WO2007047653A2 - Analogues peptidiques de liaison synthetiques hla d'une enzyme v617f jak2 mutante et leurs utilisations - Google Patents

Analogues peptidiques de liaison synthetiques hla d'une enzyme v617f jak2 mutante et leurs utilisations Download PDF

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WO2007047653A2
WO2007047653A2 PCT/US2006/040517 US2006040517W WO2007047653A2 WO 2007047653 A2 WO2007047653 A2 WO 2007047653A2 US 2006040517 W US2006040517 W US 2006040517W WO 2007047653 A2 WO2007047653 A2 WO 2007047653A2
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seq
cells
synthetic peptide
peptide
cytotoxic
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WO2007047653A3 (fr
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David A. Scheinberg
Rena J. May
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Memorial Sloan Kettering Cancer Center
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)

Definitions

  • This invention relates to the fields of immunology and therapies for neoplastic disorders. More specifically, this invention relates to the use of synthetic analogue peptides of mutant V617F JAK2 enzyme to induce heteroclitic human T cell responses against cells presenting mutant V617F JAK2 in subjects -with myeloproliferative disorders.
  • MPD polycythemia vera
  • CT essential thrombocythemia
  • IMF chronic idiopathic myelofibrosis
  • CML chronic myelogenous leukemia
  • CML is invariably associated with the Philadelphia chromosome, which is generated from the t(9,22) reciprocal chromosomal translocation.
  • the resulting bcr-abl gene product leads to a disregulated AbI protein tyrosine kinase which has been shown to be the transforming event in CML.
  • the bcr-abl fusion protein is now used as a molecular diagnostic tool and an effective target for various treatment and therapies.
  • Recently, a number of independent groups have published studies defining an amino acid point mutation (V617F) in the JAK2 protein tyrosine kinase as the molecular basis for the enhanced myeloproliferation and clonal dominance that characterizes bcr-abl negative MPDs. This mutation as been described, on average, in 85% of patients with PV, 40% of patients with ET and 45% of those with IMF, yet not in any normal patients.
  • Endogenous erythroid colony formation is a characteristic in a significant proportion of patients with a chronic myeloproliferative disorder in which erythroid progenitor cells obtained from the marrow or peripheral blood of patients proliferate in semi-solid, serum-containing cultures in the absence of exogenous EPO.
  • endogenous erythroid colony formation is the only relatively useful diagnostic test for myeloproliferative disorders.
  • the assay is technically demanding and is positive in only about 35-80% of patients with these diseases.
  • the molecular basis of the erythropoietin negative endogenous erythroid colonies is likely to lie downstream of the EPO receptor.
  • the EPO receptor belongs to a unique class of receptors that contain no cytoplasmic tyrosine kinase domains or intracellular signaling motifs. These receptors, termed type I cytokine receptors must rely on intracellular signaling molecules, such as the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. For the type I cytokine receptors, all signaling begins with the JAK molecules that are non-covalently bound to the "boxl/2 motifs present in the juxtamembrane cytoplasmic region of the receptors. Upon ligand binding, a large conformational shift of the receptor ensues, bringing the cytoplasmic domains of two receptors closer together.
  • JAK Janus kinase
  • STAT signal transducers and activators of transcription
  • JAK kinase phosphorylates tyrosine residues on the initiating receptor, which can then serve as a docking site for src homology SH2 bearing secondary signaling molecules, such as nascent transcription factors (STATs), adaptor molecules (She, Gab/IRS), and kinase regulatory subunits (p85 phosphoinositol-3-kinase), amongst others.
  • STATs nascent transcription factors
  • IVS kinase regulatory subunits
  • JAK2 is the primary tyrosine kinase associated with signaling from EPO, SCF, GM-CSF, IL-3, TPO and IGF-I receptors. Hematopoietic progenitors in myeloproliferative disorders are hypersensitive to all of these growth factors.
  • the prior art is deficient in the lack of synthetic analogue peptides of mutant V617F JAK2 enzyme that could generate an immune response that not only recognizes the immunizing epitopes, but that also cross reacts with the original mutant V617F JAK2 peptides.
  • the prior art is deficient in synthetic peptide analogs of mutant V617F JAK2 with both improved HLA binding and improved ability to elicit a greater immunogenic response against myeloproliferative disorders.
