, which may have varying degrees of sophistication, such as multicolor channels, channels that detect low-angle and obtuse-angle light scattering, and impedance channels. In other embodiments, any technique that enables the separation of the cell populations of the present invention may be used and should be considered part of the present invention.

[0225] In another embodiment, dendritic cells are obtained from a diverse population of morphologically similar cell types present in various lymphoid and non-lymphoid tissues suitable for such purposes (Steinman (1991) Ann. Rev. Immunol. 9:271-296). In another embodiment, the dendritic cells used in the present invention are isolated from bone marrow, or in another embodiment, derived from bone marrow progenitor cells, or in another embodiment, isolated / derived from peripheral blood, or in another embodiment, a certain cell line or derived from such cell line.

[0226] In another embodiment, the cell population described herein is isolated from the leukocyte fraction of a mammal such as a mouse, monkey, or human (see, for example, International Publication No. 96 / 23060). In another embodiment, the leukocyte fraction may be isolated from the peripheral blood of a mammal.

[0227] Methods for isolating dendritic cells (DCs) are known in this field. In another embodiment, the following steps are taken: (a) A step of providing a leukocyte fraction obtained from a mammalian source by a method known in the art, such as leukocyte separation, (b) A step of separating the leukocyte fraction from step (a) into four or more subfractions by countercurrent centrifugation, (c)(b) The step of stimulating the conversion of monocytes in one or more fractions obtained from step (c)(b) into dendritic cells by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF and IL-4, (d) A step of identifying the fraction in which dendritic cells are concentrated, obtained from step (c), The DC is isolated by a method comprising the step of collecting the concentrated fraction from step (e)(d), preferably at about 4°C.

[0228] In another embodiment, a fraction enriched with dendritic cells is identified by fluorescently labeled cell sorting, which identifies the simultaneous absence of at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the following markers: CD3, CD14, CD16, CD56, CD57, and CD19, CD20.

[0229] In another embodiment, the cell population includes lymphocytes, which in another embodiment are T cells, or in yet another embodiment, B cells. In other embodiments, the T cells are characterized as NK cells, helper T cells, cytotoxic T lymphocytes (CTLs), TBLs, naive T cells, or a combination thereof. It should be understood that T cells, whether primary or cell lines, clones, etc., should be considered part of the present invention. In another embodiment, the T cells are CTLs or CTL lines, CTL clones, or CTLs isolated from tumors, inflammatory infiltrations, or other infiltrations.

[0230] In another embodiment, hematopoietic stem cells or early progenitor cells comprise the cell population used in the present invention. In another embodiment, such a population is isolated or derived by leukocyte isolation. In another embodiment, leukocyte isolation is performed from bone marrow, peripheral blood (PB), or neonatal umbilical cord blood after cytokine administration. In another embodiment, stem cells or progenitor cells are characterized by cell surface expression of a surface antigen marker known as CD34+ and exclusion of expression of the surface lineage antigen marker Lin-.

[0231] In another embodiment, the subject is administered the peptide, composition, or vaccine of the present invention together with bone marrow cells. In another embodiment, the administration in combination with bone marrow cells is performed as part of a therapeutic process, following prior irradiation of the subject, to suppress, inhibit, or treat cancer in the subject.

[0232] In another embodiment, the phrases “bring cells into contact” or “bring a population into contact” refer to one method of exposure, which in other embodiments may be direct or indirect. In another embodiment, such contact includes direct injection of cells by means known in the art, such as microinjection. In another embodiment, the supply of cells may also be indirect, such as supply in a culture medium surrounding the cells, or administration to a subject via any route known in the art and as described herein.

[0233] In another embodiment, the generation of CTLs by the method of the present invention is achieved in vivo by administering antigen-presenting cells that have been in vitro contacted with the peptide of the present invention to a subject (see, for example, Paglia et al. (1996) J. Exp. Med. 183:317-322).

[0234] In another embodiment, the peptides of the method and composition of the present invention are delivered to antigen-presenting cells (APCs).

[0235] In another embodiment, the peptide is delivered to an APC in the form of cDNA encoding the peptide. In another embodiment, the term “antigen-presenting cell” (APC) refers to a dendritic cell (DC), monocyte / macrophage, B cell, or other cell type(s) expressing the required MHC / co-stimulatory molecule, which efficiently enables T cell recognition of the presented peptide. In another embodiment, the APC is a cancer cell. Each possible form represents a separate embodiment of the present invention. In each embodiment, the vaccine or APC, or any form of peptide delivery to a patient or subject, is administered together with at least one checkpoint inhibitor. As described herein, the administration of at least one checkpoint inhibitor does not need to be within the same vaccine, formulation, administration site or time of administration as the administration of the WT1 vaccine or its alternative form. As embodied herein, the administration of various forms of checkpoint inhibitors concurrently with the WT1 vaccine enhances the formation of WT1-specific CTLs in subjects requiring it.

