WO2007120603A2 - Peptides bcr-abl immunogènes et leurs méthodes d'utilisation - Google Patents

Peptides bcr-abl immunogènes et leurs méthodes d'utilisation Download PDF

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WO2007120603A2
WO2007120603A2 PCT/US2007/008747 US2007008747W WO2007120603A2 WO 2007120603 A2 WO2007120603 A2 WO 2007120603A2 US 2007008747 W US2007008747 W US 2007008747W WO 2007120603 A2 WO2007120603 A2 WO 2007120603A2
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another embodiment
peptide
bcr
abl
hla
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PCT/US2007/008747
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WO2007120603A3 (fr
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David A. Scheinberg
Rena May
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Sloan Kettering Institute For Cancer Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes

Definitions

  • This invention provides peptides, immunogenic compositions and vaccines comprising same, and methods of treating, reducing the incidence of, and inducing immune responses to a bcr-abl- expressing cancer, comprising same.
  • Leukemias including chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) are pluripotent stem cell disorders, which may be characterized by the presence of the Philadelphia chromosome (Ph). Because of the unique features, these cancers present a unique opportunity to develop therapeutic strategies using vaccination against a truly tumor specific antigen that is also the oncogenic protein required for neoplasia.
  • CML chronic myelogenous leukemia
  • AML acute myelogenous leukemia
  • ALL acute lymphocytic leukemia
  • the chimeric fusion proteins are potential antigens for two reasons.
  • the proteins are uniquely expressed in the leukemic cells in which the junctional regions contain a sequence of amino acids that is not expressed on any normal protein.
  • a new amino acid lysine in b . 3a2
  • a conserved one glutamic acid in b2a2
  • the unique amino acid sequences encompassing the b3a2 and b2a2 breakpoint region can be considered truly tumor specific antigens.
  • HLA human leukocyte antigen
  • Tumor specific, bcr-abl derived multivalent vaccine can be safely administered to patients with chronic phase CML; the vaccine reliably elicits a bcr-abl peptide specific CD4 immune response, as measured by DTH in vivo, CD4 + T cell proliferation ex vivo and gamma interferon secretion in a ELISPOT assay.
  • CD8 responses in A0201 patients were undetectable, and only weak responses in HLA A0301 patients using a sensitive gamma interferon ELISPOT assay were found.
  • CD8 responses For stimulation of responses the strength of CD8 responses depends upon the binding affinity of the target peptide to class I MHC molecules, the peptide-HLA complex stability, and the avidity of the T cell receptor binding for the peptide complex. Killing of native CML cells also requires adequate processing and presentation of the natural antigen. Therefore the lack of reproducible CD8 responses may reflect the biochemistry of the class I peptide-HLA interaction, which resulted in their weak immunogenicity to cytotoxic CD8 cells.
  • This invention provides peptides, immunogenic compositions and vaccines comprising same, and methods of treating, reducing the incidence of, and inducing immune responses to a bcr-abl- expressing cancer, comprising same.
  • the present invention provides an isolated, mutated bcr-abl peptide, comprising: (a) a binding motif of a human leukocyte antigen (HLA) Class II molecule; and (b) a binding motif of an HLA class I molecule, having a point mutation in one or more anchor residues of the binding motif of an HLA class I molecule.
  • the bcr-abl peptide is 11-30 amino acids in length.
  • the present invention provides a method of treating a subject with a bcr-abl-expressing cancer, the method comprising administering to the subject a peptide of the present invention, thereby treating a subject with a bcr-abl-expressing cancer.
  • the present invention provides a method of treating a subject with a bcr-abl-expressing cancer, the method comprising administering to the subject a vaccine of the present invention, thereby treating a subject with a bcr-abl-expressing cancer.
  • the present invention provides a method of treating a subject with a bcr-abl-expressing cancer, the method comprising administering to the subject an immunogenic composition of the present invention, thereby treating a subject with a bcr-abl- expressing cancer.
  • the present invention provides a method of suppressing or halting the progression of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject a peptide of the present invention, thereby suppressing or halting the progression of a bcr-abl- expressing cancer.
  • the present invention provides a method of suppressing or halting the progression of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject a vaccine of the present invention, thereby suppressing or halting the progression of a bcr-abl-expressing cancer.
  • the present invention provides a method of suppressing or halting the progression of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject an immunogenic composition of the present invention, thereby suppressing or halting the progression of a bcr-abl-expressing cancer
  • the present invention provides a method of inducing formation and proliferation of bcr-abl-specif ⁇ c CTL, the method comprising contacting a lymphocyte population with a peptide of the present invention, thereby inducing formation and proliferation of bcr-abl-specif ⁇ c CTL.
  • the cell is a cell of a bcr-abl-expressing leukemia.
  • the present invention provides a method of inducing formation and proliferation of (a) a CD8 + lymphocyte specific for a bcr-abl protein; and (b) a CD4 + lymphocyte specific for the bcr-abl protein, the method comprising contacting a lymphocyte population with a peptide of the present invention, thereby inducing formation and proliferation of (a) a CD8 + lymphocyte specific for a bcr-abl protein; and (b) a CD4 + lymphocyte specific for the bcr-abl protein.
  • Figure 1 T2 stabilization assay of native and synthetic WT-I peptides to HLA A0201 cells (A) and HLA A0301 cells (B-E).
  • Fluorescence index is ratio between median fluorescence with peptide tested: median fluorescence with no peptide.
  • X axis concentration per well of the peptide tested.
  • Figure 2 CD8 + /CD3 + gamma interferon (IFN) ELISPOT (A) and cytotoxicity (B) from healthy HLA A0201 donors against T2 cells pulsed with the following peptides: 1 st bar in each series: no peptide; 2 nd bar: same peptide used for stimulation; 3 rd bar: corresponding native peptide; 4 th bar: negative control peptide.
  • X axis peptides used for stimulations. Experiments were performed in triplicate and confirmed 3-5 times.
  • FIG. 3 CD8 + (A) and CD3 + (B-D) gamma IFN ELISPOT from healthy HLA A0201 donors using the indicated peptides- assignment of bars in each series is the same as for Figure 2.
  • Each subfigure in B-D represents a separate repetition of the experiment].
  • Figure4 Cytotoxicity assays using CD8 + T cells stimulated with synthetic WT-I Al peptides from a HLA A0201 donor against HLA-matched CML blasts presenting native peptide sequences.
  • A Bar graphs of results. 1" bar in each series: SKLY-16 (WTl ' ); 2 nd bar: BV173 (WTl + ); 3 rd bar: LAMA81 (WTl + ); 4 th bar: CMLA (additional negative control).
  • B Killing curves. Squares: SKLY-16. Diamonds: 697 cells.
  • G3, F4, C5, and G5 are T-cell clones generated from a healthy HLA-A0201 donor after multiple stimulations in vitro.
  • Y axis percentage of cytotoxicity.
  • X axis T cell: target cell ratio.
  • FIG. 5 part 1. Gamma interferon ELISPOT after stimulation with WTl peptides of CD3 + T cells from healthy donors with different HLA-DRBl types. Part 2. CD3 + T cells (A: HLA- DRBl*1001/1501; B: HLA-DRB 1*0701/1202; C: HLA-DRB 1*0301/901; D: HLA-
  • DRB 1*0407/1302) were stimulated twice with peptide WTlDR 328 or WTlDR 423.
  • Stimulated T cells were challenged in an IFN-gamma ELISPOT assay with the following: Grey Bars: unchallenged control; Black Bars: CD14 + cells pulsed with stimulating peptide (either WTlDR 328 or WTlDR
  • FIG. 1 Peptides with buried heteroclitic epitopes are processed, presented, and recognized by human T cells.
  • A. CD3 + T cells from an HLA A0201/301 DRBl*1301/1302 healthy donor were stimulated with autologous DCs previously incubated with 697 tumor lysates, then challenged in an IFN-gamma ELISPOT assay with autologous DCs previously incubated with either 697 tumor lysate, individual WTl peptides, control peptides or unpulsed DCs (X axis).
  • A. 1 CD3 + T cells from an HLA A0201/301 DRBl*1301/1302 healthy donor were stimulated with autologous DCs previously incubated with 697 tumor lysates, then challenged in an IFN-gamma ELISPOT assay with autologous DCs previously incubated with either 697 tumor lysate, individual WTl peptides, control peptides or unpulsed DCs (X axis).
  • CD3 + T cells from an HLA A0201/101 DRB 1*0301/1601 healthy donor were stimulated with autologous DCs previously incubated with tumor lysates from either JMN (Black Bars), or MeWo (White Bars).
  • T cells were challenged in an IFN-gamma ELISPOT assay with autologous DCs previously incubated with JMN or MeWo tumor lysates, individual WTlDR peptides, or control class II peptide (X axis). Hatched bars: background level of spots from autologous DCs incubated in the absence of T cells. * - P ⁇ 0.05 compared to control peptides.
  • Y axis number of spots per IxIO 5 CD3+ cells.
  • FIG. 7A-B A. CD3 + gamma interferon ELISPOT with peptides WTlDR 122 and WTlDR 122A 1.
  • CD3 + T cells from healthy donors with different HLA-DRB 1 types (A: HLA-DRB 1 * 1401 ; B: HLA-DRB 1*0104/1104) were stimulated twice with either peptide WTlDR 122 or WTlDR 122Al, then challenged in an IFN-gamma ELISPOT assay with the following: CD14 + cells pulsed with peptide WTlDR 122 (Grey Bars); CD14 + cells pulsed with peptide WTlDR 122Al (Black Bars); CDl 4 + cells pulsed with irrelevant CD4 peptide epitope (White Bars; RAS); unpulsed CD14 + cells (Hatched Bars). * - p ⁇ 0.05 compared to controls.
  • Y axis number of spots per 1 x 10 s CD3 + T
  • FIG. 7C WTlDR peptide 122 and 122Al stimulate CD8 + T cell responses.
  • Left panel CD3 + T cells from an HLA-A0201/DRB 1*1401 donor were stimulated twice with WTlDR 122, then challenged in an IFN-gamma ELISPOT assay with autologous CD 14 + cells.
  • Right panel CD3 + Tcells from an HLA-A0201/DRBl*1501 donor were stimulated twice with WTlDR 122Al, then challenged in an IFN-gamma ELISPOT assay with control melanoma cell line MeWo (A0201/DRBl*15XX, WTl " ).
  • * - p ⁇ 0.05 compared to no peptide controls.
  • Y axis represents the number of spots per IxIO 5 CD3 + cells.
  • X axis shows the different test peptides used in the ELISPOT.
  • FIG. 8 CD3+ gamma interferon ELISPOT against Mesothelioma cell lines.
  • Left panel Total PBMCs from an HLA-A0201 donor were stimulated twice with the different WTlDR peptides, then T cells were challenged in an IFN-gamma ELISPOT assay with the following: Mesothelioma H- Mesol A cell line (Black Bars; WTl+, A0201+); control melanoma MeWo cell line (WTl-, A0201+; Grey Bars). * - p ⁇ 0.01 compared to MeWo controls.
  • Y axis number of spots per 2xlO 5 PBMCs.
  • X axis peptide used for T cell stimulation.
  • Right panel CD3 + T cells from an HLA-A0201/DRB 1 * 1501 donor were stimulated twice with WTlDR 122Al, then T cells were challenged in an IFN-gamma ELISPOT assay with the following target cells: JMN, an A0201 /DRB 1*1505 WTl positive mesothelioma cell 1 ine or MeWo, an A0201 /DRB 1 * 15XX WT 1 negative mel anoma cell line.
  • Target cells were either pulsed with WTlDR 122Al (Black Bars) or not pulsed (Grey Bars). * p ⁇ 0.05 compared to the unpulsed Mewo target cell.
  • Y axis number of spots per lxl ⁇ s CD3 + T cells.
  • X axis cell lines used as target cells.
  • FIG. 9 left panel.
  • CD3 + T cells from an HLA-A0201/DRBl *0101/15XX donor were stimulated twice with WTlDR 122Al , then CD8 + T cells were isolated by negative selection and used as effector cells in a 51 Cr release cytotoxicity assay.
  • CD8 + T cells were incubated with radiolabeled target cells (pulsed or unpulsed 697 (A0201 + , WTl + ) or SKLY16 (A0201+, WTl-) at 3 different E:T ratios: Grey bars 100: 1 ; Black bars 30: 1 ; White bars 10:1.
  • Y axis percentage of cytotoxicity.
  • X axis target cell conditions.
  • FIG. 10 T2 stabilization assay using peptides derived from b3a2 translocation (left panel) and b2a2 translocations (right panel). Peptide sequences are delineated in Table 5. The fluorescence index is the value obtained for the ratio between median fluorescence obtained with the indicated peptide divided by background fluorescence. The X-axis represents different peptide concentrations.
  • n denotes native sequences from b3a2.
  • p210Cn, p210Dn, CMLA2, and CMLA3 are native b3a2 sequences;
  • b2a2A is the native sequence for b2a2.
  • Figure 11 gamma interferon (IFN) production detected by ELISPOT of CD8 + T cells from a healthy HLA A0201 donor following two in vitro stimulations with the peptides p210 C and F. After stimulation, CD8 + cells were challenged with the following: T2 (APC), or T2 pulsed with tested peptide (p210C or p210F), corresponding native peptide, or negative control peptide, as indicated.
  • T2 APC
  • T2 pulsed with tested peptide p210C or p210F
  • Figure 12 secretion of gamma IFN detected by ELISPOT of CD8 + T cells from an HLA A0201, chronic phase CML patient following 2 in vitro stimulations with p210C.
  • T cells were challenged with the following: media, APC T2, or T2 pulsed with p210C, corresponding native peptide, or negative control peptide.
  • Empty bars CD8+ cells plus media.
  • Diagonal bars CD8 + plus T2 pulsed with p210C.
  • Grey bars CD8 + plus T2 pulsed with irrelevant control peptide.
  • Figure 13 production of gamma IFN detected by ELISPOT of CD3 + cells of two healthy HLA A0201 donors after two in vitro stimulations.
  • T cells were challenged with the following: media, APC T2, or T2 pulsed with test peptide (b2a2 A3, A4 or A5); corresponding native peptide, or negative control peptide.
  • Dot bars CD8+ plus APC T2.
  • diagonal bars CD8+ plus T2 pulsed with tested peptide (b2a2 A3, A4 or A5).
  • black bars CD8+ plus T2 pulsed with native peptide (cross reactivity), grey bars: CD8+ plus T2 pulsed with irrelevant control peptide.
  • Figure 14 cytotoxicity assay with T cells isolated from a healthy HLA A0201 donor following 3 in vitro stimulations with ⁇ 210F.
  • Target cells used were T2 cell lines pulsed with the indicated peptides.
  • the Y-axis reflects the percent cytotoxicity, and the X-axis reflects the varied T cell/target ratio.
  • Open triangles T2 pulsed with irrelevant control peptide.
  • Figure 15 results of two cytotoxicity assays with T cells isolated from ahealthy HLA A0201 donor following five in vitro stimulations with b2a2 A3 peptide.
  • Target cells used were T2 cell line pulsed with the indicated peptides.
  • Y-axis reflects the percent cytotoxicity, and the X-axis reflects the different T cell/target ratio.
  • the present invention provides peptides, immunogenic compositions and vaccines comprising same, and methods of treating, reducing the incidence of, and inducing immune responses to a bcr-abl- expressing cancer, comprising same.