  • the present invention fulfills this longstanding need and desire in the art.
  • the present invention is directed to a synthetic peptide.
  • the synthetic peptide comprises a sequence of amino acids containing at least a segment that is an analogue of a mutant V617F JAK2 peptide that specifically binds to HLA class I or HLA class II molecules on a cell characteristic of a myeloproliferative disorder or disease in a subject.
  • the synthetic peptide further may comprise an immunogenic carrier linked thereto.
  • the present invention is directed to a related synthetic peptide that binds HLA A2 molecules.
  • the synthetic peptide has an analogues segment with an amino acid sequence of VLWYGVCFC (SEQ ID NO: 12), VLNYGVCFV (SEQ ID NO: 13), VLWYGVCFV (SEQ ID NO: 14), LNWGVCFCG (SEQ ID NO: 15), LMYGVCFCG (SEQ ID NO: 16), LLYGVCFCG (SEQ ID NO: 17), LNYGVCFCV (SEQ ID NO: 18), LNWGVCFCV (SEQ ID NO: 19), LMYGVCFCV (SEQ ID NO: 20), LLYGVCFCV (SEQ ID NO: 21), LMWGVCFCV (SEQ ID NO: 22), LLWGVCFCV (SEQ ID NO: 23), FMGDENILV (SEQ ID NO: 24), or FLGDENILV (SEQ ID NO: 25).
  • the present invention is directed to another related synthetic peptide that binds HLA A3 molecules.
  • the synthetic peptide has an analogues segment with an amino acid sequence of VLYYGVCFC (SEQ ID NO: 26), VLFYGVCFC (SEQ ID NO: 27), VLNYGVCFK (SEQ ID NO: 28), VLWYGVCFK (SEQ ID NO: 29), VLFYGVCFK (SEQ ID NO: 30), LNMGVCFCG (SEQ ID NO: 31), LNLGVCFCG (SEQ ID NO: 32), LNYGVCFCK (SEQ ID NO: 33), LLYGVCFCK (SEQ ID NO: 34), LMYGVCFCK (SEQ ID NO: 35), LNMGVCFCGD (SEQ ID NO: 36), LNLGVCFCGD (SEQ ID NO: 37), LNYGVCFCGK (SEQ ID NO: 38), LLYGVCFCGK (SEQ ID NO: 39), LMYGVCFCGK (SEQ ID NO: 40), VCFCCG
  • the present invention also is directed to a pharmaceutical composition comprising a therapeutically effective amount of the synthetic peptides described herein or a DNA encoding the synthetic peptide and a suitable carrier.
  • the present invention is directed further to an immunogenic composition comprising an immunogenically effective amount of the synthetic peptide described herein and a pharmaceutically acceptable carrier, adjuvant or diluent or a combination thereof.
  • the present invention is directed further yet to a method of inducing formation and proliferation of human cytotoxic T cells that produce a heteroclitic immune response against cells characteristic of a myeloproliferative disorder or disease.
  • the method comprises contacting human immune cells with one or more of the synthetic peptides described herein to activate the immune cells.
  • the formation and proliferation of the human cytotoxic T cells reactive against the activated cells presenting at least the analogue segment of said synthetic peptide is thereby induced such that the proliferating T cells cross react with cells characteristic of a myeloproliferative disorder or disease presenting a mutant V617F peptide from which the analogue segment is derived.
  • the human cytotoxic T cells thereby are capable of producing a heteroclitic immune response against the cells characteristic of a myeloproliferative disorder or disease.
  • the present invention is directed further still to a method of inducing a heteroclitic immune response in a subject.
  • the method comprises administering to the subject an effective amount of the immunogenic composition described herein and activating human immune cells with the immunogenic composition.
  • the formation and proliferation of human cytotoxic T cells are induced against the activated cells presenting at least the analogue segment of the synthetic peptide comprising the immunogenic composition.
  • the human cytotoxic T cells will cross- react with a cell characteristic of a chronic myeloproliferative disorder or disease presenting a mutant V617F JAK2 peptide from which the analogue segment is derived, thereby inducing the heteroclitic immune response.
  • the method may further comprise isolating the cytotoxic T-cells from the subject and donating the isolated cytotoxic T-cells to another subject having a myeloproliferative disorder or disease.