[0236] In another embodiment, CTLs are contacted with two or more APC populations along with at least one checkpoint inhibitor. In yet another embodiment, the two or more APC populations present different peptides. Each possible form represents a separate embodiment of the present invention.

[0237] In another embodiment, a method is used to deliver the peptide to an APC (e.g., DC) in which the antigen is expressed in the cytosol. Methods for expressing the antigen on an APC are known in the art. In another embodiment, the method includes (1) introducing naked DNA encoding the peptide of the present invention into the APC, (2) infecting the APC with a recombinant vector expressing the peptide of the present invention, and (3) introducing the peptide of the present 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).

[0238] In another embodiment, a foster APC (Zweerink et al. (1993) J. Immunol. 150:1763-1771), such as one derived from the human cell line 174xCEM.T2 (referred to as T2), which contains a mutation in its antigen processing pathway that restricts the association between an endogenous peptide and a cell surface MHC class I molecule, is used as illustrated herein.

[0239] In another embodiment, CTLs are induced using one of the methods described herein, and this induction is in vitro. In another embodiment, CTLs are induced ex vivo. In another embodiment, CTLs are induced in vitro. In another embodiment, the resulting CTLs are administered to a subject to treat a condition associated with the peptide, an expression product containing the peptide, or its homologue. Each possible form represents a separate embodiment of the present invention.

[0240] In another embodiment, the method involves introducing a gene sequence encoding the peptide of the present invention. In another embodiment, the method includes administering to a subject a vector containing a nucleotide sequence encoding the peptide of the present invention (Tindle, RW et al. Virology (1994) 200:54). In another embodiment, the method includes administering to a subject a naked nucleic acid (DNA or RNA) encoding the peptide, or in another embodiment, two or more peptides of the present invention (Nabel, et al. PNAS-USA (1990) 90: 11307). In another embodiment, a multi-epitope, analogue-based cancer vaccine is utilized (Fikes et al, Design of multi-epitope, analogue-based cancer vaccines. Expert Opin Biol Ther. 2003 Sep;3(6):985-93). Each possible form represents a separate embodiment of the present invention.

[0241] Nucleic acids (DNA or RNA) may be administered to a subject by means known in the art, including parenteral or intravenous administration, or, in other embodiments, by a gene gun. In other embodiments, the nucleic acid may be administered in the form of a composition, and in other embodiments, this form corresponds to any embodiment enumerated herein. DNA or RNA may be administered to a subject as naked nucleic acid or delivered by a vector.

[0242] In another embodiment, the vectors for use by the method of the present invention may include any vector that promotes or enables the expression of the peptide of the present invention (e.g., WT1 peptide) in cells of a subject in vitro or in vivo. The term “vector” is used to refer to any molecule (e.g., nucleic acid, plasmid, virus, particle) that can be used to transfer coding sequence information (e.g., a nucleic acid sequence encoding the WT1 peptide) into a cell or subject. Nucleic acid vaccines for several cancers have entered clinical trials (Wahren B et al., "DNA Vaccines: Recent Developments and the Future," Vaccines, 2014, 2:785-796; Fioretti D. et al., "DNA Vaccines: Developing New Strategies Against Cancer, Journal of Biomedicine and Biotechnology, 2010, 2010(938):174378). Strategies for expanding 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 one embodiment, the vector is a viral vector. In another embodiment, 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 (see, for example, 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-NPs) for Efficient Gene Expression," Sci Rep, 2015, 5:12737, and Ulmer JB et al., "RNA-based Vaccines," Vaccine, 2012, 30:4414-4418). In some embodiments, the “vector” includes attenuated viruses such as cowpox or fowlpox, as described, for example, in U.S. Patent No. 4,722,848, which is incorporated herein by reference. In another embodiment, the vector is BCG (Bacillus calmette-Guérin), 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) will be apparent to those skilled in the art from the description herein. Non-limiting examples of vectors that may be used to administer nucleic acid molecules to subjects in vivo and to cells in vitro include adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, poxviruses, herpesviruses, virus-like particles (VLPs), plasmids, cationic lipids, liposomes, and nanoparticles.

[0243] A "coding sequence" is a nucleic acid sequence that is transcribed into mRNA and / or translated into a polypeptide. The boundaries of a coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. Coding sequences may include, but are not limited to, mRNA, cDNA, and recombinant polynucleotide sequences. Mutants or analogs can be prepared by partial deletion, insertion, and / or substitution of one or more nucleotides within a coding sequence. Techniques for modifying nucleic acid sequences, such as site-directed mutagenesis, are known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, 1989; DNA Cloning, Vols. I and II, DN Glover ed., 1985). If necessary, the nucleic acid sequences of the present invention, and the compositions and methods of the present invention utilizing such polynucleotides, may include non-coding sequences.