  • the present invention provides an isolated, mutated bcr-abl peptide, comprising: (a) a binding motif of a human leukocyte antigen (HLA) Class II molecule; and (b) a binding motif of an HLA class I molecule, having a point mutation in one or more anchor residues of the binding motif of an HLA class I molecule.
  • the bcr-abl peptide is 11-30 amino acids in length.
  • the bcr-abl peptide is 11-30 amino acids in length. In another embodiment, the bcr-abl peptide is 11 or more amino acids in length.
  • a bcr-abl peptide of the present invention is 16-22 amino acids (AA) in length. In another embodiment, the length is 16-22 AA. In another embodiment, the length is 19 AA. In another embodiment, the bcr-abl peptide is 15-23 AA in length. In another embodiment, the length is 15-24 AA. In another embodiment, the length is 15-25 AA. In another embodiment, the length is 15-26 AA. In another embodiment, the length is 15-27 AA. In another embodiment, the length is 15-28 AA. In another embodiment, the length is 14-30 AA. In another embodiment, the length is 14-29 AA. In another embodiment, the length is 14-28 AA.
  • the length is 14-26 AA. In another embodiment, the length is 14-24 AA. In another embodiment, the length is 14-22 AA. In another embodiment, the length is 14-20 AA. In another embodiment, the length is 16-30 AA. In another embodiment, the length is 16-28 AA. In another embodiment, the length is 16-26 AA. In another embodiment, the length is 16-24 AA. In another embodiment, the length is 16-22 AA. In another embodiment, the length is 18-30 AA. In another embodiment, the length is 18-28 AA. In another embodiment, the length is 18-26 AA. In another embodiment, the length is 18-24 AA. In another embodiment, the length is 18-22 AA. In another embodiment, the length is 18-20 AA.
  • the length is 20-30 AA. In another embodiment, the length is 20-28 AA. In another embodiment, the length is 20-26 AA. In another embodiment, the length is 20-24 AA. In another embodiment, the length is 22-30 AA. In another embodiment, the length is 22-28 AA. In another embodiment, the length is 22-26 AA. In another embodiment, the length is 24-30 AA. In another embodiment, the length is 24-28 AA. In another embodiment, the length is 24-26 AA.
  • a bcr-abl peptide of methods and compositions of the present invention is longer than the minimum length for binding to an HLA class II molecule, which is, in another embodiment, about 12 AA.
  • increasing the length of the HLA class II- binding bcr-abl peptide enables binding to more than one HLA class II molecule.
  • increasing the length enables binding to an HLA class II molecule whose binding motif is not known.
  • increasing the length enables binding to an HLA class I molecule.
  • the binding motif of the HLA class I molecule is known.
  • the binding motif of the HLA class I molecule is not known.
  • the "point mutation,” in another embodiment, indicates that the fragment is mutated with respect to the native sequence of the protein, thus creating the HLA class I molecule binding motif. In another embodiment, the "point mutation” strengthens the binding capacity of an HLA class I molecule binding motif present in the native sequence.
  • the point mutation is in 1-3 anchor residues of the HLA class I molecule binding motif. In another embodiment, the point mutation is in 1 anchor residue of the HLA class I molecule binding motif. In another embodiment, the point mutation is in 2 anchor residues of the HLA class I molecule binding motif. In another embodiment, the point mutation is in 1-2 anchor residues of the HLA class I molecule binding motif. In another embodiment, the point mutation is in 2- 3 anchor residues of the HLA class I molecule binding motif. In another embodiment, the point mutation is in 1-4 anchor residues of the HLA class I molecule binding motif. Each possibility represents a separate embodiment of the present invention.
  • Peptide in another embodiment of methods and compositions of the present invention, refers to a compound of two or more subunit AA connected by peptide bonds.
  • the term is used hereinbelow to refer to peptides (e.g. peptides of the present invention).
  • the peptide comprises an AA analogue.
  • the AA analogue is one of those enumerated below.
  • the peptide is a peptidomimetic.
  • the subunits are, in another embodiment, linked by peptide bonds.
  • the subunit is linked by another type of bond, e.g. ester, ether, etc .
  • a peptide of the present invention is one of the types of peptidomimetics enumerated below. Each possibility represents a separate embodiment of the present invention.
  • HLA molecules known in another embodiment as major histocompatibility complex (MHC) molecules, bind peptides and present them to immune cells.
  • MHC major histocompatibility complex
  • the immunogenicity of a peptide is partially determined by its affinity for HLA molecules.
  • HLA class I molecules interact with CD8 molecules, which are generally present on cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • HLA class II molecules interact with CD4 molecules, which are generally present on helper T lymphocytes.
  • a peptide of the present invention is immunogenic.
  • the term "immunogenic" refers to an ability to stimulate, elicit or participate in an immune response.
  • the immune response elicited is a cell-mediated immune response.
  • the immune response is a combination of cell-mediated and humoral responses.
  • T cells that bind to the MHC molecule-peptide complex become activated and induced to proliferate and lyse cells expressing a protein comprising the peptide.
  • T cells are typically initially activated by "professional" antigen presenting cells ("APC"; e.g. dendritic cells, monocytes, and macrophages), which present costimulatory molecules that encourage T cell activation as opposed to anergy or apoptosis.
  • APC antigen presenting cells
  • the response is heteroclitic, as described herein, such that the CTL lyses a cell expressing a protein which has an AA sequence homologous to a peptide of this invention, or a different peptide than that used to first stimulate the T cell.
  • an encounter of a T cell with a peptide of this invention induces its differentiation into an effector and/or memory T cell. Subsequent encounters between the effector or memory T cell and the same peptide, or, in another embodiment, with a related peptide of this invention, leads to a faster and more intense immune response. Such responses are gauged, in one embodiment, by measuring the degree of proliferation of the T cell population exposed to the peptide. In another embodiment, such responses are gauged by any of the methods enumerated hereinbelow.
  • the subject is exposed to a peptide, or a composition/cell population comprising a peptide of this invention, which differs from the native protein expressed, wherein subsequently a host immune response cross-reactive with the native protein/antigen develops.
  • peptides, vaccines, and compositions of this invention stimulate an immune response that results in lysis of a tumor cell.
  • the HLA class I molecule binding motif of a peptide of the present invention is contained within the HLA class II molecule binding motif of the peptide.
  • the HLA class I molecule binding motif overlaps with the HLA class 11 molecule binding motif.
  • the HLA class I molecule binding motif does not overlap with the HLA class II molecule binding motif.
  • the HLA class ⁇ molecule whose binding motif is contained in a peptide of the present invention is, in another embodiment, an HLA-DR molecule.
  • the HLA class II molecule is an HLA-DP molecule.
  • the HLA class II molecule is an HLA-DQ molecule.
  • the HLA class II molecule is an HLA-DRB molecule. In another embodiment, the HLA class II molecule is DRBlOl. 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 Il molecule is DRB701. In another embodiment, the HLA class II molecule is DRBIlOl. In another embodiment, the HLA class II molecule is DRB1501. In another embodiment, the HLA class II molecule is any other HLA-DRB molecule known in the art. In another embodiment, the HLA class II molecule is an HLA-DRA molecule.
  • the HLA class II molecule is an HLA-DQAl molecule. In another embodiment, the HLA class II molecule is an HLA-DQB 1 molecule. In another embodiment, the HLA class II molecule is an HLA-DPA 1 molecule. In another embodiment, the HLA class II molecule is an HLA-DPBl molecule. In another embodiment, the HLA class II molecule is an HLA-DMA molecule. In another embodiment, the HLA class TI 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 Il-molecule known in the art.
  • a peptide of the present invention binds to 2 distinct HLA class II molecules. In another embodiment, the peptide binds to three distinct HLA class II molecules. In another embodiment, the peptide binds to four distinct HLA class II molecules. In another embodiment, the peptide binds to five distinct HLA class II molecules. In another embodiment, the peptide binds to six distinct HLA class II molecules. In another embodiment, the peptide binds to more than six distinct HLA class II molecules.
  • the HLA class II molecules that are bound by a peptide of the present invention are encoded by two or more distinct alleles at a given HLA class II locus.
  • the HLA class II molecules are encoded by three distinct alleles at a locus.
  • the HLA class II molecules are encoded by four distinct alleles at a locus.
  • the HLA class II molecules are encoded by five distinct alleles at a locus.
  • the HLA class II molecules are encoded by six distinct alleles at a locus.
  • the HLA class II molecules are encoded by more than six distinct alleles at a locus.
  • the HLA class II molecules bound by the peptide are encoded by HLA class ⁇ genes at two distinct loci.
  • the HLA class II molecules are encoded by HLA class II genes at 2 or more distinct loci.
  • the HLA class II molecules are encoded by HLA class II genes at 3 distinct loci.
  • the HLA class II molecules are encoded by HLA class II genes at 3 or more distinct loci.
  • the HLA class II molecules are encoded by HLA class II genes at 4 distinct loci.
  • the HLA class II molecules are encoded by HLA class II genes at 4 or more distinct loci.
  • the HLA class II molecules are encoded by HLA class II genes at 5 distinct loci.
  • the HLA class II molecules are encoded by HLA class H genes at 5 or more distinct loci. In another embodiment, the HLA class II molecules are encoded by HLA class II genes at 6 distinct loci. In another embodiment, the HLA class II molecules are encoded by HLA class II genes at 6 or more distinct loci. In another embodiment, the HLA class II molecules are encoded by HLA class II genes at more than 6 distinct loci. Each possibility represents a separate embodiment of the present invention.
  • a peptide of the present invention binds to 2 distinct HLA-DRB molecules. In another embodiment, the peptide binds to three distinct HLA-DRB molecules. In another embodiment, the peptide binds to four distinct HLA-DRB molecules. In another embodiment, the peptide binds to five distinct HLA-DRB molecules. In another embodiment, the peptide binds to six distinct HLA-DRB molecules. In another embodiment, the peptide binds to more than six distinct HLA-DRB molecules.
  • a peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by three distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by four distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by five distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by six distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by more than six distinct HLA-DRB alleles. Each possibility represents a separate embodiment of the present invention.
  • a peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles selected from DRB 101, DRB 301 , DRB 401 , DRB 701, DRB 1101, and DRB 1501.
  • the peptide binds to HLA-DRB molecules encoded by 3 distinct HLA-DRB alleles selected from DRB 101, DRB 301 , DRB 401 , DRB 701, DRB 1101 , and DRB 1501.
  • the peptide binds to HLA-DRB molecules encoded by 4 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the peptide binds to HLA-DRB molecules encoded by 5 distinct HLA-DRB alleles selected from DRB 101, DRB 301 , DRB 401, DRB 701, DRB 1 101, and DRB 1501. In another embodiment, the peptide binds to HLA-DRB molecules encoded by each of the following HLA-DRB alleles: DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. Each possibility represents a separate embodiment of the present invention.
  • HLA class II molecule Each of the above HLA class II molecule, types, classes, and combinations thereof represents a separate embodiment of the present invention.
  • the HLA class I molecule whose binding motif is contained in a peptide of the present invention is, in another embodiment, an HLA-A molecule.
  • the HLA class I molecule is an HLA-B molecule.
  • the HLA class I molecule is an HLA-C molecule.
  • the HLA class I molecule is an HLA-A0201 molecule.
  • the molecule is HLA Al .
  • the HLA class I molecule is HLA A2.
  • the HLA class I molecule is HLA A2.1.
  • the HLA class I molecule is HLA A3.
  • the HLA class I molecule is HLA A3.2.
  • the HLA class I molecule is HLA Al 1. In another embodiment, the HLA class I molecule is HLA A24. In another embodiment, the HLA class I molecule is HLA B7. In another embodiment, the HLA class I molecule is HLA B27. In another embodiment, the HLA class I molecule is HLA B8. Each possibility represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention binds to a superfamily of HLA class I molecules.
  • the superfamily is the A2 superfamily.
  • the superfamily is the A3 superfamily.
  • the superfamily is the A24 superfamily.
  • the superfamily is the B7 superfamily.
  • the superfamily is the B27 superfamily.
  • the superfamily is the B44 superfamily.
  • the superfamily is the Cl superfamily. Tn another embodiment, the superfamily is the C4 superfamily.
  • the superfamily is any other superfamily known in the art. Each possibility represents a separate embodiment of the present invention.
  • an HLA class I molecule binding motif of a peptide of the present invention exhibits an increased affinity for the HLA class I molecule.
  • the point mutation increases the affinity of the mutated peptide for the HLA class I molecule.
  • the increase in affinity is relative to the affinity (for the same HLA class I molecule) of the unmutated protein fragment wherefrom the mutated peptide was derived.
  • a peptide of the present invention retains ability to bind multiple HLA class ⁇ molecules, as exhibited by the isolated unmutated protein fragment wherefrom the peptide of the present invention was derived.
  • the bcr-abl protein of methods and compositions of the present invention can be any bcr-abl protein known in the art.
  • the bcr-abl protein comprises one of the sequences set forth a GenBank sequence entry having one of the following Accession Numbers: X02596, NM_004327, X02596, U07000, Y00661, X06418, NM_005157, NM_007313, U07563, M15025, BAB62851, AAL05889, AAL99544, CAA10377, CAA10376, AAD04633, M14752, M14753, AAA35592, AAA35594, AAA87612, AAA88013, 1314255A, AAF61858, AAA35596, AAF89176, AAD04633.
  • the bcr-abl protein has one of the sequences set forth in a GenBank sequence entry having one of the following Accession Numbers: X02596, NM_004327, X02596, U07000, Y00661, X06418, NM_005157, NM_007313, U07563, M15025, BAB62851, AAL05889, AAL99544, CAA10377, CAA10376, AAD04633, M14752, M14753, AAA35592, AAA35594, AAA87612, AAA88013, 1314255A, AAF61858, AAA35596, AAF89176, AAD04633.
  • the bcr-abl protein has any other bcr-abl sequence known in the art.
  • a peptide of methods and compositions of the present invention is derived from a fragment of a bcr-abl protein.
  • the process of derivation comprises introduction of the point mutation in the anchor residues of the HLA class I molecule binding motif.
  • the process of derivation consists of introduction of the point mutation in the anchor residues of the HLA class I molecule binding motif.
  • a peptide of the present invention differs from the corresponding fragment of a antigenic protein only by the point mutation in the HLA class I molecule binding motif anchor residue.
  • an HLA class I molecule binding motif of a peptide of the present invention differs from the corresponding wild-type protein sequence only by the point mutation in the anchor residue.
  • the process of derivation of a peptide of the present invention further comprises one or more modifications of an amino acid (AA) to an AA analogue.
  • the process of derivation further comprises a modification of one or more peptide bond connecting two or more of the AA.
  • the AA analogue or peptide bond modification is one of the AA analogues or peptide bond modifications enumerated below. Each possibility represents a separate embodiment of the present invention.
  • the unmutated fragment of a bcr-abl protein wherefrom a peptide of the present invention is derived has the sequence IVHSATGFKQSSKALQRPVASDFEP (SEQ ID NO: 62) and VHSIPLTINKEEALQRPVASDFE (SEQ ID No: 63).
  • the unmutated bcr-abl fragment is any other bcr-abl fragment that contains an HLA class II molecule binding motif.
  • the unmutated bcr-abl fragment is any other bcr-abl fragment that contains an HLA-DR molecule binding motif.
  • the unmutated bcr-abl fragment contains multiple HLA-DR molecule binding motifs. In another embodiment, the unmutated bcr-abl fragment is any other bcr-abl fragment that contains an HLA-DRB molecule binding motif. In another embodiment, the unmutated bcr-abl fragment contains multiple HLA-DRB molecule binding motifs.