  • Figures 1A-1D show T2 stabilization assays using peptides predicted to bind to HLA A0201 and HLA A0301 molecules.
  • the peptides are derived from mutant V617F JAK2 peptides of SEQ ID NO: 3 ( Figure IA) and SEQ ID NO: 7 ( Figure IB) for HLA A0301 molecules and peptides of SEQ ID NO: 3 ( Figure 1C) and SEQ ID NO: 5 ( Figure ID) for HLA A0301 molecules.
  • Fluorescence index represents the median fluorescence on the peptide tested divided by no peptide as a function of peptide concentration.
  • Figures 2A-2E depict a gamma interferon ELISPOT assay using
  • CD3+ T cells from normal HLA A0201 ( Figures 2A-2B) and HLA A0301 ( Figures 2C-2D) donors.
  • CD3+ T cells were stimulated three times with JAK2 heteroclitic peptides and challenged with CD 14+ cells pulsed with the stimulating heteroclitic peptide (black), CD14+ cells pulsed with the corresponding native JAK2 peptide (white), CE14+ cells pulsed with the corresponding V617F mutant peptide (hatched), CD 14+ cells pulsed with an irrelevant A0201 peptide (grey). Unchallenged cells are represented by white bars.
  • CD3+ cells pulsed with J2.5 did not grow and were therefore not tested.
  • Figure 4 depicts a gamma interferon ELISPOT assay using CD3+ T cells from an HLA DRB1*7O1/11XX donor.
  • CD3+ T cells were stimulated three times with JAK2 mutant and WT class II peptides and challenged with CD 14+ cells pulsed with JAK2DR WT peptide (white), the JAK2DR V617F mutant peptide (hatched), CD14+ cells pulsed with an Irrelevant class II peptide (grey). Unchallenged cells are represented by white bars and HEL (HLADRBl*701/1303) V617F+ cell line is represented by black bars.
  • a synthetic peptide comprising a sequence of amino acids containing at least a segment that is an analogue of a mutant V617F JAK2 peptide that specifically binds to HLA class I or HLA class II molecules on a cell characteristic of a myeloproliferative disorder or disease in a subject.
  • the method may comprise an immunogenic carrier linked thereto.
  • the immunogenic carrier may be a protein, a peptide or an antigen-presenting cell.
  • Examples of a protein or of a peptide may be keyhole limpet hemocyanin, an albumin or a polyamino acid.
  • An example of an antigen-presenting cell is a dendritic cell.
  • a total number of amino acids in the analogue segment is about 70% to about 130% of a total number of amino acids in the mutant V617F JAK2 peptide.
  • the analogue segment may have about 8 to about 12 amino acids and may bind to HLA A2 or to HLA A3 molecules.
  • An example of an HLA A2 molecule is HLA A0201 and an example of an HLA A3 molecule is HLA A0301.
  • the myeloproliferative disorder or disease may be polycythemia vera (PV), essential thrombocythemia (ET), chronic idiopathic myelofibrosis (IMF), chronic myelogenous leukemia, chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia (CNL) 5 hypereosinophilic syndrome (HES), systemic mastocytosis (SM), or myelodysplastic syndrome (MDS).
  • PV polycythemia vera
  • EMF essential thrombocythemia
  • IMF chronic idiopathic myelofibrosis
  • CMML chronic myelogenous leukemia
  • CMML chronic myelomonocytic leukemia
  • CTL chronic neutrophilic leukemia
  • HES hypereosinophilic syndrome
  • SM systemic mastocytosis
  • MDS myelodysplastic syndrome
  • VLWYGVCFC SEQ ID NO: 12
  • VLNYGVCFV SEQ ID NO: 13
  • VLWYGVCFV SEQ ID NO: 14
  • LNWGVCFCG SEQ ID NO: 15
  • LMYGVCFCG SEQ ID NO: 16
  • LLYGVCFCG SEQ ID NO: 17
  • LNYGVCFCV SEQ ID NO: 18
  • LNWGVCFCV SEQ ID NO: 19
  • LMYGVCFCV SEQ ID NO: 20
  • LLYGVCFCV SEQ ID NO: 21
  • LMWGVCFCV SEQ ID NO: 22
  • LLWGVCFCV SEQ ID NO: 23
  • FMGDENILV SEQ ID NO: 24
  • FLGDENILV FLGDENILV
  • VLYYGVCFC SEQ ID NO: 26
  • VLFYGVCFC SEQ ID NO: 27
  • VLNYGVCFK SEQ ID NO: 28
  • VLWYGVCFK SEQ ID NO: 29
  • VLFYGVCFK SEQ ID NO: 30
  • LNMGVCFCG SEQ ID NO: 31
  • LNLGVCFCG SEQ ID NO: 32
  • LNYGVCFCK SEQ ID NO: 33
  • LLYGVCFCK SEQ ID NO: 34
  • LMYGVCFCK SEQ ID NO: 35
  • LNMGVCFCGD SEQ ID NO: 36
  • LNLGVCFCGD SEQ ID NO: 37
  • LNYGVCFCGK SEQ ID NO: 38
  • LLYGVCFCGK SEQ ID NO: 39
  • LMYGVCFCGK SEQ ID NO: 40
  • VCFCCGDENK SEQ ID NO: 41
  • VMFCGDENI SEQ ID NO: 42
  • the analogue segment may have about 14 to about 25 amino acids and may bind to HLA DRB molecules.