[0244] The term “operably linked” is used herein to refer to the arrangement of flanking control sequences such that the flanking sequences described herein are configured or assembled to perform their normal functions. Thus, flanking control sequences operably linked to a coding sequence can replicate, transcribe, and / or translate 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 induce transcription of that coding sequence. The flanking sequence does not need to be contiguous with the coding sequence in order to function correctly. Thus, for example, an intervening sequence that is not translated but is transcribed may be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence. Each nucleic acid sequence encoding a polypeptide (e.g., WT1 peptide) typically has its own operably linked promoter sequence.

[0245] In another embodiment, the vector further encodes an immunomodulatory compound described herein. In another embodiment, the subject is administered the further vector encoding the immunomodulatory compound either before or after administration of the vector encoding the peptide of the present invention, simultaneously with the administration of the vector encoding the peptide of the present invention to the subject.

[0246] In another embodiment, the peptides, compositions, and vaccines of the present invention are administered to a subject in combination with other anticancer compounds and chemotherapeutic agents, including alternative cancer antigens, or, in another embodiment, monoclonal antibodies that target an epitope consisting of an AA sequence corresponding to, or partially corresponding to, an AA sequence from which the peptides of the present invention are derived, or are utilized in the manner of the present invention. This is done in addition to the use of at least one checkpoint inhibitor in various embodiments of the present invention.

[0247] In another embodiment, the present invention comprises the step of administering the peptide, vaccine, or immunogenic composition of the present invention to a subject, wherein the subject has WT1-specific CD4 + The present invention provides a method for detecting T cell responses. In another embodiment, WT1-specific CD4 + A delayed-type hypersensitivity test is performed to detect the T cell response. In another embodiment, the peptide of the present invention is used in a subject with CD4 + It is superior to its unmutated counterpart in inducing a T cell response. Each possible form represents a separate embodiment of the present invention.

[0248] As used herein, the terms “patient,” “subject,” and “individual” are interchangeable and are intended to include human and non-human animal species. For example, a subject may be a human or a non-human mammal. In some embodiments, a subject is a non-human animal model or an animal patient. A subject may be of any age or sex.

[0249] In another embodiment, the immunogenic composition of the method and composition of the present invention comprises an APC associated with the peptide of the present invention. In another embodiment, the immunogenic composition comprises an APC associated with a mixture of the peptide of the present invention. In another embodiment, the immunogenic composition consists of an APC associated with the peptide of the present invention. In another embodiment, the immunogenic composition consists of an APC associated with a mixture of the peptide of the present invention. Each possible form represents a separate embodiment of the present invention.

[0250] The compositions of the methods of the present invention are, in other embodiments, immunogenic compositions. In other embodiments, the compositions are pharmaceutical compositions. In other embodiments, the compositions are any other type of composition known in the art. Each possible form represents a separate embodiment of the present invention. Each composition further comprises at least one checkpoint inhibitor.

[0251] Various embodiments of the dose range are intended by the present invention. In another embodiment, the dose is 20 μg / peptide / day. In another embodiment, the dose is 10 μg / peptide / day. In another embodiment, the dose is 30 μg / peptide / day. In another embodiment, the dose is 40 μg / peptide / day. In another embodiment, the dose is 60 μg / peptide / day. In another embodiment, the dose is 80 μg / peptide / day. In another embodiment, the dose is 100 μg / peptide / day. In another embodiment, the dose is 150 μg / peptide / day. In another embodiment, the dose is 200 μg / peptide / day. In another embodiment, the dose is 300 μg / peptide / day. In another embodiment, the dose is 400 μg / peptide / day. In another embodiment, the dose is 600 μg / peptide / day. In another embodiment, the dose is 800 μg / peptide / day. In another embodiment, the dose is 1000 μg / peptide / day.

[0252] In another embodiment, the dose is 10 μg / peptide / dose. In another embodiment, the dose is 30 μg / peptide / dose. In another embodiment, the dose is 40 μg / peptide / dose. In another embodiment, the dose is 60 μg / peptide / dose. In another embodiment, the dose is 80 μg / peptide / dose. In another embodiment, the dose is 100 μg / peptide / dose. In another embodiment, the dose is 150 μg / peptide / dose. In another embodiment, the dose is 200 μg / peptide / dose. In another embodiment, the dose is 300 μg / peptide / dose. In another embodiment, the dose is 400 μg / peptide / dose. In another embodiment, the dose is 600 μg / peptide / dose. In another embodiment, the dose is 800 μg / peptide / dose. In another embodiment, the dose is 1000 μg / peptide / dose.