  • a peptide of the present invention retains the ability to bind an HLA class II molecule, as exhibited by the unmutated protein fragment wherefrom the peptide was derived. In another embodiment, a peptide of the present invention retains ability to bind multiple HLA class II molecules, as exhibited by the unmutated protein fragment.
  • Each possibility represents a separate embodiment of the present invention.
  • the HLA class I molecule binding motif contained in a peptide of the present invention in another embodiment, has the sequence YLKALQRPV (SEQ ID No: 45). In another embodiment, the HLA class I molecule binding motif has the sequence KQSSKALQV (SEQ ID No: 47). In another embodiment, the HLA class 1 molecule binding motif has the sequence KLSSKALQV (SEQ ID No: 48). In another embodiment, the HLA class I molecule binding motif has the sequence KLLQRPVAV (SEQ ID No: 50). In another embodiment, the HLA class I molecule binding motif has the sequence TLFKQSSKV (SEQ ID No: 52).
  • the HLA class I molecule binding motif has the sequence YLFKQSSKV (SEQ ID No: 53). In another embodiment, the HLA class I molecule binding motif has the sequence LLINKEEAL (SEQ ID No: 55). In another embodiment, the HLA class I molecule binding motif has the sequence LTINKVEAL (SEQ ID No: 56). In another embodiment, the HLA class 1 molecule binding motif has the sequence YLlNKEEAL (SEQ ID No: 57). In another embodiment, the HLA class 1 molecule binding motif has the sequence YLINKEEAV (SEQ ID No: 58). In another embodiment, the HLA class I molecule binding motif has the sequence YLINKVEAL (SEQ ID No: 59). In another embodiment, the HLA class I molecule binding motif is any other HLA class I motif known in the art. In another embodiment, the motif is any other HLA-A motif known in the art. Each possibility represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention binds with high affinity to the HLA class I molecule whose binding motif is contained therein.
  • the HLA class I molecule is any HLA class I molecule enumerated herein.
  • the peptide binds to the HLA class I molecule with- medium affinity.
  • the peptide binds to the HLA class I molecule with significant affinity.
  • the peptide binds to the HLA class I molecule with measurable affinity.
  • the peptide exhibits stable binding to the HLA class I molecule.
  • a peptide of methods and compositions of the present invention binds with high affinity to the HLA class II molecule whose binding motif is contained therein.
  • the HLA class II molecule is any HLA class II molecule enumerated herein.
  • the peptide binds with high affinity to more than 1 HLA class II molecules, (give example)
  • the peptide binds to the HLA class II molecule with medium affinity.
  • the peptide binds with medium affinity to more than 1 HLA class II molecules.
  • the peptide binds to the HLA class II molecule with significant affinity.
  • the peptide binds with significant affinity to more than 1 HLA class II molecules. In another embodiment, the peptide binds to the HLA class II molecule with measurable affinity. In another embodiment, the peptide binds with measurable affinity to more than 1 HLA class II molecules. In another embodiment, the peptide exhibits stable binding to the HLA class II molecule. In another embodiment, the peptide exhibits stable binding to more than 1 HLA class II molecules. Each possibility represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention binds to both an HLA class I molecule and an HLA class II molecule with significant affinity.
  • the peptide binds to both an HLA class I molecule and an HLA class II molecule with high affinity.
  • the peptide binds to both an HLA class I molecule and an HLA class II molecule with medium affinity.
  • the peptide binds to both an HLA class I molecule and an HLA class II molecule with measurable affinity.
  • “Fragment,” in another embodiment, refers to a peptide of 11 or more AA in length.
  • a peptide fragment of the present invention is 16 or more AA long.
  • the fragment is 12 or more AA long.
  • the fragment is 13 or more AA.
  • the fragment is 14 or more AA.
  • the fragment is 15 or more AA.
  • the fragment is 17 or more AA.
  • the fragment is 18 or more AA.
  • Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a composition comprising an isolated peptide of the invention in combination with at least one additional bcr-abl peptide.
  • a composition comprising at least two different isolated peptides of the present invention is provided.
  • a composition comprising at least three or at least four different isolated peptides of the present invention is provided. Each possibility represents a separate embodiment of the present invention.
  • the composition of the present invention is a vaccine.
  • the additional bcr-abl peptide in another embodiment, has the sequence IVHSATGFKQSSKALQRPVASDFEP; SEQ ID NO: 62) are utilized.
  • the additional bcr-abl peptide comprises the sequence IVHSATGFKQSSKALQRPVASDFEP.
  • the additional bcr-abl peptide has the sequence VHSIPLTINKEEALQRPVASDFE
  • the additional bcr-abl peptide comprises the sequence VHSIPLTINKEEALQRPVASDFE.
  • the additional bcr-abl peptide has a sequence selected from the sequences set forth in SEQ ID No: 44-59.
  • the additional bcr-abl peptide is a heteroclitic peptide having a sequence selected from SEQ ID No: 44-59.
  • the additional bcr-abl peptide is a wild-type peptide having a sequence selected from SEQ ID No: 44-59.
  • the additional bcr-abl peptide is another heteroclitic bcr-abl peptide.
  • the additional bcr-abl peptide is another wild-type bcr-abl peptide. In another embodiment, the additional bcr-abl peptide is any other bcr-abl peptide known in the art. Each possibility represents a separate embodiment of the present invention.
  • the additional bcr-abl peptide has a length of 8-22 AA. In another embodiment, the additional bcr-abl peptide has a length of 8-30 AA. In another embodiment, the additional bcr-abl peptide has a length of 11 -30 AA. In another embodiment, the length is 16-22 AA. In another embodiment, the length is 19 AA. In another embodiment, the peptide is 15-23 AA in length. In another embodiment, the length is 15-24 AA. In another embodiment, the length is 15-25 AA. In another embodiment, the length is 15-26 AA. In another embodiment, the length is 15-27 AA. In another embodiment, the length is 15-28 AA.
  • the length is 14-30 AA. In another embodiment, the length is 14-29 AA. In another embodiment, the length is 14-28 AA. In another embodiment, the length is 14-26 AA. In another embodiment, the length is 14-24 AA. In another embodiment, the length is 14-22 AA. In another embodiment, the length is 14-20 AA. In another embodiment, the length is 16-30 AA. In another embodiment, the length is 16-28 AA. In another embodiment, the length is 16-26 AA. In another embodiment, the length is 16-24 AA. In another embodiment, the length is 16-22 AA. In another embodiment, the length is 18-30 AA. In another embodiment, the length is 18-28 AA. In another embodiment, the length is 18-26 AA.
  • the length is 18-24 AA. In another embodiment, the length is 18-22 AA. In another embodiment, the length is 18-20 AA. In another embodiment, the length is 20-30 AA. In another embodiment, the length is 20-28 AA. In another embodiment, the length is 20-26 AA. In another embodiment, the length is 20-24 AA. In another embodiment, the length is 22-30 AA. In another embodiment, the length is 22-28 AA. In another embodiment, the length is 22-26 AA. In another embodiment, the length is 24-30 AA. In another embodiment, the length is 24-28 AA. In another embodiment, the length is 24-26 AA. In another embodiment, the length is 24-26 AA.
  • the additional bcr-abl peptide has any other length.
  • Each possibility represents a separate embodiment of the present invention.
  • affinity refers to the concentration of peptide necessary for inhibiting binding of a standard peptide to the indicated MHC molecule by fifty percent.
  • “high affinity” refers to an affinity is such that a concentration of about 100 nanomolar (nM) or less of the peptide is required for inhibition of binding of a standard peptide.
  • the binding affinity is 80 nM.
  • the binding affinity is 60 nM.
  • the binding affinity is 40 nM.
  • the binding affinity is 30 nM.
  • the binding affinity is 20 nM.
  • the binding affinity is 15 nM.
  • the binding affinity is 10 nM.
  • 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.
  • high affinity refers to a binding affinity of 0.5-100 nM.
  • the binding affinity is 1-100 nM. In another embodiment, the binding affinity is 1.5-200 nM. In another embodiment, the binding affinity is 2-100 nM. In another embodiment, the binding affinity is 3-100 nM. In another embodiment, the binding affinity is 4-100 nM. In another embodiment, the binding affinity is 6-100 nM. In another embodiment, the binding affinity is 10-100 nM. In another embodiment, the binding affinity is 30-100 nM. In another embodiment, the binding affinity is 3-80 nM. In another embodiment, the binding affinity is 4-60 nM. In another embodiment, the binding affinity is 5-50 nM.
  • the binding affinity is 6-50 nM. In another embodiment, the binding affinity is 8-50 nM. In another embodiment, the binding affinity is 10-50 nM. In another embodiment, the binding affinity is 20-50 nM. In another embodiment, the binding affinity is 6-40 nM. In another embodiment, the binding affinity is 8-30 nM. In another embodiment, the binding affinity is 10-25 nM. In another embodiment, the binding affinity is 15-25 nM.
  • Each affinity and range of affinities represents a separate embodiment of the present invention.
  • “medium affinity” refers to a binding affinity of 100-500 nM. In another embodiment, the binding affinity is 100-300 nM. In another embodiment, the binding affinity is 100-200 nM. In another embodiment, the binding affinity is 50-100 nM. In another embodiment, the binding affinity is 50-80 nM. In another embodiment, the binding affinity is 50-60 nM.
  • Each affinity and range of affinities represents a separate embodiment of the present invention.
  • Signal affinity refers, in another embodiment, to sufficient affinity to mediate recognition of a target cell by a T cell carrying a T cell receptor (TCR) that recognizes the MHC molecule-peptide complex.
  • TCR T cell receptor
  • the term refers to sufficient affinity to mediate recognition of a cancer cell by a T cell carrying a TCR that recognizes the MHC molecule-peptide complex.
  • the term refers to sufficient affinity to mediate activation of a naive
  • the term refers to sufficient affinity to mediate activation of a naive T cell by an APC presenting the peptide. In another embodiment, the term refers to sufficient affinity to mediate re-activation of a memory T cell by a dendritic cell presenting the peptide. In another embodiment, the term refers to sufficient affinity to mediate re-activation of a memory T cell by an APC presenting the peptide. In another embodiment, the term refers to sufficient affinity to mediate re-activation of a memory T cell by a somatic cell presenting the peptide. Each possibility represents a separate embodiment of the present invention. 7 008747
  • “Measurable affinity” refers, in another embodiment, to sufficient affinity to be measurable by an immunological assay.
  • the immunological assay is any assay enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention binds to a superfamily of HLA molecules.
  • Superfamilies of HLA molecules share very similar or identical binding motifs.
  • the superfamily is a HLA class I superfamily.
  • the superfamily is a HLA class II superfamily. Each possibility represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention is heteroclitic.
  • Heteroclitic refers, in one embodiment, to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived (e.g. the peptide not containing the anchor residue mutations).
  • original peptide refers to a fragment of bcr-abl.
  • WTl 122Al having the sequence SGQAYMFPNAPYLPSCLES (SEQ ID NO: 41), was generated from the wild-type WTl peptide SGQARMFPNAPYLPSCLES (SEQ ID NO: 39) by mutation of residue 5 to arginine (Example 6).
  • the mutation introduced the CD8 + heteroclitic WTlAl peptide YMFPNAPYL (SEQ ID No: 6) into the WT 1 peptide.
  • heteroclitic refers to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide.
  • a “heteroclitic” immune response refers to an immune response that recognizes the original peptide from which the improved peptide was derived (e.g. the peptide not containing the anchor residue mutations).
  • a "heteroclitic" immune response refers to an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide.
  • a heteroclitic peptide of the present invention induces an immune response that is increased at least 2-fold relative to the peptide from which the heteroclitic peptide was derived ("native peptide").
  • the increase is 3-fold relative to the native peptide.
  • the increase is 5-fold relative to the native peptide.
  • the increase is 7-fold relative to the native peptide.
  • the increase is 10-fold relative to the native peptide.
  • the increase is 15-fold relative to the native peptide.
  • the increase is 20-fold relative to the native peptide.
  • the increase is 30-fold relative to the native peptide.
  • the increase is 50-fold relative to the native peptide. In another embodiment, the increase is 100-fold relative to the native peptide. In another embodiment, the increase is 150-fold relative to the native peptide. In another embodiment, the increase is 200-fold relative to the native peptide. In another embodiment, the increase is 300-fold relative to the native peptide. In another embodiment, the increase is 500-fold relative to the native peptide. In another embodiment, the increase is 1000-fold relative to the native peptide. In another embodiment, the increase is more than 1000-fold relative to the native peptide. Each possibility represents a separate embodiment of the present invention.
  • anchor motifs or “anchor residues” refers, in one embodiment, to one or a set of preferred residues at particular positions in an HLA-binding sequence. For example, residues at positions 1 , 2, 3, 6, and 9 are used as anchor residues in the Examples herein.
  • the HLA-binding sequence is an HLA class Il-binding sequence.
  • the HLA-binding sequence is an HLA class I-binding sequence.
  • the positions corresponding to the anchor motifs are those that play a significant role in binding the HLA molecule.
  • the anchor residue is a primary anchor motif.
  • the anchor residue is a secondary anchor motif. Each possibility represents a separate embodiment of the present invention.
  • anchor residues are residues in positions 1 , 3, 6, and 9 of the HLA class I binding motif.
  • the term refers to positions 1, 2, 6, and 9 of the HLA class I binding motif.
  • the term refers to positions 1 , 6, and 9 of the HLA class I binding motif.
  • the term refers to positions 1 , 2, and 9 of the HLA class I binding motif.
  • the term refers to positions 1, 3, and 9 of the HLA class I binding motif.
  • the term refers to positions 2 and 9 of the HLA class I binding motif.
  • the term refers to positions 6 and 9 of the HLA class I binding motif.
  • MHC class II epitopes are well known in the art.
  • the MHC class II epitope is predicted using TEPITOPE (Meister GE, Roberts CG et al, Vaccine 1995 13: 581-91).
  • the MHC class II epitope is identified using EpiMatrix (De Groot AS, Jesdale BM et al, AIDS Res Hum Retroviruses 1997 13: 529-31 ).
  • the MHC class II epitope is identified using EpiMatrix (De Groot AS, Jesdale BM et al, AIDS Res Hum Retroviruses 1997 13: 529-31 ).
  • MHC class II epitope is identified using the Predict Method (Yu K, Petrovsky N et al, MoI Med.2002 8: 137-48). In another embodiment, the MHC class II epitope is identified using the SYFPEITHI 47
  • SYFPEITHI is a database comprising more than 4500 peptide sequences known to bind class I and class II MHC molecules. SYFPEITHI provides a score based on the presence of certain amino acids in certain positions along the MHC-binding groove. Ideal amino acid anchors are valued at 10 points, unusual anchors are worth 6-8 points, auxiliary anchors are worth 4-6 points, preferred residues are worth 1-4 points; negative amino acid effect on the binding score between —1 and -3. The maximum score for HLA-A*0201 is 36.
  • the MHC class II epitope is identified using Rankpep.
  • Rankpep uses position specific scoring matrices (PSSMs) or profiles from sets of aligned peptides known to bind to a given MHC molecule as the predictor of MHC-peptide binding.
  • PSSMs position specific scoring matrices
  • Rankpep includes information on the score of the peptide and the % optimum or percentile score of the predicted peptide relative to that of a consensus sequence that yields the maximum score, with the selected profile.