  • An example of an HLA DRB molecule is HLA DRB 1501.
  • the amino acid sequence of these analogue segments binding to HLA DRB molecules may be G V C F C G D E N I L V Q E F (SEQ ID NO: 46).
  • a synthetic peptide with an amino acid sequence VLWYGVCFC (SEQ ID NO: 12), VLNYGVCFV (SEQ ID NO: 13), VLWYGVCFV (SEQ ID NO: 14), LNWGVCFCG (SEQ ID NO: 15), LMYGVCFCG (SEQ ID NO: 16), LLYGVCFCG (SEQ ID NO: 17), LNYGVCFCV (SEQ ID NO: 18), LNWGVCFCV (SEQ ID NO: 19), LMYGVCFCV (SEQ ID NO: 20), LLYGVCFCV (SEQ ID NO: 21), LMWGVCFCV (SEQ ID NO: 22), LLWGVCFCV (SEQ ID NO: 23), FMGDENILV (SEQ ID NO: 24), or FLGDENILV (SEQ ID NO: 25) or combinations thereof.
  • VLYYGVCFC SEQ ID NO: 26
  • VLFYGVCFC SEQ ID NO: 27
  • VLNYGVCFK SEQ ID NO: 28
  • VLWYGVCFK SEQ ID NO: 29
  • VLFYGVCFK SEQ ID NO: 30
  • LNMGVCFCG SEQ ID NO: 31
  • LNLGVCFCG SEQ ID NO: 32
  • LNYGVCFCK SEQ ID NO: 33
  • LLYGVCFCK SEQ ID NO: 34
  • LMYGVCFCK SEQ ID NO: 35
  • LNMGVCFCGD SEQ ID NO: 36
  • LNLGVCFCGD SEQ ID NO: 37
  • LNYGVCFCGK SEQ ID NO: 38
  • LLYGVCFCGK SEQ ID NO: 39
  • LMYGVCFCGK SEQ ID NO: 40
  • VCFCCGDENK SEQ ID NO: 41
  • VMFCGDENI SEQ ID NO: 42
  • a synthetic peptide with an amino acid sequence GVCFCGDENILVQEF (SEQ ID NO: 46).
  • a pharmaceutical composition comprising a therapeutically effective amount of the synthetic peptide described supra or a DNA encoding the synthetic peptide; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a DNA encoding the synthetic peptide
  • the DNA may be inserted into a vector or into an antigen-presenting cell.
  • An example of an antigen presenting cell is a dendritic cell.
  • an immunogenic composition comprising an immunogenically effective amount of the synthetic peptides described supra and a pharmaceutically acceptable carrier, adjuvant or diluent or a combination thereof.
  • the carrier may be a protein, a peptide or an antigen-presenting cell linked to the synthetic peptide.
  • a protein or peptide carrier are keyhole limpet hemocyanin, an albumin or a polyamino acid.
  • An example of an antigen-presenting cell is a dendritic cell.
  • the synthetic peptides and analogue segments comprising the synthetic peptides are as described supra. In this embodiment, the analogue segments may be derived from the mutant V617F JAK2 peptide described supra.