[0253] In another embodiment, the dose is 10-20 μg / peptide / dose. In another embodiment, the dose is 20-30 μg / peptide / dose. In another embodiment, the dose is 20-40 μg / peptide / dose. In another embodiment, the dose is 30-60 μg / peptide / dose. In another embodiment, the dose is 40-80 μg / peptide / dose. In another embodiment, the dose is 50-100 μg / peptide / dose. In another embodiment, the dose is 50-150 μg / peptide / dose. In another embodiment, the dose is 100-200 μg / peptide / dose. In another embodiment, the dose is 200-300 μg / peptide / dose. In another embodiment, the dose is 300-400 μg / peptide / dose. In another embodiment, the dose is 400-600 μg / peptide / dose. In another embodiment, the dose is 500-800 μg / peptide / dose. In another embodiment, the dose is 800-1000 μg / peptide / dose.

[0254] In another embodiment, the total amount of peptides / dose or peptides / day is one of the above amounts. In another embodiment, the total peptide dose / dose is one of the above amounts.

[0255] Each of the above dosages represents a separate embodiment of the present invention.

[0256] In another embodiment, the present invention provides a kit comprising a peptide, composition, or vaccine of the present invention together with at least one checkpoint inhibitor. In another embodiment, the kit further comprises a label or accompanying documentation. In another embodiment, the kit is used for the detection of a WT1-specific CD4 response by a delayed-type hypersensitivity test. In another embodiment, the kit is used in any other method enumerated herein. In another embodiment, the kit is used in any other method known in the art. Each possible form represents a separate embodiment of the present invention.

[0257] Examples

[0258] Evaluation of the efficacy of WT1 peptide vaccine administered with nivolumab in ovarian cancer patients.

[0259] Eligible patients diagnosed with ovarian cancer will begin the vaccination schedule within four months of completing chemotherapy. Patients will first receive six doses of WT1 peptide over 12 weeks, followed by seven infusions of the immune checkpoint inhibitor nivolumab over 14 weeks. Toxicity assessments will be performed at each vaccine dose and three weeks after the completion of treatment at week 15. Patients will be monitored by the study staff for up to 30 minutes post-treatment. Dose increases are not planned. Periodic toxicity assessments will continue throughout the study period.

[0260] Patients who show no disease progression at the 15-week evaluation are permitted to receive four additional doses of the vaccine approximately every eight weeks. This maintenance vaccination course begins at week 19.

[0261] The immune response will be assessed from 40 ml heparinized blood samples at six separate time points: baseline (at the time of consent to determine baseline variation, and before the first dose), before vaccines 5 and 6, and 3 weeks after the last nivolumab infusion. If possible, additional blood will be collected at a 3-month follow-up.

[0262] ELISA is used to measure antibody levels generated against the four WT1 peptides in the vaccine. Antibodies are generally produced upon completion of the fourth vaccination. T cell proliferation response assays are performed on peripheral blood lymphocytes, including: flow cytometry for phenotypic analysis using FACS, including leukocyte subset analysis; T regulatory cell assays (including CD3, CD4, CD8, FOXP3, ICOS, and PD1); and myeloid-derived suppressor cells (MDSCs, CD14+HLA-DRlow cells) in peripheral blood and tumors (if biopsies are obtained as needed). WT1 T cell-specific CD4 and CD8 proliferation responses are measured using cytotoxicity assays with a functional Meso Scale Discovery System, measured by multifunctional intracellular cytokine staining (ICS) and IFN-gamma production based on flow cytometry. Detailed procedures for blood sample preparation, T cell monitoring, antibody ELISA, and multifunctional T cell assays are described in

[29] .

[0263] Baseline values ​​and T-cell response results correlate with the duration of clinical remission.

[0264] If a patient leaves the study before 15 weeks, blood will be obtained for post-study immunological investigation. CT scans will be performed at baseline and at 15 weeks (or earlier if medically deemed necessary), and thereafter every 3 months for up to 1 year until disease progression. Abdominal and pelvic MRI may be used instead of abdominal and pelvic CT. A reference radiologist will use immune-related response criteria to determine disease progression

[57] . CA125 will be obtained at baseline, at 6 and 15 weeks, and thereafter every 3 months for up to 1 year until disease progression. CA125 will not be used to determine disease progression because inflammation in vaccinated patients may be confused with CA125. Patients will remain in the study until progression, development of unacceptable toxicity, completion of vaccination, or time of patient withdrawal.

[0265] WT1 vaccine: The vaccine used in this study contains four distinct WT1 peptides: • YMFPNAPYL (SEQ ID NO124; WT1-A1): An HLA class I peptide containing the mutant amino acid R126Y that stimulates the CD8+ response. • SGQAYMFPNAPYLPSCLES (SEQ ID NO125; WT1-122A1 long): An HLA class II peptide containing an embedded WT1-A1 heterocritic sequence that stimulates both CD4+ and CD8+ responses, according to data from preclinical and phase I trials. RSDELVRHHNMHQRNMTKL (SEQ ID NO1; WT1-427 long) and PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO2; WT1-331 long): HLA class II peptides that induce CD4+ responses that may aid in long-lasting CD8+ T cell responses.