  • Rankpep includes a selection of 102 and 80 PSSMs for the prediction of peptide binding to MHC I and MHC II molecules, respectively.
  • PSSMs for the prediction of peptide binders of different sizes are usually available for each MHC I molecule.
  • the MHC class II epitope is identified using SVMHC (Donnes P, Elofsson A. Prediction of MHC class I binding peptides, using SVMHC. BMC Bioinformatics.2002 Sep 11 ;3:25).
  • the MHC class II.epitope is identified using any other method known in the art. The above methods are utilized, in another embodiment, to identify MHC class II binding will be perturbed by introduction of an MHC class I anchor residue mutation into the sequence. Each possibility represents a separate embodiment of the present invention.
  • MHC class I epitopes are well known in the art.
  • the MHC class I epitope is predicted using BIMAS software (Example 1 ).
  • the MHC class I epitope is identified using SYFPEITHI.
  • the MHC class I epitope is identified using SVMHC (Donnes P, Elofsson A. Prediction of MHC class I binding peptides, using SVMHC. BMC Bioinformatics. 2002 Sep 11 ;3:25).
  • the MHC class I epitope is identified using NetMHC-2.0 (Sensitive quantitative predictions of peptide-MHC binding by a 'Query by Committee 1 artificial neural network approach.
  • the MHC class I epitope is identified using any other method known in the art. The above methods are utilized, in another embodiment, to identify MHC class I epitopes that can be created by introduction of an anchor residue mutation into the bcr-abl sequence. Each possibility represents a separate embodiment of the present invention.
  • the mutation that enhances MHC binding is in the residue at position 1 of the HLA class I binding motif.
  • the residue is changed to tyrosine. In another embodiment, the residue is changed to glycine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to phenylalanine. In another embodiment, the residue is changed to any other residue known in the art. In another embodiment, a substitution in position 1 (e.g. to tyrosine) stabilizes the binding of the position 2 anchor residue.
  • the mutation is in position 2 of the HLA class I binding motif.
  • the residue is changed to leucine.
  • the residue is changed to valine.
  • the residue is changed to isoleucine.
  • the residue is changed to methionine.
  • the residue is changed to any other residue known in the art.
  • the mutation is in position 6 of the HLA class I binding motif.
  • the residue is changed to valine.
  • the residue is changed to cysteine.
  • the residue is changed to glutamine.
  • the residue is changed to histidine.
  • the residue is changed to any other residue known in the art.
  • the mutation is in position 9 of the HLA class I binding motif.
  • the mutation changes the residue at the C-terminal position thereof.
  • the residue is changed to valine.
  • the residue is changed to threonine.
  • the residue is changed to isoleucine.
  • the residue is changed to leucine.
  • the residue is changed to alanine.
  • the residue is changed to cysteine.
  • the residue is changed to any other residue known in the art.
  • the point mutation is in a primary anchor residue.
  • the HLA class I primary anchor residues are positions 2 and 9.
  • the point mutation is in a secondary anchor residue.
  • the HLA class I secondary anchor residues are positions 1, 3, 6, and 7.
  • the point mutation is in a position selected from positions 4, 5, and 8. Each possibility represents a separate embodiment of the present invention.
  • the point mutation is in one or more residues in positions selected from positions 1, 3, 6, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in one or more residues in positions selected from positions 1, 2, 6, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in one or more residues in positions selected from positions 1 , 6, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in one or more residues in positions selected from positions 1 , 2, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in one or more residues in positions selected from positions 1, 3, and 9 of the HLA class I binding motif.
  • the point mutation is in one or more residues in positions selected from positions 2 and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in one or more residues in positions selected from positions 6 and 9 of the HLA class I binding motif.
  • the mutation is in the 4 position of the HLA class I binding motif. In another embodiment, the mutation is in the 5 position of the HLA class I binding motif. In another embodiment, the mutation is in the 7 position of the HLA class I binding motif. In another embodiment, the mutation is in the 8 position of the HLA class I binding motif.
  • peptides of the present invention exhibited significant ability to stimulate both CD4 + and CD8 + T cells (Examples). Moreover, the peptides exhibited enhanced immuno- stimulating activity, relative to the native peptides from which they were derived.
  • the HLA class II binding site in a peptide of the present invention is created or improved by mutation of an HLA class II motif anchor residue.
  • the anchor residue that is modified is in the Pl position (e.g. a position corresponding to F263 of the CII(259-273) peptide).
  • the anchor residue is in the P2 position (e.g. a position corresponding to K264 of the CII(259-273) peptide).
  • the anchor residue is in the P6 position.
  • the anchor residue is in the P9 position.
  • the anchor residue is selected from the Pl, P2, P6, and P9 positions.
  • the HLA class II motif anchor residue is the P3 position. In another embodiment, the HLA class II motif anchor residue is the P4 position. In another embodiment, the HLA class II motif anchor residue is the P5 position. In another embodiment, the HLA class II motif anchor residue is the P6 position. In another embodiment, the HLA class II motif anchor residue is the P8 position. In another embodiment, the HLA class II motif anchor residue is the PlO position. In another embodiment, the HLA class II motif anchor residue is the PI l position. In another embodiment, the HLA class II motif anchor residue is the Pl 2 position. In another embodiment, the HLA class II motif anchor residue is the Pl 3 position.
  • the anchor residue is any other anchor residue of an HLA class II molecule that is known in the art.
  • residues other than Pl, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding.
  • a peptide of the present invention is homologous to a peptide enumerated in the Examples.
  • the terms "homology,” “homologous,” etc, when in reference to any protein or peptide, refer, in one embodiment, to a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art.
  • Homology is, in one embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art.
  • computer algorithm analysis of nucleic acid sequence homology includes the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.
  • homology refers to identity to a sequence of a peptide of the present invention of greater than 70%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 72%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 75%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 78%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 80%.
  • homology refers to identity to a sequence of a peptide of the present invention of greater than 82%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 83% . 08747
  • “homology” refers to identity to a sequence of a peptide of the present invention of greater than 85%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 87% . In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 88%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 90%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 92%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 93%.
  • “homology” refers to identity to a sequence of a peptide of the present invention of greater than 95%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 96%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 97%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 98%. In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of greater than 99% . In another embodiment, “homology” refers to identity to a sequence of a peptide of the present invention of 100%. Each possibility represents a separate embodiment of the present invention.
  • homology is determined is via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y).
  • methods of hybridization are carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide.
  • Hybridization conditions being, for example, overnight incubation at 42 0 C in a solution comprising: 10-20 % formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 X Denhardt's solution, 10 % dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • the present invention provides a composition comprising a peptide of this invention.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the composition further comprises an adjuvant.
  • the composition comprises two or more peptides of the present invention.
  • the composition further comprises any of the additives, compounds, or excipients set forth hereinbelow.
  • the adjuvant is QS21, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG or alum.
  • the carrier is any carrier enumerated herein.
  • the adjuvant is any adjuvant enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • this invention provides a vaccine comprising a peptide of the present invention.
  • the vaccine further comprises a carrier.
  • the vaccine further comprises an adjuvant.
  • the vaccine further comprises a combination of a carrier and an adjuvant.
  • the vaccine is an antigen presenting cell (APC) associated with a mixture of peptides of the present invention.
  • APC antigen presenting cell
  • this invention provides an immunogenic composition comprising a peptide of the present invention.
  • the immunogenic composition further comprises a carrier.
  • the immunogenic composition further comprises an adjuvant.
  • the immunogenic composition further comprises a combination of a carrier and an adjuvant.
  • the term "vaccine” refers to a material or composition that, when introduced into a subject, provides a prophylactic or therapeutic response for a particular disease, condition, or symptom of same.
  • this invention comprises peptide-based vaccines, wherein the peptide comprises any embodiment listed herein, including immunomodulating compounds such as cytokines, adjuvants, etc.
  • a vaccine of methods and compositions of the present invention further comprises an adjuvant.
  • the adjuvant is Montanide ISA 51.
  • Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier.
  • the adjuvant is GM-CSF.
  • Recombinant GM-CSF is a human protein grown, in one embodiment, in a yeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, APC, and dendritic cells and T cells.
  • the adjuvant is a cytokine. In another embodiment, the adjuvant is a growth factor. In another embodiment, the adjuvant is a cell population. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is Freund's incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. In another embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is BCG. In another embodiment, the adjuvant is alum. 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 vaccine comprises two of the above adjuvants. In another embodiment, the vaccine comprises more than two of the above adjuvants. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a cell comprising a peptide of the present invention.
  • the cell is an antigen-presenting cell (APC).
  • the present invention provides a vaccine comprising an APC of the present invention.
  • the present invention provides a nucleic acid molecule encoding a peptide of the present invention.
  • the present invention provides a vaccine comprising a nucleic acid molecule of the present invention.
  • the present invention provides a vector comprising a nucleic acid • molecule of the present invention.
  • the present invention provides a vaccine comprising a vector of the present invention.
  • a vaccine or composition of the present invention comprises any of the embodiments of peptides of the present invention and combinations thereof. Each possibility represents a separate embodiment of the present invention.
  • a composition of the present invention contains two peptides that are derived from the same protein fragment, each containing a different HLA class I heteroclitic peptide.
  • the two HLA class I heteroclitic peptides contain mutations in different HLA class I molecule anchor residues.
  • the two HLA class I heteroclitic peptides contain different mutations in the same anchor residue(s). Each possibility represents a separate embodiment of the present invention.
  • the peptides in a composition of the present invention bind to two distinct HLA class II molecules. In another embodiment, the peptides bind to three distinct HLA class II molecules. In another embodiment, the peptides bind to four distinct HLA class II molecules. In another embodiment, the peptides bind to five distinct HLA class II molecules. In another embodiment, the peptides bind to more than five distinct HLA class II molecules. In another embodiment, the peptides in the composition bind to the same HLA class H molecules.
  • each of the peptides in a composition of the present invention binds to a set of HLA class II molecules. In another embodiment, each of the peptides binds to a distinct 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 of the peptides bind to a distinct but overlapping set of HLA class II molecules. In another embodiment, two or more of the peptides bind to the same set of HLA class II molecules, while another of the peptides binds to a distinct set. In another embodiment, two or more of the peptides bind to an overlapping set of HLA class II molecules, while another of the peptides binds to a distinct set.
  • the peptides in a composition of the present invention bind to two distinct HLA class I molecules. In another embodiment, the peptides bind to three distinct HLA class I molecules. In another embodiment, the peptides bind to four distinct HLA class I molecules. In another embodiment, the peptides bind to five distinct HLA class I molecules. In another embodiment, the peptides bind to more than five distinct HLA class I molecules. In another embodiment, the peptides in the composition bind to the same HLA class I molecules.
  • each of the peptides in a composition of the present invention binds to a set of HLA class I molecules. In another embodiment, each of the peptides binds to a distinct set of HLA class I molecules. In another embodiment, the peptides in the composition bind to the same set of HLA class I molecules. In another embodiment, two of the peptides bind to a distinct but overlapping set of HLA class I molecules. In another embodiment, two or more of the peptides bind to the same set of HLA class I molecules, while another of the peptides binds to a distinct set. In another embodiment, two or more of the peptides bind to an overlapping set of HLA class I molecules, while another of the peptides binds to a distinct set.
  • a "set of HLA class ⁇ molecules" or “set of HLA class I molecules” refers to the HLA molecules encoded by different alleles at a particular locus.
  • the term refers to HLA molecules with a particular binding specificity.
  • the term refers to HLA molecules with a particular peptide consensus sequence.
  • the term refers to a superfamily of HLA class II molecules. Each possibility represents a separate embodiment of the present invention. 47
  • compositions and types of compositions represents a separate embodiment of the present invention.
  • any embodiments described herein, regarding peptides, vaccines and compositions of this invention can be employed in any of the methods of this invention.
  • Each combination of peptide, vaccine, or composition with a method represents an embodiment thereof.
  • the present invention provides a method of treating a subject with a bcr-abl-expressing cancer, the method comprising administering to the subject a peptide of the present invention, thereby treating a subject with a bcr-abl-expressing cancer.
  • the present invention provides a method of treating a subject with a bcr-abl-expressing cancer, the method comprising administering to the subject a vaccine of the present invention, thereby treating a subject with a bcr-abl-expressing cancer.
  • the present invention provides a method of treating a subject with a bcrrabl-expressing cancer, the method comprising administering to the subject an immunogenic composition of the present invention, thereby treating a subject with a bcr-abl- expressing cancer.
  • the present invention provides a method of suppressing or halting the progression of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject a peptide of the present invention, thereby suppressing or halting the progression of a bcr-abl- expressing cancer.
  • the present invention provides a method of suppressing or halting the progression of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject a vaccine of the present invention, thereby suppressing or halting the progression of a bcr-abl-expressing cancer.
  • the present invention provides a method of suppressing or halting the progression of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject an immunogenic composition of the present invention, thereby suppressing or halting the progression of a bcr-abl-expressing cancer
  • the present invention provides a method of reducing the incidence of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject a peptide of the present invention, thereby reducing the incidence of a bcr-abl-expressing cancer in a subject.
  • the present invention provides a method of reducing the incidence of a bcr-abl- expressing cancer in a subject, the method comprising administering to the subject a vaccine of the present invention, thereby reducing the incidence of a bcr-abl-expressing cancer in a subject.
  • the present invention provides a method of reducing the incidence of a bcr-abl- expressing cancer in a subject, the method comprising administering to the subject an immunogenic composition of the present invention, thereby reducing the incidence of abcr-abl-expressing cancer in a subject.
  • the present invention provides a method of reducing the incidence of relapse of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject a peptide of the present invention, thereby reducing the incidence of relapse of a bcr-abl-expressing cancer in a subject.
  • the present invention provides a method of reducing the incidence of relapse of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject a vaccine of the present invention, thereby reducing the incidence of relapse of a bcr-abl- expressing cancer in a subject.
  • the present invention provides a method of reducing the incidence of relapse of a bcr-abl-expressing cancer in a subject, the method comprising administering to the subject an immunogenic composition of the present invention, thereby reducing the incidence of relapse of a bcr-abl-expressing cancer in a subject
  • the present invention provides a method of overcoming a T cell tolerance of a subject to a bcr-abl-expressing cancer, the method comprising administering to the subject a peptide of the present invention, thereby overcoming a T cell tolerance to a bcr-abl- expressing cancer.
  • the present invention provides a method of overcoming a T cell tolerance of a subject to a bcr-abl-expressing cancer, the method comprising administering to the subject a vaccine of the present invention, thereby overcoming a T cell tolerance to a bcr-abl- expressing cancer.
  • the present invention provides a method of overcoming a T cell tolerance of a subject to a bcr-abl-expressing cancer, the method comprising administering to the subject an immunogenic composition of the present invention, thereby overcoming a T cell tolerance to a bcr-abl-expressing cancer
  • the present invention provides a method of treating a subject having a bcr-abl-expressing cancer, comprising (a) inducing in a donor formation and proliferation of human cytotoxic T lymphocytes (CTL) that recognize a malignant cell of the cancer by a method of the present invention; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer.
  • CTL cytotoxic T lymphocytes
  • the present invention provides a method of treating a subject having a bcr-abl-expressing cancer, comprising (a) inducing ex vivo formation and proliferation of human CTL that recognize a malignant cell of the cancer by a method of the present invention, wherein the human immune cells are obtained from a donor; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer.