  • a method of inducing formation and proliferation of human cytotoxic T cells that produce a heteroclitic immune response against cells characteristic of a myeloproliferative disorder or disease comprising contacting human immune cells with one or more of the synthetic peptides of described supra to activate the immune cells; and inducing formation and proliferation of the human cytotoxic T cells reactive against the activated cells presenting at least the analogue segment of the synthetic peptide, where the proliferating T cells will cross react with cells characteristic of a myeloproliferative disorder or disease presenting a mutant V617F peptide from which the analogue segment is derived, said human cytotoxic T cells thereby capable of producing a heteroclitic immune response against the cells characteristic of a myeloproliferative disorder or disease.
  • the method comprises providing a DNA encoding the synthetic peptide and expressing the DNA.
  • the DNA may be inserted into a vector or into an antigen presenting cell.
  • the human immune cells may be contacted in vivo in a subject having a myeloproliferative disorder or disease.
  • the human immune cells may be contacted in vivo in a donor where the method further comprises obtaining the cytotoxic T cells from the donor; and infusing the cytotoxic T cells into the subject having a chronic myeloproliferative disorder or disease.
  • the human immune cells are contacted ex vivo where the method further comprises obtaining human immune cells from a donor prior to the contacting step; and infusing the activated immune cells into a subject having a myeloproliferative disorder or disease prior to the inducing step.
  • the human immune cells are contacted ex vivo where the method further comprises obtaining human immune cells from a donor prior to the contacting step, where, after the contacting step, formation and proliferation of the cytotoxic T-cells occurs ex vivo; and infusing the cytotoxic T-cells into a subject having a myeloproliferative disorder or disease.
  • the human immune cells may be peripheral blood mononuclear cells, bone marrow cells, dendritic cells, or macrophages.
  • the cytotoxic T cells may be CD8+ or CD4+ or a combination thereof.
  • the myeloproliferative disorder or disease may be polycythemia vera (PV), essential thrombocythemia (ET), chronic idiopathic myelofibrosis (IMF), chronic myelogenous leukemia, chronic myelomonocytic leukemia (CMML) 5 chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES), systemic mastocytosis (SM), or myelodysplastic syndrome (MDS).
  • PV polycythemia vera
  • EDF essential thrombocythemia
  • IMF chronic idiopathic myelofibrosis
  • CML chronic myelogenous leukemia
  • CML chronic myelomonocytic leukemia
  • the cytotoxic T cells may CD8+ T cells formed against one or more of the synthetic peptide analogue segments with an amino acid sequence of VLWYGVCFC (SEQ ID NO: 12), VLNYGVCFV (SEQ ID NO: 13), VLWYGVCFV (SEQ ID NO: 14), LNWGVCFCG (SEQ ID NO: 15), LMYGVCFCG (SEQ ID NO: 16), LLYGVCFCG (SEQ ID NO: 17), LNYGVCFCV (SEQ ID NO: 18), LNWGVCFCV (SEQ ID NO: 19), LMYGVCFCV (SEQ ID NO: 20), LLYGVCFCV (SEQ ID NO: 21), LMWGVCFCV (SEQ ID NO: 22), LLWGVCFCV (SEQ ID NO: 23), VLYYGVCFC (SEQ ID NO: 26), VLFYGVCFC (SEQ ID NO: 27), VLNYGVCFK (SEQ ID NO: 28), VLWYGVCFK (SEQ ID NO:
  • a method of treating a myelogenous disorder or disease in a subject comprising administering the pharmaceutical composition described supra to the subject; and inducing a heteroclitic response by cytotoxic T-cells that recognize at least the analogue segment of the synthetic peptide against cells characteristic of a myelogenous disorder or disease presenting a mutant V617F JAK2 peptide from which the analogue segment is derived such that the cytotoxic T-cells recognize or kill the characteristic cells thereby treating the myelogenous disorder or disease.
  • the cytotoxic T cells may CD8+ or CD4+ or a combination thereof. These CD8+ and/or CD4+ cytotoxic T cells may recognize the synthetic peptide analogue segments with the amino acid sequences described supra. Also, the myeloproliferative disorder or disease is as described supra.