[0266] Drug product: To produce the vaccine product ("WT1 Vax"), four peptides are provided in a sterile solution with phosphate-buffered saline. Each vial contains 280 mcg of each peptide in a total volume of 0.7 ml (0.4 mg / ml of each peptide, 40% overfill). Vial filling 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 immunoadjuvant montanide ISA 51 VG.

[0267] Intended dose: A dose of 200 mcg is selected for each peptide as it falls within the range of safe and effective doses used by others. Peptide vaccines have produced immune and clinical responses at a wide range of doses (100–2000 mcg infusions) without clear evidence of a dose-response relationship. There is a theoretical possibility that higher doses may stimulate lower affinity TCRs on T cells and reduce the response [30, 33, 34]. Vial size: Each single-dose vial contains 0.7 ml. Route of administration: Subcutaneous route.

[0268] Nivolumab: Planned dose: 3 mg / kg; Vial size: 10 mL; Route of administration: Intravenous. Nivolumab is administered intravenously at a dose of 3 mg / kg every two weeks as a 60-minute IV infusion. At the end of the infusion, the tube is flushed with a sufficient amount of saline. If the patient's body weight differs by >10% from the previous weight used to calculate the required dose, the required dose, or corrected dose, should be calculated. Dosage increases or decreases of nivolumab are not permitted. There are no recommended premedications for the initial nivolumab treatment.

[0269] The drug can be administered at least 12 days after the previous nivolumab dose, and may be administered within 3 days after the scheduled dose date. Administration after a 3-day period is considered a delayed administration. The treatment can be postponed for up to 6 weeks from the previous dose.

[0270] Tumor evaluation using CT or MRI should be continued according to the protocol, even if medication administration is delayed.

[0271] Treatment / Intervention Plan • Treat the patient as an outpatient. ・The WT1 vaccine is administered at weeks 0, 2, 4, 6, 8, and 10. All injections should be administered subcutaneously to the areas that rotate between the limbs. All patients should receive a subcutaneous injection of salglamostim (GM-CSF) 70 mcg on days 0 and 2. Patients may self-administer GM-CSF if they have been appropriately instructed on SQ injection administration. Patients should be informed of any expected reactions, such as irritation at the injection site. Patients should keep a record of the time and location of the injection. • In addition, administer 1.0 ml of WT1 peptide emulsion to the patient along with montanide. The nurse should administer it subcutaneously at the same anatomical site as GM-CSF (self-administration is not required). Observe the patient for approximately 30 minutes after vaccination. · Nivolumab is administered intravenously as a 60-minute infusion at weeks 0, 2, 4, 6, 8, 10, and 12. It can be administered at least 12 days after the previous nivolumab administration, and may be administered within 3 days after the scheduled administration date. Administration after a 3-day period is considered a dosing delay. Treatment can be postponed up to a maximum of 6 weeks from the previous administration.

[0272] The combination therapy of WT1 vaccine and nivolumab is expected to increase the WT1-specific CTL population of patients and increase the activity against WT1-expressing tumors compared to WT1 vaccine inoculation alone or nivolumab therapy alone.

[0273] (References) [1] Siegel, R., D. Naishadham, and A. Jemal, Cancer statistics, 2012. CA Cancer J Clin, 2012. 62(1): p. 10-29 [2] Hoskins, W.J., C.A. Perez, and R.C. Young, Principles and practice of gynecologic oncology. 3rd ed. 2000, Philadelphia: Lippincott Williams & Wilkins. xxi, 1268 p. [3] Barnhill, D.R., et al., The second-look surgical reassessment for epithelial ovarian carcinoma. Gynecol Oncol, ********. 19(2): p. 148-54. [4] Rubin, S.C., et al., Recurrence after negative second-look laparotomy for ovarian cancer: analysis of risk factors. Am J Obstet Gynecol, 1988. 159(5): p. 1094-8. [5]Markman, M., et al., Second-line platinum therapy in patients with ovarian cancer previously treated with cisplatin. J Clin Oncol, 1991. 9(3): p. 389-93. [6]Zhang, H., et al., Antibodies against GD2 ganglioside can eradicate syngeneic cancer micrometastases. Cancer Res, 1998. 58(13): p. 2844-9. [7]Zhang, L., et al., Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med, 2003. 348(3): p. 203-13. [8]Curiel, T.J., et al., Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med, 2004. 10(9): p. 942-9. [9]Iasonos, A., et al., Identifying clinical improvement in consolidation single-arm phase 2 trials in patients with ovarian cancer in second or greater clinical remission. Int J Gynecol Cancer, 2012. 22(1): p. 63-9.

[10] Berek, J.S., et al., Randomized, placebo-controlled study of oregovomab for consolidation of clinical remission in patients with advanced ovarian cancer. J Clin Oncol, 2004. 22(17): p. 3507-16.