  • the present invention provides a method of inducing formation and proliferation of bcr-abl-specif ⁇ c CTL, the method comprising contacting a lymphocyte population with a vaccine of the present invention, thereby inducing formation and proliferation of bcr-abl-specific CTL.
  • the present invention provides a method of inducing formation and proliferation of bcr-abl-specific CTL, the method comprising contacting a lymphocyte population with a peptide of the present invention, thereby inducing formation and proliferation of bcr-abl-specific CTL.
  • the present invention provides a method of inducing formation and proliferation of bcr-abl-specific CTL, the method comprising contacting a lymphocyte population with an immunogenic composition of the present invention, thereby inducing formation and proliferation of bcr-abl-specific CTL.
  • the target cell is a cell of a bcr-abl-expressing leukemia. In another embodiment, the target cell is a cell of a bcr-abl-expressing cancer.
  • the present invention provides a method of inducing in a subject formation and proliferation of bcr-abl-specific CTL, the method comprising contacting the subject with a vaccine of the present invention, thereby inducing in a subject formation and proliferation of bcr-abl-specif ⁇ c CTL.
  • the present invention provides a method of inducing in a subject formation and proliferation of bcr-abl-specif ⁇ c CTL, the method comprising contacting the subject with a peptide of the present invention, thereby inducing in a subject formation and proliferation of bcr-abl-specific CTL.
  • the present invention provides a method of inducing in a subject formation and proliferation of bcr-abl-specific CTL, the method comprising contacting the subject with an immunogenic composition of the present invention, thereby inducing in a subject formation and proliferation of bcr-abl-specific CTL.
  • the target cell is a cell of a bcr-abl-expressing leukemia. In another embodiment, the target cell is a cell of a bcr-abl- expressing cancer.
  • the present invention provides a method of inducing formation and proliferation of both (a) a CD8+ lymphocyte specific for abcr-abl protein; and (b) aCD4+ lymphocyte specific for the bcr-abl protein, the method comprising contacting a lymphocyte population with a vaccine of the present invention, thereby inducing formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein.
  • the present invention provides a method of inducing formation and proliferation of both (a) a CD8+ lymphocyte specific for abcr-abl protein; and (b) a CD4+ lymphocyte 08747
  • the method comprising contacting a lymphocyte population with a peptide of the present invention, thereby inducing formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein.
  • the present invention provides a method of inducing formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein, the method comprising contacting a lymphocyte population with a immunogenic composition of the present invention, thereby inducing formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein.
  • the present invention provides a method of inducing in a subject formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein, the method comprising administering to the subject with a vaccine of the present invention, thereby inducing in a subject formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein.
  • the present invention provides a method of inducing in a subject formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein, the method comprising administering to the subject with a peptide of the present invention, thereby inducing in a subject formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein.
  • the present invention provides a method of inducing in a subject formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein, the method comprising administering to the subject with a immunogenic composition of the present invention, thereby inducing in a subject formation and proliferation of both (a) a CD8+ lymphocyte specific for a bcr-abl protein; and (b) a CD4+ lymphocyte specific for the bcr-abl protein.
  • this invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against a bcr-abl- expressing cancer, the method comprising administering to the subject a peptide of the present invention, thereby generating a heteroclitic immune response.
  • this invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against a bcr-abl-expressing cancer, the method comprising administering to the subject a vaccine of the present invention, thereby generating a heteroclitic immune response.
  • this invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against a bcr-abl-expressing cancer, the method comprising administering to the subject an immunogenic composition of the present invention, thereby generating a heteroclitic immune response.
  • An immunogenic composition of the present invention comprises, in another embodiment, an APC associated with a peptide of the present invention.
  • the immunogenic composition comprises an APC associated with a mixture of peptides of the present invention.
  • the immunogenic composition consists of an APC associated with a peptide of the present invention.
  • the immunogenic composition consists of an APC associated with a mixture of peptides of the present invention.
  • a target cell of an immune response elicited by a method of the present invention presents the peptide of the present invention, or a corresponding bcr-abl fragment, on an HLA class I molecule.
  • the HLA molecule is an HLA class I molecule.
  • the HLA molecule is an HLA class II molecule.
  • the peptide or a fragment thereof is presented on both an HLA class I molecule and an HLA class II molecule.
  • the HLA class I molecule is any HLA class I subtype or HLA class I molecule known in the art.
  • the HLA class I molecule is any HLA class I subtype or HLA class I molecule enumerated herein.
  • the HLA class II molecule is any HLA class II subtype or HLA class II molecule known in the art.
  • the HLA class II molecule is any HLA class II subtype or HLA class II molecule enumerated herein.
  • the immune response against the peptide or fragment is a heteroclitic immune response. Each possibility represents a separate embodiment of the present invention.
  • the bcr-abl-expressing cancer is a bcr-abl-expressing leukemia.
  • the bcr-abl-expressing cancer is acute myeloid leukemia (AML).
  • the bcr-abl-expressing cancer is chronic myeloid leukemia (CML).
  • the bcr-abl-expressing cancer is acute lymphoblastic leukemia (ALL).
  • ALL acute lymphoblastic leukemia
  • the bcr-abl-expressing cancer is any other bcr-abl-expressing cancer known in the art. Each possibility represents a separate embodiment of the present invention. 47
  • multiple peptides of this invention are used to stimulate an immune response in methods of the present invention.
  • heteroclitic peptides that elicit antigen-specific CD8 + T cell responses can be created using methods of the present invention (Examples 1-2 and 11-14). As provided in Examples 3-4, peptides that elicit CD4 + T cell responses to multiple HLA class II molecules can be identified. CD4 + T cells recognize peptides bound to the HLA class II molecule on APC. In another embodiment, antigen-specific CD4 + T cell responses assist in induction andmaintenance of CD8 + cytotoxic T cell (CTL) responses (Examples 7-10 and 16-19).
  • CTL cytotoxic T cell
  • peptides of the present invention exhibit an enhanced ability to elicit CTL responses, due to their ability to bind both HLA class I and HLA class II molecules.
  • vaccines of the present invention have the advantage of activating or eliciting both CD4 + and CD8 + T cells that recognize bcr-abl antigens. In another embodiment, activation or eliciting both
  • CD4 + and CD8 + T cells provides a synergistic anti-bcr-abl immune response, relative to activation of either population alone.
  • the enhanced immunogenicity of peptides of the present invention is exhibited in individuals of multiple HLA class II subtypes, due to the ability of peptides of the present invention to bind multiple HLA class II subtypes. Each possibility represents a separate embodiment of the present invention.
  • activated CD4 + cells enhance immunity by licensing dendritic cells, thereby sustaining the activation and survival of the cytotoxic T cells.
  • activated CD4 + T cells induce tumor cell death by direct contact with the tumor cell or by activation of the apoptosis pathway.
  • Mesothelioma tumor cells for example, are able to process and present antigens in the context of HLA class I and class II molecules.
  • peptides, vaccines, and/or immunogenic compositions of the present invention have the advantage of activating or eliciting bcr-abl-specific CD4 + T cells containing multiple different HLA class II alleles.
  • the vaccines have the advantage of activating or eliciting bcr-abl-specific CD4 + T cells in a substantial proportion of the population.
  • the peptides activate bcr-abl-specific CD4 + T cells in 10% of the population.
  • the peptides activate bcr-abl-specific CD4 + T cells in 15% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 20% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 25% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 30% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 35% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 40% of the population.
  • the peptides activate bcr-abl-specific CD4 + T cells in 45% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 50% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 55% of the population. In another embodiment, the peptides activate bcr-abl-specif ⁇ c CD4 + T cells in 60% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 70% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 75% of the population.
  • the peptides activate bcr-abl-specific CD4 + T cells in 80% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 85% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 90% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in 95% of the population. In another embodiment, the peptides activate bcr-abl-specific CD4 + T cells in greater than 95% of the population. In another embodiment, the vaccines activate or elicit bcr-abl-specific CD4 + T cells in a substantial proportion of a particular population (e.g. American Caucasians). Each possibility represents a separate embodiment of the present invention.
  • peptides, vaccines, and/or immunogenic compositions of the present invention elicit enhanced bcr-abl-specific CTL responses in individuals carrying both the HLA class I molecule and the HLA class 11 molecule whose binding motifs are present in the peptide.
  • the peptides due to the binding of multiple HLA class I molecules and/or multiple HLA class II molecules, the peptides elicit enhanced bcr-abl-specific CTL responses in a substantial proportion of the population.
  • the peptides elicit enhanced bcr-abl-specific CTL responses in 10% of the population.
  • the peptides elicit enhanced bcr-abl-specific CTL responses in 15% of the population.
  • the peptides elicit enhanced bcr-abl- specific CTL responses in 20% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 25% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 30% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 35% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 40% of the population. In 08747
  • the peptides elicit enhanced bcr-abl-specific CTL responses in 45% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 50% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 55% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 60% of the population . In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 70% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 75% of the population.
  • the peptides elicit enhanced bcr-abl-specific CTL responses in 80% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 85% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 90% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in 95% of the population. In another embodiment, the peptides elicit enhanced bcr-abl-specific CTL responses in greater than 95% of the population. In another embodiment, the vaccines activate or elicit bcr-abl-specific CD4 + T cells in a substantial proportion of a particular population (e.g. American Caucasians). Each possibility represents a separate embodiment of the present invention.
  • a particular population e.g. American Caucasians
  • methods of the present invention provide for an improvement in an immune response that has already been mounted by a subject.
  • methods of the present invention comprise administering the peptide, composition, or vaccine 2 or more times.
  • the peptides are varied in their composition, concentration, or a combination thereof.
  • the peptides provide for the initiation of an immune response against bcr-abl in a subject in which an immune response against bcr-abl has not already been initiated.
  • reference to modulation of the immune response involves, either or both the humoral and cell-mediated arms of the immune system, which is accompanied by the presence of Th2 and Th 1 T helper cells, respectively, or in another embodiment, each arm individually.
  • the methods affecting the growth of a tumor result in (1) the direct inhibition of tumor cell division, or (2) immune cell mediated tumor cell lysis, or both, which leads to a suppression in the net expansion of tumor cells.
  • the direct inhibition of tumor cell division or (2) immune cell mediated tumor cell lysis, or both, which leads to a suppression in the net expansion of tumor cells.
  • tumor inhibition is determined by measuring the actual tumor size over a period of time.
  • tumor inhibition can be determined by estimating the size of a tumor (over a period of time) utilizing methods well known to those of skill in the art. More specifically, a variety of radiologic imaging methods (e.g., single photon and positron emission computerized tomography; see generally, “Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.) Springer-Verlag, New York, 1986), can be utilized to estimate tumor size.
  • radiologic imaging methods e.g., single photon and positron emission computerized tomography; see generally, "Nuclear Medicine in Clinical Oncology," Winkler, C. (ed.) Springer-Verlag, New York, 1986
  • imaging agents can also utilize a variety of imaging agents, including for example, conventional imaging agents (e.g., Gallium-67 citrate), as well as specialized reagents for metabolite imaging, receptor imaging, or immunologic imaging (e.g., radiolabeled monoclonal antibody specific tumor markers).
  • conventional imaging agents e.g., Gallium-67 citrate
  • immunologic imaging e.g., radiolabeled monoclonal antibody specific tumor markers
  • non-radioactive methods such as ultrasound (see, "Ultrasonic Differential Diagnosis of Tumors", Kossoff and Fukuda, (eds.), Igaku- Shoin, New York, 1984), can also be utilized to estimate the size of a tumor.
  • in vitro methods can be utilized in order to determine in vivo tumor inhibition.
  • Representative examples include lymphocyte mediated anti-tumor cytolytic activity determined for example, by a 51 Cr release assay (Examples), tumor dependent lymphocyte proliferation (Ioannides, et al., J. Immunol. 146(5):1700-1707, 1991), in vitro generation of tumor specific antibodies (Herlyn, et al., J. Immunol. Meth.
  • cell e.g., CTL, helper T-cell
  • humoral e.g., antibody
  • methods of suppressing tumor growth indicate a growth state that is curtailed compared to growth without contact with, or exposure to a peptide of this invention.
  • Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a 3 H-thymidine incorporation assay, or counting tumor cells.
  • "Suppressing" tumor cell growth refers, in other embodiments, to slowing, delaying, or stopping tumor growth, or to tumor shrinkage. Each possibility represents a separate embodiment of the present invention.
  • lymphocyte proliferation assays wherein T cell uptake of a radioactive substance, e.g. 3 H-thymidine is measured as a function of cell proliferation.
  • detection of T cell proliferation is accomplished by measuring increases in interleukin-2 (IL-2) 007/008747
  • CTL stimulation is determined by means known to those skilled in the art, including detection of cell proliferation, cytokine production and others.
  • Analysis of the types and quantities of cytokines secreted by T cells upon contacting ligand-pulsed targets can be a measure of functional activity.
  • Cytokines can be measured by ELISA, ELISPOT assays or fluorescence-activated cell sorting (FACS) to determine the rate and total amount of cytokine production. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160:181; Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259).
  • CTL activity is determined by 51 Cr-release lysis assay. Lysis of peptide-pulsed 51 Cr-labeled targets by antigen-specific T cells can be compared for target cells pulsed with control peptide.
  • T cells are stimulated with a peptide of this invention, and lysis of target cells expressing the native peptide in the context of MHC can be determined. The kinetics of lysis as well as overall target lysis at a fixed timepoint (e.g., 4 hours) are used, in another embodiment, to evaluate ligand performance. (Ware C. F. et al. (1983) J Immunol 131: 1312).
  • affinity is determined by TAP stabilization assays (Examples).
  • affinity is determined by competition radioimmunoassay.
  • the following protocol is utilized: Target cells are washed two times in PBS with 1% bovine serum albumin (BSA; Fisher Chemicals, Fairlawn, NJ). Cells are resuspended at 10 7 /ml on ice, and the native cell surface bound peptides are stripped for 2 minutes at 0° C using citrate-phosphate buffer in the presence of 3 mg/ml bcta 2 microglobulin.
  • BSA bovine serum albumin
  • the pellet is resuspended at 5 x 10 6 cells/ml in PBS/1 % BSA in the presence of 3 mg/ml beta 2 microglobulin and 30 mg/ml deoxyribonuclease, and 200 ml aliquots are incubated in the presence or absence of HLA-specific peptides for 10 min at 20 0 C, then with 125 I- labeled peptide for 30 min at 20°C. Total bound 125 I is determined after two washes with
  • Relative affinities are determined by comparison of escalating concentrations of the test peptide versus a known binding peptide.
  • a specificity analysis of the binding of peptide to HLA on surface of live cells is conducted to confirm that the binding is to the appropriate HLA molecule and to characterize its restriction.
  • This assay is performed, in one embodiment, on live fresh or 0.25% paraformaldehyde-fixed human PBMC, leukemia cell lines and EBV-transformed T-cell lines of specific HLA types.
  • the relative avidity of the peptides found to bind MHC molecules on the specific cells are assayed by competition assays as described above against 125 I-labeled peptides of known high affinity for the relevant HLA molecule, e.g., tyrosinase or HBV peptide sequence.
  • a peptide of methods and compositions of the present invention comprises a non-classical amino acid such as: l,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991) J. Am Chem. Soc.
  • histidine isoquinoline carboxylic acid Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); and HIC (histidine cyclic urea), (Dharanipragada et al.(1993) Int. J. Pep. Protein Res. 42(l):68-77) and ((1992) Acta. Crst., Crystal Struc. Comm. 48(IV): 1239-124).