[11] Reinartz, S., et al., Vaccination of patients with advanced ovarian carcinoma with the anti-idiotype ACA125: immunological response and survival (phase Ib / II). Clin Cancer Res, 2004. 10(5): p. 1580-7.

[12] Bookman, M.A., et al., Evaluation of monoclonal humanized anti-HER2 antibody, trastuzumab, in patients with recurrent or refractory ovarian or primary peritoneal carcinoma with overexpression of HER2: a phase II trial of the Gynecologic Oncology Group. J Clin Oncol, 2003. 21(2): p. 283-90.

[13] Allavena, P., et al., Intraperitoneal recombinant gamma-interferon in patients with recurrent ascitic ovarian carcinoma: modulation of cytotoxicity and cytokine production in tumor-associated effectors and of major histocompatibility antigen expression on tumor cells. Cancer Res, 1990. 50(22): p. 7318-23.

[14] Pujade-Lauraine, E., et al., Intraperitoneal recombinant interferon gamma in ovarian cancer patients with residual disease at second-look laparotomy. J Clin Oncol, 1996. 14(2): p. 343-50.

[15] Recchia, F., et al., Interleukin-2 and 13-cis retinoic acid as maintenance therapy in advanced ovarian cancer. Int J Oncol, 2005. 27(4): p. 1039-46.

[16] Sabbatini, P.J., et al., Immunization of ovarian cancer patients with a synthetic Lewis(y)-protein conjugate vaccine: a phase 1 trial. Int J Cancer, 2000. 87(1): p. 79-85.

[17] Nicholson, S., et al., A phase I trial of idiotypic vaccination with HMFG1 in ovarian cancer. Cancer Immunol Immunother, 2004. 53(9): p. 809-16.

[18] Diefenbach, C.S., et al., Safety and immunogenicity study of NY-ESO-1b peptide and montanide ISA-51 vaccination of patients with epithelial ovarian cancer in high-risk first remission. Clin Cancer Res, 2008. 14(9): p. 2740-8.

[19] Keilholz, U., et al., Wilms' tumour gene 1 (WT1) in human neoplasia. Leukemia, 2005. 19(8): p. 1318-23.

[20] Oji, Y., et al., Expression of the Wilms' tumor gene WT1 in solid tumors and its involvement in tumor cell growth. Jpn J Cancer Res, 1999. 90(2): p. 194-204.

[21] Scharnhorst, V., et al., Internal translation initiation generates novel WT1 protein isoforms with distinct biological properties. J Biol Chem, 1999. 274(33): p. 23456-62.

[22] Haber, D.A., et al., Alternative splicing and genomic structure of the Wilms tumor gene WT1. Proc Natl Acad Sci U S A, 1991. 88(21): p. 9618-22.

[23] Mundlos, S., et al., Nuclear localization of the protein encoded by the Wilms' tumor gene WT1 in embryonic and adult tissues. Development, 1993. 119(4): p. 1329-41.

[24] Buckler, A.J., et al., Isolation, characterization, and expression of the murine Wilms' tumor gene (WT1) during kidney development. Mol Cell Biol, 1991. 11(3): p. 1707-12.

[25] Fraizer, G.C., et al., Expression of the tumor suppressor gene WT1 in both human and mouse bone marrow. Blood, 1995. 86(12): p. 4704-6.

[26] Al-Hussaini, M., et al., WT1 assists in distinguishing ovarian from uterine serous carcinoma and in distinguishing between serous and endometrioid ovarian carcinoma. Histopathology, 2004. 44(2): p. 109-15.

[27] Pinilla-Ibarz, J., et al., Improved human T-cell responses against synthetic HLA-0201 analog peptides derived from the WT1 oncoprotein. Leukemia, 2006. 20(11): p. 2025-33.

[28] May, R.J., et al., Peptide epitopes from the Wilms' tumor 1 oncoprotein stimulate CD4+ and CD8+ T cells that recognize and kill human malignant mesothelioma tumor cells. Clin Cancer Res, 2007. 13(15 Pt 1): p. 4547-55.

[29] Krug, L.M., et al., WT1 peptide vaccinations induce CD4 and CD8 T cell immune responses in patients with mesothelioma and non-small cell lung cancer. Cancer Immunol Immunother, 2010. 59(10): p. 1467-79.

[30] Oka, Y., et al., Induction of WT1 (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression. Proc Natl Acad Sci U S A, 2004. 101(38): p. 13885-90.

[31] Letsch, A., et al., Effect of vaccination of leukemia patients with a MHC class I peptide of Wilms tumor gene 1 (WT1) peptide with unspecific T helper stimulation on WT1-specific IgM responses and on IgG responses. J Clin Oncol, 2008. 26: p. Abstr 3054.

[32] Ohno, S., et al., Wilms' tumor 1 (WT1) peptide immunotherapy for gynecological malignancy. Anticancer Res, 2009. 29(11): p. 4779-84.