  • a peptide of methods and compositions of the present invention comprises an AA analog or is a peptidomimetic, which, in other embodiments, induces or favors specific secondary structures.
  • Such peptides comprise, in other embodiments, the following: LL- Acp (LL-3-amino-2- ⁇ ropenidone-6-carboxylic acid), a ⁇ -turn inducing dipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838); ⁇ -sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5081-5082); ⁇ -turn inducing analogs (Kemp et al.
  • a peptide of this invention is conjugated to one of various other molecules, as described hereinbelow, which can be via covalent or non-covalent linkage (complexed), the nature of which varies, in another embodiment, depending on the particular purpose.
  • the peptide is covalently or non-covalently complexed to a macromolecular carrier, (e.g. an immunogenic carrier), including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids.
  • a peptide of this invention is linked to a substrate.
  • the peptide is conjugated to a fatty acid, for introduction into a liposome (U.S. Pat. No. 5,837,249).
  • a peptide of the invention is complexed covalently or non-covalently with a solid support, a variety of which are known in the art.
  • linkage of the peptide to the carrier, substrate, fatty acid, or solid support serves to increase an elicited an immune response.
  • the carrier is thyroglobulin, an albumin (e.g. human serum albumin), tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), an influenza protein, hepatitis B virus core protein, keyhole limpet hemocyanin, an albumin, or another carrier protein or carrier peptide; hepatitis B virus recombinant vaccine, or an APC.
  • albumin e.g. human serum albumin
  • polyamino acids such as poly (lysine: glutamic acid)
  • influenza protein hepatitis B virus core protein
  • keyhole limpet hemocyanin an albumin
  • hepatitis B virus recombinant vaccine or an APC.
  • amino acid refers to a natural or, in another embodiment, an unnatural or synthetic AA, and can include, in other embodiments, glycine, D- or L optical isomers, AA analogs, peptidomimetics, or combinations thereof.
  • cancer in another embodiment, the terms “cancer,” “neoplasm,” “neoplastic” or “tumor,” are used interchangeably and refer to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a tumor is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation, and in another embodiment, is identified by biochemical or immunologic findings, the latter which is used to identify cancerous cells, as well, in other embodiments.
  • peptides of this invention are synthesized using an appropriate solid-state synthetic procedure (see for example,
  • the peptides of this invention are purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • immuno- affinity chromatography is used, whereby an epitope is isolated by binding it to an affinity column comprising antibodies that were raised against that peptide, or a related peptide of the invention, and were affixed to a stationary support.
  • affinity tags such as hexa-His (Invitxogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Meth. Enzymol. 194:508- 509), glutathione-S -transferase, or others, are attached to the peptides of this invention to allow easy purification by passage over an appropriate affinity column.
  • Isolated peptides can also be physically characterized, in other embodiments, using such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallography.
  • the peptides of this invention are produced by in vitro translation, through known techniques, as will be evident to one skilled in the art.
  • the peptides are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).
  • the peptides of this invention further comprise a detectable label, which in one embodiment, is fluorescent, or in another embodiment, luminescent, or in another embodiment, radioactive, or in another embodiment, electron dense.
  • the dectectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase, 32 P, 125 1, 3 H and 14 C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin or any number of other such labels known to one skilled in the .art.
  • the particular label used will depend upon the type of immunoassay used.
  • a peptide of this invention is linked to a substrate, which, in one embodiment, serves as a carrier. In one embodiment, linkage of the peptide to a substrate serves to increase an elicited an immune response. [00166] In one embodiment, peptides of this invention are linked to other molecules, as described herein, using conventional cross-linking agents such as carbodimides.
  • carbodimides examples include l-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), l-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and l-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.
  • the cross-linking agents comprise cyanogen bromide, glutaraldehyde and succinic anhydride.
  • any of a number of homo-bifunctional agents including a homo- bifunctional aldehyde, a homo-bifunctional epoxide, a homo-bifunctional imido-ester, a homo- bifunctional N-hydroxysuccinimide ester, a homo-bifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyl disulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo-bifunctional diazonium derivative and a homo-bifunctional photoreactive compound can be used.
  • hetero-bifunctional compounds for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.
  • the homo-bifunctional cross-linking agents include the bifunctional N- hydroxysuccinimide esters dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers l,4-di-[3'-(2'- pyridyldithio)propionamido]butane, bismaleimidohexane, and bis-N-maleimido-l,8-octane; the bifunctional aryl halides 1 ,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'-dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[
  • hetero-bifunctional cross-linking agents used to link the peptides to other molecules include, but are not limited to, SMCC (succinimidyl-4-(N- maleimidomethyl)cyclohexane-l-carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p- 007/008747
  • GMBS N-(.ga ⁇ una.-maleimidobutyryloxy)succinimi(ie ester)
  • MPBH 4- (4-N-maleimidopohenyl) butyric acid hydrazide
  • M2C2H 4-(N-maleimidomethyl) cyclohexane-1- carboxyl-hydrazide
  • SMPT succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • the peptides of the invention are formulated as non-covalent attachment of monomers through ionic, adsorptive, or biospecific interactions.
  • Complexes of peptides with highly positively or negatively charged molecules can be accomplished, in another embodiment, through salt bridge formation under low ionic strength environments, such as in deionized water.
  • Large complexes can be created, in another embodiment, using charged polymers such as poiy-(L- glutamic acid) or poly-(L-lysine), which contain numerous negative and positive charges, respectively.
  • peptides are adsorbed to surfaces such as microparticle latex beads or to other hydrophobic polymers, forming non-covalently associated peptide-superantigen complexes effectively mimicking cross-linked or chemically polymerized protein, in other embodiments.
  • peptides are non-covalently linked through the use of biospecific interactions between other molecules. For instance, utilization of the strong affinity of biotin for proteins such as avidin or streptavidin or their derivatives could be used to form peptide complexes.
  • the peptides can be modified to possess biotin groups using common biotinylation reagents such as the N-hydroxysuccinimidyl ester of D-biotin (NHS-biotin), which reacts with available amine groups.
  • biotinylation reagents such as the N-hydroxysuccinimidyl ester of D-biotin (NHS-biotin), which reacts with available amine groups.
  • the peptides are linked to carriers.
  • the peptides are any that are well known in the art, including, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine:gluta ⁇ nic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like.
  • thyroglobulin albumins such as human serum albumin, tetanus toxoid
  • polyamino acids such as poly (lysine:gluta ⁇ nic acid)
  • influenza hepatitis B virus core protein
  • hepatitis B virus recombinant vaccine hepatitis B virus recombinant vaccine
  • the peptides of this invention are conjugated to a lipid, such as P3 CSS. In another embodiment, the peptides of this invention are conjugated to a bead.
  • compositions of this invention further comprise immunomodulating compounds.
  • the immunomodulating compound is a cytokine, chemokine, or complement component that enhances expression of immune system accessory or adhesion molecules, their receptors, or combinations thereof.
  • the immunomodulating compound include interleukins, for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte-macrophage colony stimulating factor 007/008747
  • the immunomodulating compound stimulate expression, or enhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-I, 2 or 3, cytokine receptors, or combination thereof.
  • the immunomodulatory compound induces or enhances expression of co- stimulatory molecules that participate in the immune response, which include, in some embodiments, CD40 or its ligand, CD28, CTLA-4 or a B7 molecule.
  • the immunomodulatory compound induces or enhances expression of a heat stable antigen (HSA) (Liu Y. et al. (1992) J. Exp. Med. 175:437-445), chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas M. F. et al. (1993) Cell 74:257-268), or an intracellular adhesion molecule 1 (ICAM-I) (Van R. H. (1992) Cell 71:1065-1068), which assists, in another embodiment, co-stimulation by interacting with their cognate ligands on the T cells.
  • HSA heat stable antigen
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • the composition comprises a solvent, including water, dispersion media, cell culture media, isotonic agents and the like.
  • the solvent is an aqueous isotonic buffered solution with a pH of around 7.0.
  • the composition comprises a diluent such as water, phosphate buffered saline, or saline.
  • the composition comprises a solvent, which is non-aqueous, such as propyl ethylene glycol, polyethylene glycol and vegetable oils.
  • the composition is formulated for administration by any of the many techniques known to those of skill in the art.
  • this invention provides for administration of the pharmaceutical composition parenterally, intravenously, subcutaneously, intradermally, intramucosally, topically, orally, or by inhalation.
  • the vaccine comprising a peptide of this invention further comprises a cell population, which, in another embodiment, comprises lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells or combinations thereof, which, in another embodiment are autologous, syngeneic or allogeneic, with respect to each other.
  • the cell population comprises a peptide of the present invention.
  • the cell population takes up the peptide.
  • the cell populations of this invention are obtained from in vivo sources, such as, for example, peripheral blood, leukopheresis blood product, apheresis blood product, peripheral lymph nodes, gut associated lymphoid tissue, spleen, thymus, cord blood, mesenteric lymph nodes, liver, sites of immunologic lesions, e.g. synovial fluid, pancreas, cerebrospinal fluid, tumor samples, granulomatous tissue, or any other source where such cells can be obtained.
  • the cell populations are obtained from human sources, which are, in other embodiments, from human fetal, neonatal, child, or adult sources.
  • the cell populations of this invention are obtained from animal sources, such as, for example, porcine or simian, or any other animal of interest. In another embodiment, the cell populations of this invention are obtained from subjects that are normal, or in another embodiment, diseased, or in another embodiment, susceptible to a disease of interest.
  • the cell populations of this invention are separated via affinity-based separation methods.
  • Techniques for affinity separation include, in other embodiments, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or use in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and "panning" with an antibody attached to a solid matrix, such as a plate, or any other convenient technique.
  • separation techniques include the use of fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • any technique that enables separation of the cell populations of this invention can be employed, and is to be considered as part of this invention.
  • the dendritic cells are from the diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, qualified as such (Steinman (1991) Ann. Rev. Immunol.9:271-296).
  • the dendritic cells used in this invention are isolated from bone marrow, or in another embodiment, derived from bone marrow progenitor cells, or, in another embodiment, from isolated from/derived from peripheral blood, or in another embodiment, derived from, or are a cell line.
  • the cell populations described herein are isolated from the white blood cell fraction of a mammal, such as a murine, simian or a human (See, e.g., WO 96/23060).
  • the white blood cell fraction can be, in another embodiment, isolated from the peripheral blood of the mammal.
  • the DC are isolated via a method which includes the following steps: (a) providing a white blood cell fraction 47
  • step (d) obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation; (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF and IL-4, (d) identifying the dendritic cell-enriched fraction from step (c); and (e) collecting the enriched fraction of step (d), preferably at about 4° C.
  • the dendritic cell-enriched fraction is identified by fluorescence- activated cell sorting, which identifies, in another embodiment, at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absence of the following markers: CD3, CD14, CD16, 56, 57, and CD 19, 20.
  • the cell population comprises lymphocytes, which are, in one embodiment, T cells, or in another embodiment, B cells.
  • the T cells are, in other embodiments, characterized as NK cells, helper T cells, cytotoxic T lymphocytes (CTL), TILs, na ⁇ ve T cells, or combinations thereof.
  • CTL cytotoxic T lymphocytes
  • TILs na ⁇ ve T cells, or combinations thereof.
  • T cells which are primary, or cell lines, clones, etc. are to be considered as part of this invention.
  • the T cells are CTL, or CTL lines, CTL clones, or CTLs isolated from tumor, inflammatory, or other infiltrates.
  • hematopoietic stem or early progenitor cells comprise the cell populations used in this invention.
  • populations are isolate or derived, by leukapheresis.
  • the leukapheresis follows cytokine administration, from bone marrow, peripheral blood (PB) or neonatal umbilical cord blood.
  • the stem or progenitor cells are characterized by their surface expression of the surface antigen marker known as CD34 + , and exclusion of expression of the surface lineage antigen markers, Lin-.
  • the subject is administered a peptide, composition or vaccine of this invention, in conjunction with bone marrow cells.
  • the administration together with bone marrow cells embodiment follows previous irradiation of the subject, as part of the course of therapy, in order to suppress, inhibit or treat cancer in the subject.
  • the phrase "contacting a cell” or “contacting a population” refers to a method of exposure, which can be, in other embodiments, direct or indirect.
  • such contact comprises direct injection of the cell through any means well known in the art, such as microinjection.
  • supply to the cell is indirect, such as via provision in a culture medium that surrounds the cell, or administration to a subject, via any route well known in the art, and as described herein.
  • CTL generation of methods of the present invention is accomplished in vivo, and is effected by introducing into a subject an antigen presenting cell contacted in vitro with a peptide of this invention (See for example Paglia et al. (1996) J. Exp. Med. 183:317-322).
  • the peptides of methods and compositions of the present invention are delivered to antigen-presenting cells (APC).
  • APC antigen-presenting cells
  • the peptides are delivered to APC in the form of cDNA encoding the peptides.
  • the term "antigen-presenting cells” refers to dendritic cells (DC), monocytes/macrophages, B lymphocytes or other cell type(s) expressing the necessary MHC/co- stimulatory molecules, which effectively allow for T cell recognition of the presented peptide.
  • the APC is a cancer cell. Each possibility represents a separate embodiment of the present invention.
  • the CTL are contacted with two or more APC populations.
  • the two or more APC populations present different peptides. Each possibility represents a separate embodiment of the present invention.
  • techniques that lead to the expression of antigen in the cytosol of APC are used to deliver the peptides to the APC.
  • Methods for expressing antigens on APC are well known in the art.
  • the techniques include (1) the introduction into the APC of naked DNA encoding a peptide of this invention, (2) infection of APC with recombinant vectors expressing a * peptide of this invention, and (3) introduction of a peptide of this invention into the cytosol of an APC using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med. 184:465-472; Rouse et al. (1994) J. Virol. 68:5685-5689; and Nair ct al. (1992) J. Exp. Med. 175:609-612).
  • foster antigen presenting cells such as those derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763-1771), are used, as exemplified herein.
  • any of the methods described herein is used to elicit CTL, which are elicited in vitro.
  • the CTL are elicited ex-vivo.
  • the CTL are elicited in vitro.
  • the resulting CTL are, in another embodiment, administered to the subject,
  • the method entails introduction of the genetic sequence that encodes the peptides of this invention.
  • the method comprises administering to the subject a vector comprising a nucleotide sequence, which encodes a peptide of the present invention (Tindle, R.
  • the method comprises administering to the subject naked DNA which encodes a peptide, or in another embodiment, two or more peptides of this invention (Nabel, et al. PNAS-USA (1990) 90: 11307).
  • label, et al. PNAS-USA (1990) 90: 11307 In another embodiment, multi-epitope, analogue-based cancer vaccines are utilized (Fikes et al, ibid). Each possibility represents a separate embodiment of the present invention.
  • Nucleic acids can be administered to a subject via any means as is known in the art, including parenteral or intravenous adminstration, or in another embodiment, by means of a gene gun. In another embodiment, the nucleic acids are administered in a composition, which correspond, in other embodiments, to any embodiment listed herein.
  • Vectors for use according to methods of this invention can comprise, in another embodiment, any vector that facilitates or allows for the expression of a peptide of this invention.
  • "vectors” includes attenuated viruses, such as vaccinia or fowlpox, such as described in, e.g., U.S. Pat. No. 4,722,848, incorporated herein by reference.
  • the vector is BCG (Bacille Calmette Guerin), such as described in Stover et al. (Nature 351:456-460 (1991)).
  • BCG Bacille Calmette Guerin
  • Other vectors useful for therapeutic administration or immunization of the peptides of the invention e.g., Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.
  • the vector further encodes for an immunomodulatory compound, as described herein.
  • the subject is administered an additional vector encoding same, concurrent, prior to or following administration of the vector encoding a peptide of this invention to the subject.
  • the peptides, compositions and vaccines of this invention are administered to a subject, or utilized in the methods of this invention, in combination with other anti- cancer compounds and chemotherapeutics, including monoclonal antibodies directed against alternate cancer antigens, or, in another embodiment, epitopes that consist of an AA sequence which US2007/008747
  • the dosage is 20 ⁇ g per peptide per day. In another embodiment, the dosage is 10 ⁇ g/peptide/day. In another embodiment, the dosage is 30 ⁇ g/peptide/day. In another embodiment, the dosage is 40 ⁇ g/peptide/day. In another embodiment, the dosage is 60 ⁇ g/peptide/day. In another embodiment, the dosage is 80 ⁇ g/peptide/day. In another embodiment, the dosage is 100 ⁇ g/peptide/day. In another embodiment, the dosage is 150 ⁇ g/peptide/day. In another embodiment, the dosage is 200 ⁇ g/peptide/day. In another embodiment, the dosage is 300 ⁇ g/peptide/day.
  • the dosage is 400 ⁇ g/peptide/day. In another embodiment, the dosage is 600 ⁇ g/peptide/day. In another embodiment, the dosage is 800 ⁇ g/peptide/day. In another embodiment, the dosage is 1000 ⁇ g/peptide/day.
  • the dosage is 10 ⁇ g/peptide/dose. In another embodiment, the dosage is 30 ⁇ g/peptide/dose. In another embodiment, the dosage is 40 ⁇ g/peptide/dose. In another embodiment, the dosage is 60 ⁇ g/peptide/dose.. In another embodiment, the dosage is 80 ⁇ g/peptide/dose. In another embodiment, the dosage is 100 ⁇ g/peptide/dose. In another embodiment, the dosage is 150 ⁇ g/peptide/dose. In another embodiment, the dosage is 200 ⁇ g/peptide/dose. In another embodiment, the dosage is 300 ⁇ g/peptide/dose. In another embodiment, the dosage is 400 ⁇ g/peptide/dose. In another embodiment, the dosage is 600 ⁇ g/peptide/dose. In another embodiment, the dosage is 800 ⁇ g/peptide/dose. In another embodiment, the dosage is 1000 ⁇ g/peptide/dose.
  • the dosage is 10-20 ⁇ g/peptide/dose. In another embodiment, the dosage is 20-30 ⁇ g/peptide/dose. In another embodiment, the dosage is 20-40 ⁇ g/peptide/dose. lri another embodiment, the dosage is 30-60 ⁇ g/peptide/dose. In another embodiment, the dosage is 40-80 ⁇ g/peptide/dose. In another embodiment, the dosage is 50-100 ⁇ g/peptide/dose. In another embodiment, the dosage is 50-150 ⁇ g/peptide/dose. In another embodiment, the dosage is 100-200 ⁇ g/peptide/dose. In another embodiment, the dosage is 200-300 ⁇ g/peptide/dose.
  • the dosage is 300-400 ⁇ g/peptide/dose. In another embodiment, the dosage is 400-600 ⁇ g/peptide/dose. In another embodiment, the dosage is 500-800 ⁇ g/peptide/dose. In another embodiment, the dosage is 800-1000 ⁇ g/peptide/dose.
  • the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts.
  • EXAMPLE 1 BINDING OF HLA-A0201 AND -A0301 BY SYNTHETIC PEPTIDE ANALOGUES DERIVED FROM WTl
  • Peptides were synthesized by Genemed Synthesis Inc, CA using fluorenylmethoxycarbonyl chemistry and solid phase synthesis, and were purified by high pressure liquid chromatography (HPLC). The quality of the peptides was assessed by HPLC analysis, and the expected molecular weight was measured using matrix-assisted laser desorption mass spectrometry. Peptides were sterile and > 90% pure. The peptides were dissolved in DMSO and diluted in PBS atpH 7.4 or saline solution to yield a concentration of 5 milligrams per milliliter (mg/ml) and were stored at -80° C. For in vitro experiments, an irrelevant control peptide, HLA A24 consensus, was used.
  • Cell lines were cultured in RPMI 1640 medium supplemented with 5% FCS, penicillin, streptomycin, 2mM glutamine and 2-mercaptoethanol at 37 0 C in humidified air containing 5% CO 2 .
  • T2 is a human cell line lacking TAP 1 and TAP2 and therefore unable to present peptides derived from cytosolic proteins.
  • Raji cells are a human Burkitt lymphoma cells that exhibit a high level of TAP expression.
  • T2 assay for peptide binding and stabilization of HLA A0201 molecules T2 cells lack TAP function and consequently are defective in properly loading class I molecules with antigenic peptides generated in the cytosol. The association of exogenously added peptides with thermolabile, empty HLA- A2 molecules stabilizes them and results in an increase in the level of surface HLA-A0201 recognizable by specific mAb such as BB7.2.
  • T2 cells (TAP-, HLA- A0201 + ) were incubated overnight at 27° C at a concentration of 1 x 10 6 cells/ml in FCS-free RPMI medium supplemented with 5 ⁇ g/ml human ⁇ 2 m (Sigma, St Louis, MO) in the absence (negative control) or presence of either a positive reference tyrosinase peptide or test peptides at various final concentrations (50, 10, 1, and 0.1 micrograms ( ⁇ g)/ml).
  • T2 cells were labeled for 30 minutes at 4 0 C with a saturating concentration of anti-HLA-A2.1 (BB7.2) mAb, then washed twice.
  • Cells were then incubated for 30 minutes, 4° C with a saturating concentration of FITC-conjugated goat IgG F(ab')2 anti-mouse Ig (Caltag, San Francisco, CA), washed twice, fixed in PBS/1% paraformaldehyde and analyzed using a FACS Calibur® cytofluorometer (Becton Dickinson, Immunocytometry Systems, San Jose, CA).
  • MIF mean intensity of fluorescence
  • Stabilization assays were performed similarly. Following initial evaluation of peptide binding at time 0, cells were washed in RPMI complete medium to remove free peptides and incubated in the continuous presence of 0.5 ⁇ g/ml brefeldin-A for 2, 4, 6 or 8 hours.
  • the number of stable peptide-HLA-A2.1 complexes was estimated as described above by immunofluorescence.
  • [0021 1] Peptides having predicted affinity for HLA-A0201 and HLA-A0301 molecules were identified from the WTl sequence. These WTl native peptides were modified to generate heteroclitic peptides with increased predicted binding to HLA-A0201 and HLA-A0301 molecules, as shown in Tables 1-2. Several of the heteroclitic peptides significantly stabilized HLA-A0201 and HLA-A0301 molecules in thermostabilization assays using a TAP 1/2 negative cell line (T2) and Raji HLA-A0301 cells. Specifically, WTl-Al, Bl, and Cl exhibited similar or increased binding compared to the corresponding native peptides WTl-A, B, and C. WTl-Dl exhibited similar or increased binding compared to corresponding native peptide WTl-D ( Figure 1 A). A comparison of
  • heteroclitic WTl peptides of the present invention exhibit enhanced binding to HLA class I molecules.
  • EXAMPLE 2 INDUCTION OF IMMUNE RESPONSES AGAINST SYNTHETIC PEPTIDE ANALOGUES DERIVED FROM WTl
  • PBMC peripheral blood dendritic cells
  • IL interleukin-4
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • T lymphocytes were isolated from the same donors by use of negative selection by depletion with an anti-CDl lb, anti-CD56 and CD19 monoclonal antibody (Miltenyi, CA).
  • 1 x 10 ⁇ 6 T lymphocytes were cultured with 1 x 10 ⁇ 5 autologous DC in RPMI 1640 containing 5% heat- inactivated human autologous plasma with 10 ⁇ g/mL peptide and 2 ⁇ g/ml ⁇ 2 microglobulin, 5 ng/mL recombinant human IL-7 (Genzyme), and 0.1 ng/ml IL- 12 in 24 well plates.
  • heteroclitic WT I peptides of the present invention are able to generate T cells that (a) secrete inflammatory cytokines and (b) perform cytolysis in response to cells presenting WTl peptides.
  • the T cells generated by the heteroclitic WTl peptides recognize both native and heteroclitic WTl peptides
  • EXAMPLE 3 SELECTION OF SYNTHETIC WTl PEPTIDES THAT BIND HLA CLASS II MOLECULES
  • Table 3 WTl native peptides predicted binding to HLA-DR alleles based on SYFPEITHI algorithm (0 (low)- 28 (high)).
  • AA sequences of the peptides in Table 3 are LVRHHNMHQRNMTKL (427); RSDELVRHHNMHQRNMTKL (427 long); NKRYFKLSHLQMHSR (331); and PGCNKRYFKLSHLQMHSRKHTG (331 long).
  • HLA class II-binding WTl peptides of the present invention bind to HLA class II molecules in a large percentage of the population.
  • EXAMPLE 4 HLA CLASS II MOLECULE-BINDING. WTl PEPTIDES STIMULATE
  • PBMC peripheral blood mononuclear cells
  • Ex-Vivo-15® medium BioWhittaker, Walkersville, MD
  • media containing 1 x 10 3 IU/ml GM-CSF and 0.0032 IU/ml IL-4 On day 2 and 4, the same media was added as re-feed (i.e., V2 the volume of media, containing enough cytokines for the entire dish, was added).
  • V2 the volume of media, containing enough cytokines for the entire dish, was added.
  • 10 ⁇ g/ml of peptide was added.
  • the maturation cocktail consisted of: 4 x 10 2 IU/ml IL-I -beta, 0.0032 IU/ml IL-4, 1 x 10 3 IU/ml IL-6, 1 x 10 3 IU/ml GMCSF, 10 ⁇ g/ml TNF-alpha, and 1 ⁇ g/ml PGE2.
  • PBMC peripheral blood mononuclear cells
  • DC were isolated from the adherent fraction and prepared as described above for the second stimulation of the effector cells on day 14.
  • CD3 + cells were isolated from the non-adherent fraction by negative selection and stimulated with the previously prepared DC by re-suspending the CD3 + cells at a concentration of 2 x I O 6 cells/ml in RPMI + 5% autologous plasma, and adding DC at an effecto ⁇ DC ratio of 20: 1 and 10 ng/ml DL-15. Cells were then plated in 2-ml and co-incubated at 37 °C and 5% CO 2 for 1 week.
  • the CD3 + cells were stimulated a second time with the second batch of DC in the same manner, except that 1 x 10 6 cells/ml were mixed with DC at an effecto ⁇ DC ratio of 50: 1.
  • the same media was added as re-feed.
  • the DC from the previous generation were defrosted and incubated in maturation cytokines in X-vivol5 media.
  • the ELISPOT assay was conducted.
  • ELISPOT assay Plates were pre- wet with 30 ⁇ l/well 70% alcohol, shaking the plates to ensure coverage of the entire surface area, washed 3 times with 150 ⁇ l/well sterile PBS, then incubated overnight at 4°C with
  • solution # 1 (acetate buffer), 23.4 ml dd H 2 O, 2.3 ml 0.1 N Acetic Acid, and 5.5 ml 0.1 N Sodium Acetate were mixed.
  • 1 tablet of AEC (Sigma) was dissolved in 2.5 ml of dimethylformamide. Then 1.25ml of solution #2 was mixed with 23.7 ml of solution #1, 13 ⁇ l of 30% H 2 O 2 was added, and the resulting solution mixed well and filtered using a 0.45 ⁇ m filter.
  • the CD4 + PBMC s ⁇ bpopulations of healthy donors were isolated and stimulated with autologous monocyte-derived, peptide-pulsed DC, then re-stimulated with peptide- pulsed CD 14 + monocytes.
  • Antigen-specific CD4 + T cells recognizing each of the HLA class II-binding WTl peptides were generated, as shown by IFN- ⁇ ELISPOT ( Figure 5). As expected, cells stimulated with RAS (irrelevant control peptide) or with APC alone did not produce IFN- ⁇ over background levels.
  • HLA class II-binding WTl peptides of the present invention are able to generate T cells that recognize cells presenting WTl peptides.
  • HLA class II molecule-binding WTl peptides are modified by mutation of HLA class II molecule anchor residues, using methods and algorithms described herein, to generate heteroclitic HLA class II WTl peptides.
  • the HLA binding and immunogenicity of the heteroclitic peptides are tested using methods known in the art (e.g. methods described in the previous Examples).
  • the heteroclitic peptides are found to bind HLA class II molecules and stimulate WTl -specific immune responses.
  • Various heteroclitic peptides exhibit enhanced affinity for HLA class II molecules and/or expanded repertoire of HLA class II molecules that are bound, relative to the WTl peptides from which the heteroclitic peptides were derived.
  • the heteroclitic peptides are used to increase the immunogenicity of vaccines of the present invention and the range of subjects for which the vaccines are effective.
  • a WTl peptide spanning residues 122-140, having the sequence SGQARMFPNAPYLPSCLES (SEQ ID No: 39) was generated and designated "WTl 122.”
  • Binding affinity of WTl 122 for common HLA DRB molecules was predicted using the SYFPEITHI epitope prediction algorithm (Rammensee H et al, SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 Nov;50(3- 4):213-9).4 of the 6 HLA-DR types showed improved predicted binding relative to a shorter peptide, WTl 124, having the sequence Q ARMFPNAP YLPS CL (SEQ ID No: 40) (Table 4).
  • WTl 122Al a peptide termed "WTl 122Al” was generated, comprising the CD8 + heteroclitic WTlAl peptide YMFPNAPYL (Example 1 ; SEQ ID No: 6) nestled inside the elongated CD4 + peptide epitope and having the sequence SGQAYMFPNAPYLPSCLES (SEQ ID No: 41).
  • WTl 122Al also exhibited improved predicted binding over WTl 124 to a broad array of HLA-DR types (Table 4).
  • the average score of WTl 122Al was 19, with a binding score over 14 (the halfway mark) for all 6 HLA-DR types, compared to an average score of 12 with only one HLA-DR type over 14.
  • Predicted WTl 122Al binding to the HLA-DR types was also superior to a shorter peptide containing the WTlAl peptide, "124Al,” having the sequence QAYMFPNAPYLPSCL (SEQ ID No: 42).
  • WTl 244 a WTl peptide spanning residues 247-261 , having the sequence GATLKGVAAGSSSSVKWT (SEQ ID NO: 43) was generated and designated "WTl 244." Binding affinity of WTl 244 for common HLA DRB molecules was predicted as described above for WT 122. Several HLA-DR types showed improved predicted binding relative to a shorter peptide, WTl 247, having the sequence LKGVAAGSSSSVKWT (SEQ ID No: 64) (Table 4). [00236] Table 4. Predicted binding identification of WTl peptides to class 2 HLA-DR types.
  • DCs from healthy A0201 + donors were incubated for 18 hours with the tumor lysates and used to stimulate autologous CD3 + T cells. Following 3 stimulations, the T cells were tested for their reactivity to autologous DCs pulsed with the WTl peptides. T cells that had been stimulated with WTl positive tumor lysates recognized the individual HLA class II peptides ( Figure 6A-B), while T cells stimulated by DCs pulsed with MeWo lysate did not stimulate WTl specific T cells. In addition, T cells stimulated with DCs pulsed with 697 tumor lysate recognized the native short class I peptide WTlA (126-134) and the analog WTlAl peptide. These experiments were repeated in 5 separate donors.