[33] Schaed, S.G., et al., T-cell responses against tyrosinase 368-376(370D) peptide in HLA*A0201+ melanoma patients: randomized trial comparing incomplete Freund's adjuvant, granulocyte macrophage colony-stimulating factor, and QS-21 as immunological adjuvants. Clin Cancer Res, 2002. 8(5): p. 967-72.

[34] Slingluff, C.L., Jr., et al., Clinical and immunologic results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J Clin Oncol, 2003. 21(21): p. 4016-26.

[35] Faries, M.B., et al., Effect of granulocyte / macrophage colony-stimulating factor on vaccination with an allogeneic whole-cell melanoma vaccine. Clin Cancer Res, 2009. 15(22): p. 7029-35.

[36] Keilholz, U., et al., A clinical and immunologic phase 2 trial of Wilms tumor gene product 1 (WT1) peptide vaccination in patients with AML and MDS. Blood, 2009. 113(26): p. 6541-8.

[37] Weber, J., et al., Granulocyte-macrophage-colony-stimulating factor added to a multipeptide vaccine for resected Stage II melanoma. Cancer, 2003. 97(1): p. 186-200.

[38] Keir, M.E., et al., PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol, 2008. 26: p. 677-704.

[39] Freeman, G.J., et al., Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med, 2000. 192(7): p. 1027-34.

[40] Latchman, Y., et al., PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol, 2001. 2(3): p. 261-8.

[41] Hamanishi, J., et al., Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer. Proc Natl Acad Sci U S A, 2007. 104(9): p. 3360-5.

[42] Mu, C.Y., et al., High expression of PD-L1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation. Med Oncol, 2011. 28(3): p. 682-8.

[43] Pardoll, D.M., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012. 12(4): p. 252-64.

[44] Nivolumab (BMS-936558) Investigator Brochure, Version 12. 2013.

[45] Hwang, W.T., et al., Prognostic significance of tumor-infiltrating T cells in ovarian cancer: a meta-analysis. Gynecol Oncol, 2012. 124(2): p. 192-8.

[46] Matsuzaki, J., et al., Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer. Proc Natl Acad Sci U S A, 2010. 107(17): p. 7875-80.

[47] Brahmer, J.R., et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med, 2012. 366(26): p. 2455-65.

[48] Page, D.B., et al., Immune modulation in cancer with antibodies. Annu Rev Med, 2014. 65: p. 185-202.

[49] Harrison, M.L., et al., Duration of second or greater complete clinical remission in ovarian cancer: exploring potential endpoints for clinical trials. Gynecol Oncol, 2007. 106(3): p. 469-75.

[50] Juretzka, M., et al., A phase 2 trial of oral imatinib in patients with epithelial ovarian, fallopian tube, or peritoneal carcinoma in second or greater remission. Eur J Gynaecol Oncol, 2008. 29(6): p. 568-72.

[51] Levine, D., et al., A phase II evaluation of goserelin and bicalutamide in patients with ovarian cancer in second or higher complete clinical disease remission. Cancer, 2007. 110(11): p. 2448-56.

[52] Walter, S., et al., Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival. Nat Med, 2012.

[53] Wolchok, J.D., et al., Development of ipilimumab: a novel immunotherapeutic approach for the treatment of advanced melanoma. Ann N Y Acad Sci, 2013. 1291(1): p. 1-13.

[54] Hodi, F.S., et al., Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients. Proc Natl Acad Sci U S A, 2008. 105(8): p. 3005-10.

[55] Quezada, S.A., et al., CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells. J Clin Invest, 2006. 116(7): p. 1935-45.

[56] Duraiswamy, J., et al., Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumors. Cancer Res, 2013. 73(12): p. 3591-603.

[57] Wolchok, J.D., et al., Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria. Clin Cancer Res, 2009. 15(23): p. 7412-20.

[58] Dupont, J., et al., Wilms Tumor Gene (WT1) and p53 expression in endometrial carcinomas: a study of 130 cases using a tissue microarray. Gynecol Oncol, 2004. 94(2): p. 449-55.

[59] Eisenhauer, E.A., et al., New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer, 2009. 45(2): p. 228-47.

Claims

1. A composition used to treat WT1-expressing cancer, reduce the incidence of WT1-expressing cancer, or induce an immune response against WT1-expressing cancer, (a) A combination of WT1 peptides comprising YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRRKHTG (SEQ ID NO: 2), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), or one or more WT1 delivery agents that deliver cytotoxic T cells (CTLs) specific to the aforementioned combination of WT1 peptides, (i) at least one of the above combinations of WT1 peptides, (ii) A nucleic acid that encodes at least one of the above combinations of WT1 peptides, or (iii) One or more WT1 delivery agents comprising at least one of the aforementioned combinations of WT1 peptides, or a nucleic acid encoding at least one of the aforementioned combinations of WT1 peptides, or an immune cell presenting such a nucleic acid, (b) at least one anti-PD-1 antibody used in combination with the WT1 delivery agent and A composition containing the following:

2. The composition according to claim 1, characterized in that the CTL is produced in vitro or ex vivo, or obtained from a donor.