  • Stimulated T cells recognized WTlDR peptide 328 and WTlDR peptide 122Al in 3/5 experiments and recognized WTlDR 427 in all experiments. Therefore, despite the low expression of WTl transcript in the mesothelioma cell lines, WTl CD4 epitopes of the present invention were processed and presented by HLA class II molecules of mesothelioma cells.
  • peptides comprising a heteroclitic MHC class I peptide that is associated with an MHC class II peptide are (a) taken up and presented by APC in an antigenic form; and (b) are presented by APC exposed to WTl -expressing tumor cells; and (c) APC exposed to WTl 122 and 122Al peptides elicit the formation of T cells that recognize WTl -expressing tumor cells.
  • WTl -expressing cells including mesothelioma and leukemia cells, process and present peptides comprising a heteroclitic MHC class I peptide that is associated with an MHC class II peptide.
  • CD3 + cells from healthy donors were isolated and stimulated 2 times with peptide, and then recognition of WTl + JMN cells or WTl " Mewo cells, either alone or with the indicated peptides, was determined by gamma IF 7 N ELISPOT, using the methods described in Example 4.
  • T cells were stimulated with autologous, monocyte-deri ved DC pulsed with WTl 122, 122Al , or negative control peptide, re-stimulated with CDl 4 + monocytes pulsed with the same peptide, then assayed for formation of antigen-specific T cells by IFN- ⁇ ELISPOT. Stimulation with WTl 122 or 122Al, but not negative control peptide, generated CD4 + T cells that recognized targets pulsed with peptides containing the respective CD4 + epitopes, but not targets pulsed with negative control peptide ( Figure 7A-B).
  • both WTl DR 122 and WTl DR 122 A 1 were able to activate CD8 + T cells against the native short epitope WTl A (amino acids 126-134 ( Figure 7C); WTl DR 122Al was a more potent stimulator.
  • WTlDR 328 stimulated peptide specific T cell responses in 11 / 15 experiments; WTl DR 423 in 3 / 14 experiments; WTlDR 122 in 2 / 5 experiments; and WTlDR 122Al stimulated T cells that recognized WTlDR 122Al and WTlDR 122 peptide in 6 / 9 experiments.
  • stimulation with WTl 122 or 122Al generates antigen-specific CD4 + and CD8 + T cells.
  • stimulation with WT 1 122A 1 generates CD8 + T cells that recognize the heteroclitic CD8 + peptide and its native counterpart, whether buried in a longer peptide (WTl 122 or WTl 122Al, respectively) or alone.
  • EXAMPLE 9 ANTIGEN-SPECIFIC CD4* T CELLS GENERATED BY PEPTmES COMPRISING A HETEROCLITIC MHC CLASS I PEPTIDE ASSOCIATED WITH AN MHC CLASS ⁇ PEPTIDE RECOGNIZE WTl-EXPRESSING TUMOR CELLS
  • peptide-stimulated T cells were challenged in an IFN-gamma ELTSPOT with WT-I + and - negative tumor cells.
  • a sufficient amount of WTl peptide was presented on the surface of the WTl + mesothelioma tumor cell for T cells stimulated with individual WTlDR peptides to recognize mesothelioma tumor cells, compared to the control WTl negative melanoma cells ( Figure 8, left panel).
  • T cells were stimulated by the mutated WTlDR 122Al and challenged with pulsed and unpulsed targets.
  • control WTl negative target cells were pulsed with additional WTlDR 122Al peptide
  • IFN-gamma production increased.
  • WTl positive target cells were pulsed with additional WTlDR 122Al peptide
  • production did not increase, showing that a maximal response was achieved with the native processed peptides ( Figure 8, right panel).
  • vaccination with peptides comprising a heteroclitic MHC class I peptide that is associated with an MHC class II peptide results in generation of antigen-specific T cells with activity against WTl -expressing tumors.
  • CD3 + cells from healthy donors were isolated and stimulated with autologous, monocyte-derived DC pulsed with WTlDR 122Al, WTlDR 122, or negative control peptide, re- stimulated with CD14 + monocytes pulsed with the same peptide, then assayed by IFN- ⁇ ELISPOT for formation of antigen-specific T cells that recognized WTl + JMN cells.
  • WTlDR 122Al but not WTlDR 122, stimulated a sufficient number of CD8 + cells to be cytotoxic to 697, a WTl + leukemia cell line.
  • the CD8 + T cells did not recognize SKLY16, a WTl negative B cell lymphoma, unless it was pulsed with WTlA ( Figure 9, left panel), showing antigen specificity of the immune response. Similar results were observed in 3/4 different A0201 + donors, each with a distinct HLA-DRBl type. As expected, the negative control peptides generated no antigen- specific CD8 + T cells.
  • CD3 + T cells generated by stimulation with WTl 122Al or WTlAl recognized JMN cells but not negative control MeWo cells, whether alone or pulsed with WTl 122Al peptide (Figure 9, right panel).
  • CD4 + cells stimulated with WTlDR 122Al showed no cytotoxicity to either WTl+ mesothelioma or WTl- melanoma cells.
  • Human T cells stimulated 2 times with either the native WTlA or the analog WTlAl peptide were able to lyse human WTl + mesothelioma cell lines compared to WTl " control cell lines (9.2% lysis of MeWo vs. 19% lysis of JMN for WTlA stimulated T cells; 22.2% lysis of MeWo vs. 44.8% lysis of JMN for WTlAl stimulated T cells).
  • EXAMPLE 11 IDENTIFICATION AND GENERATION OF BCR-ABL BREAKPOINT PEPTIDES WITH A HIGH PROBABILITY OF HLA A0201 BINDING
  • HLA-A0201 is expressed in about 40% of the Caucasian population.
  • the BIMAS algorithm assumes that each amino acid in the peptide contributes independently to binding to the class I molecule. Dominant anchor residues, which are critical for binding, have coefficients in the tables that are significantly higher than 1. Unfavorable amino acids have positive coefficients that are less than 1. If an amino acid is not known to make either a favorable or unfavorable contribution to binding, then is assigned the value 1. All the values assigned to the amino acids are multiplied and the resulting running score is multiplied by a constant to yield an estimate of half-time of dissociation.
  • Table 5 The AA sequences of native breakpoint peptides and synthetic analogues and their predicted score for binding to HLA A0201, generated by two BIMAS and SYFPEITHI.
  • Residues in bold represent the amino acid at the fusion breakpoint. Residues underlined represent modifications from the native sequence.
  • p210C has a high BIMAS score that correlated with T2 binding assay data ( Figure 10, left panel).
  • p21 OF is a peptide derived from CMLA2 (Table 5), shown to be a weak binder in the T2 assay. In this case, the two serine residues in positions 1 and 2 were substituted for a tyrosine and a leucine, respectively, with the intent of increasing peptide binding and stabilization to HLA A0201, while retaining the amino-acids for the TCR interaction.
  • the BIMAS prediction was increased 700-fold, and high avidity for HLA A0201 molecules was demonstrated by binding to T2 cells.
  • EXAMPLE 13 PEPTIDE ANALOGUE DISSOCIATION FROM HLA A02Q1
  • the immunogenicity of peptide antigens is, under the conditions utilized herein, related to their low dissociation rate from MHC molecule-peptide complexes.
  • the stability of complexes formed between HLA-A0201 and the b3a2 analogue peptides was therefore assayed with T2 cells, as a function of time. Overnight incubation of T2 cells with saturating amounts of HLA-A0201 binding peptides and human ⁇ 2 microglobulin resulted in increased surface HLA-A0201 expression. After removal of unbound peptide and addition of brefeldin A to inhibit protein synthesis, the number of HLA-A0201 molecules remaining at the T2 cell surface was determined.
  • HLA-A0201 complexes with p210C, p210D, p210E and p210F formed complexes that were stable over 6-8 hours.
  • ⁇ 210A and p210B were less stable, reaching background levels in less than 1 hour of incubation.
  • EXAMPLE 14 P21Q PEPTIDE STIMULATION OF CD8 + IMMUNE RESPONSES: T CELLS GENERATED BY SYNTHETIC ANALOGUES RECOGNIZED NATIVE
  • the next experiment measured the ability of bcr-abl peptides to induce reactive precursor T cells with cognate T cell receptors.
  • synthetic b3a2 and b2a2 analogues were evaluated for their ability to stimulate peptide-specif ⁇ c CTL.
  • Cells from ten healthy HLA A0201 donors and 4 patients with chronic phase CML were assayed.
  • the peptides used were heteroclitic peptides p210A, p210B, p210C, p210D, and p210E, and CMLA3, p210Cn, p201Dn, and CMLA2, the native sequences corresponding to p210A-B, p210C, p210D, and p210E, respectively (Table 5).
  • the T cells elicited by p210C and p210F vaccination were able to recognize their respective native sequences (Figure 11).
  • the peptide CMLA2 the native sequence corresponding to p21 OF, is a weak MHC binder, and is expressed in the surface of CML blasts.
  • T cell lines obtained after several stimulations with p210C and b2a2A3 were assayed by chromium-51 release assays using peptide-pulsed target cell lines.
  • the cells were able to kill T2 cells pulsed with the heteroclitic peptides.
  • the cells were able to recognize and kill cells expressing the native peptide from which the heteroclitic peptide was derived ( Figures 14-15). As expected, the cells did not lyse T2 cells without peptide or T2 cells with control peptide, showing the specificity of the assay.
  • heteroclitic peptides of the present invention exhibit increased immunogenicity relative to the corresponding unmutated ("native") sequences in both healthy subjects and CML subjects;
  • T cells generated with the heteroclitic peptides recognize MHC molecules bearing the native peptides, even when the native peptide is a weak binder, and lyse target cells bearing the corresponding peptides;
  • Bcr-abl peptides are tested for ability to bind HLA class II molecules, and thus stimulate CD4 + T cells, as described in Example 6.
  • b3a2- bcr-abl derived peptides e.g. IVHS ATGFKQSSKALQRPVASDFEP; SEQ ID No: 62
  • b2a2-bcr-abl derived peptides e.g. VHSIPLTINKEEALQRPV ASDFE; SEQ ID No: 63
  • Wild-type bcr-abl peptides with ability to bind HLA class II molecules are identified.
  • EXAMPLE 16 MUTATION OF BCR-ABL HLA CLASS II PEPTIDES TO CONTAIN HETEROCLITIC HLA CLASS I MOLECULE EPITOPES [00265]
  • Bcr-abl peptides identified in the previous Example are mutated to contain buried heteroclitic HLA class I molecule epitopes, as described in Example 6.
  • the heteroclitic HLA class I molecule epitope introduced into the peptides is one of the sequences set forth in SEQ ID No: 45, 47-48, 50, 52-53, and 55-59.
  • bcr-abl peptides containing buried, heteroclitic HLA class I epitopes is tested, as described in Examples 7-8.
  • the peptides exhibit the ability to stimulate bcr-abl-specific T cells.
  • addition of the peptides to naive PMBC generates antigen-specific CD4 + T cells and CD8 + T cells. Both the CD4 + T cells and the CD8 + T cells recognize both the heteroclitic bcr-abl peptides and their native counterparts.
  • bcr-abl peptides of the present invention are immunogenic.
  • EXAMPLE 18 BCR-ABL PEPTIDES CONTAINING BURIED HLA CLASS I EPITOPES RECOGNIZE ANTIGEN-PRESENTING CELLS EXPOSED TO BCR-ABL-EXPRESSING TUMOR CELLS
  • CD4 + T cells from the previous Example are incubated with DC pulsed with tumor lysate from CML cells, then target recognition is measured by IFN- ⁇ ELISPOT.
  • CD4 + T cells generated by vaccination with bcr-abl peptides of the present invention recognize the peptide-pulsed DC, but not DC pulsed with bcr-abl-negative tumor cells.
  • vaccination with bcr-abl peptides of the present invention results in generation of antigen-specific T cells with activity against bcr-abl- expressing tumors.
  • EXAMPLE 19 ANTIGEN-SPECIFIC CD8* T CELLS GENERATED BY BCR-ABL PEPTIDES OF THE PRESENT INVENTION RECOGNIZE BCR-ABL-EXPRESSING
  • Antigen-specific CD8 + T cells are generated by incubating PBMC with either bcr-abl peptides containing buried, heteroclitic HLA class I epitopes, the corresponding wild-type bcr-abl peptides, the heteroclitic HLA class I epitope alone, or negative control peptide, as described in Example 8, then are incubated with bcr-abl-expressing tumor cells or bcr-abl-negative tumor cells and target recognition is determined by IFN- ⁇ ELISPOT or in vitro cytolysis assay.
  • CD8 + T cells generated by vaccination with either (a) peptides containing buried, heteroclitic HLA class I epitopes or (b) the heteroclitic HLA class I epitope alone recognize the DC, but not DC pulsed with bcr-abl-negative tumor cells, while vaccination with the corresponding wild-type bcr-abl peptides is less effective.
  • peptides containing buried, heteroclitic HLA class I epitopes are more potent and/or stronger than the heteroclitic HLA class I epitope alone in induction of antigen-specific CD8 + T cells.

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Abstract

L'invention concerne des peptides, des compositions immunogènes et des vaccins les comprenant, ainsi que des méthodes destinées à traiter un cancer exprimant bcr-abl, à réduire l'incidence de ce cancer et à induire des réponses immunitaires contre ledit cancer.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1951281A2 (fr) * 2005-10-17 2008-08-06 Sloan-Kettering Institute For Cancer Research Peptides de liaison wt1 hla de classe ii, compositions et methodes associees comprenant ces peptides
US9919037B2 (en) 2013-01-15 2018-03-20 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof
US10100087B2 (en) 2012-01-13 2018-10-16 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof
US10815273B2 (en) 2013-01-15 2020-10-27 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof
US11414457B2 (en) 2006-04-10 2022-08-16 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof

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US6156316A (en) * 1995-05-08 2000-12-05 Sloan-Kettering Institute For Cancer Research Oncogene fusion protein peptide vaccines

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US6156316A (en) * 1995-05-08 2000-12-05 Sloan-Kettering Institute For Cancer Research Oncogene fusion protein peptide vaccines

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1951281A2 (fr) * 2005-10-17 2008-08-06 Sloan-Kettering Institute For Cancer Research Peptides de liaison wt1 hla de classe ii, compositions et methodes associees comprenant ces peptides
EP1951281A4 (fr) * 2005-10-17 2009-10-28 Sloan Kettering Inst Cancer Peptides de liaison wt1 hla de classe ii, compositions et methodes associees comprenant ces peptides
EP2565201A1 (fr) * 2005-10-17 2013-03-06 Sloan-Kettering Institute For Cancer Research Peptides de liaison WT1 HLA de classe II et compositions et procédés comprenant ceux-ci
US10221224B2 (en) 2005-10-17 2019-03-05 Memorial Sloan Kettering Cancer Center WT1 HLA class II-binding peptides and compositions and methods comprising same
US11548924B2 (en) 2005-10-17 2023-01-10 Memorial Sloan Kettering Cancer Center WT1 HLA class II-binding peptides and compositions and methods comprising same
US11414457B2 (en) 2006-04-10 2022-08-16 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof
US10100087B2 (en) 2012-01-13 2018-10-16 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof
US10815274B2 (en) 2012-01-13 2020-10-27 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof
US9919037B2 (en) 2013-01-15 2018-03-20 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof
US10815273B2 (en) 2013-01-15 2020-10-27 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof

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