3. The composition according to claim 1, further comprising a carrier, an excipient, or a diluent.

4. The composition according to claim 1, further comprising an adjuvant.

5. The composition according to claim 1, characterized in that the anti-PD-1 antibody is nivolumab, pembrolizumab, pizilizumab, or MEDI-0680 (AMP-514), or any combination thereof.

6. The composition according to claim 1, characterized in that the one or more WT1 delivery agents and at least one anti-PD-1 antibody are administered simultaneously, in overlapping administration schedules, or the anti-PD-1 antibody is administered after the administration of the WT1 delivery agent.

7. The composition according to claim 1, characterized in that the WT1-expressing cancer is ovarian cancer, mesothelioma, leukemia, Wilms' tumor, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), melanoma, gastric cancer, prostate cancer, biliary tract cancer, urinary tract cancer, glioblastoma, soft tissue sarcoma, osteosarcoma, or non-small cell lung cancer (NSCLC).

8. The composition according to claim 4, characterized in that the adjuvant is QS21, montanide, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG, cytokine, or alum.

9. The composition according to claim 1, characterized in that 200 mcg of each peptide is emulsified with montanide ISA 51 VG and administered subcutaneously at weeks 0, 2, 4, 6, 8 and 10.

10. The composition according to claim 5, characterized in that the anti-PD-1 antibody is nivolumab.

11. The composition according to claim 10, characterized in that 3 mg / kg of nivolumab is administered intravenously at weeks 0, 2, 4, 6, 8, 10 and 12.

12. The composition according to claim 1, characterized in that it has a greater effect in treating WT1-expressing cancer, reducing the incidence of WT1-expressing cancer, or inducing an immune response against WT1-expressing cancer compared to the administration of the WT1 delivery agent alone or the administration of the anti-PD-1 antibody alone.

13. If the CTL is produced in vitro or ex vivo, or obtained from a donor, The composition according to claim 2, characterized in that the anti-PD-1 antibody is included in vitro or ex vivo, or administered to the donor.

14. The composition according to claim 13, characterized in that the subject is further administered the anti-PD-1 antibody.

15. The composition according to claim 1, characterized in that the WT1 delivery agent includes the combination of the WT1 peptides in (i) above.

16. The composition according to claim 1, characterized in that it comprises the WT1 delivery agent and the nucleic acid encoding the combination of the (ii) WT1 peptide.

17. The composition according to claim 1, characterized in that the nucleic acid encoding the aforementioned combination of WT1 peptides is located within the vector.

18. The composition according to claim 17, characterized in that the vector is a viral vector selected from the group consisting of adenovirus, adeno-associated virus, retrovirus, lentivirus, poxvirus, and herpesvirus.

19. The composition according to claim 17, characterized in that the vector is a non-viral vector selected from the group consisting of plasmids, cationic lipids, liposomes, and virus-like particles.

20. The composition according to claim 17, characterized in that the vector is a non-viral vector selected from the group consisting of autologous cells, allogeneic cells, cell lines, dendritic cells, antigen-presenting cells, and any combination thereof.

21. The composition according to claim 1, characterized in that the immune cells are antigen-presenting cells or professional antigen-presenting cells.

22. The composition according to claim 21, characterized in that the antigen-presenting cells or professional antigen-presenting cells are dendritic cells, macrophages, monocytes, or B cells.

23. A composition used to treat WT1-expressing cancer, reduce the incidence of WT1-expressing cancer, or induce an immune response against WT1-expressing cancer, A combination of WT1 peptides including YMFPNAPYL (SEQ ID NO: 124), RSDELVRHHNMHQRNMTKL (SEQ ID NO: 1), PGCNKRYFKLSHLQMHSRRKHTG (SEQ ID NO: 2), and SGQAYMFPNAPYLPSCLES (SEQ ID NO: 125), A composition characterized by comprising at least one anti-PD-1 antibody used in combination with the aforementioned combination of WT1 peptides.

24. The composition according to claim 23, characterized in that the anti-PD-1 antibody is nivolumab, pembrolizumab, pizilizumab, MEDI-0680 (AMP-514), or any combination thereof.

25. The composition according to claim 1, characterized in that one or more WT1 delivery agents and at least one anti-PD-1 antibody are each administered individually.

26. The composition according to claim 23, characterized in that the cancer is ovarian cancer, mesothelioma, leukemia, Wilms' tumor, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), melanoma, gastric cancer, prostate cancer, biliary tract cancer, urinary tract cancer, glioblastoma, soft tissue sarcoma, osteosarcoma, or non-small cell lung cancer (NSCLC).

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