  • a method of inducing a heteroclitic immune response in a subject comprising administering to the subject an effective amount of the immunogenic composition described supra; activating human immune cells with the immunogenic composition; and inducing formation and proliferation of human cytotoxic T cells against the activated cells presenting at least the analogue segment of the synthetic peptide comprising the immunogenic composition, where the human cytotoxic T cells will cross-react with a cell characteristic of a myeloproliferative disorder or disease presenting a mutant V617F JAK2 peptide from which the analogue segment is derived, thereby inducing the heteroclitic immune response.
  • the method may comprise isolating the cytotoxic T-cells from the subject and donating the isolated cytotoxic T-cells to another subject having a myeloproliferative disorder or disease.
  • the cytotoxic T cells may CD8+ or CD4+ or a combination thereof. These CD8+ and/or CD4+ cytotoxic T cells form against the synthetic peptide analogue segments with the amino acid sequences described supra.
  • the myeloproliferative disorder or disease is as described supra.
  • the term, "a” or “an” refers to one or more.
  • the words "a” or “an” refers to one or more than one.
  • the term “heteroclitic response” refers to cross- reaction of cytotoxic T cells activated with an analogue peptide segment from mutant V617F JAK2 protein with mutant V617 JAK2 protein presented on antigen presenting cells without cross-reaction with native JAK2 protein by these cytotoxic T cells.
  • the term "contacting" in terms of activating target immune cells to elicit a subsequent immune response refers to any suitable delivery method of bringing an immunogenic agent into contact with the target cells. In vitro or ex vivo this is achieved by exposing the target cells to the agent in a suitable medium. For in vivo applications, any known method of administration is suitable as described herein.
  • Neoplastic disease refers to a disease or disorder associated with or caused by a mass of tissue or cells or neoplasm characterized by, inter alia, abnormal cell proliferation.
  • the abnormal cell proliferation results in growth of these tissues or cells that exceeds and is uncoordinated with that of the normal tissues or cells and persists in the same excessive manner after the stimuli which evoked the change ceases or is removed.
  • Neoplastic tissues or cells show a lack of structural organization and coordination relative to normal tissues or cells which usually results in a mass of tissues or cells which can be either benign or malignant. As would be apparent to one of ordinary skill in the art, a neoplastic disease may be benign or malignant.
  • the terms “treating” or “treatment” includes prophylactic treatment as well as alleviation of ongoing or intermittent symptoms occurring in a neoplastic disease or disorder, such as, preferably, a myeloproliferative or myelodysplastic disease or disorder.
  • the terms “effective amount” or “therapeutically effective amount” are interchangeable and refer to an amount that results in an improvement or remediation of the symptoms of the disease or condition. Those of skill in the art understand that the effective amount may improve the patient's or subject's condition, but may not be a complete cure of the disease and/or condition.
  • the term “subject” refers to any target of an immunotherapeutic treatment, preferably a mammal, more preferably a human.
  • synthetic immunogenic peptides with an amino acid sequence containing at least a highly homologous analogue segment of a V617F JAK2 peptide that demonstrates improved binding over the mutant peptide to human class I, i.e., HLA-A2 and HLA-A3, and class II HLA, i.e., HLA-DRB, molecules.
  • These synthetic peptides or analogue segments can stimulate T-cells to cross-react with the mutant V617F JAK2 peptide, thus eliciting a heteroclitic immune response that will recognize or kill cells presenting the mutant V617F JAK2 peptide.
  • T cells stimulated or activated by the synthetic immunogenic peptides do not recognize the native, nonmutated JAK2 kinase.
  • the V617F mutation is not found in T lymphocytes, leaving them fully functional. It is contemplated that at least the analogue segments comprising the synthetic peptides will bind with more affinity to the HLA class I and class II molecules that are instrumental in presenting the analogue segments to the T-cells than the mutant V617F peptide itself.
  • Such cells are characteristic of a neoplastic disease or neoplastic disorder, for example, a myeloproliferative or myelodysplastic disease or disorder, including chronic, acute and atypical leukemias.
  • Peptides were identified based on their predictive binding affinity to HLA molecules using a peptide library based algorithm which ranks the binding of peptides on a predicted half-time coefficient from common HLA class molecules.
  • Any predictive algorithms available publicly or commercially may be utilized, for example, the online software BIMAS and/or SYFPEITHI, as more fully described in Example 1, may be used to predict binding scores to HLA A0201 and HLA A0301 molecules or to HLA DRB molecules.
  • Such synthetic analog peptides are generated by introducing amino acid point mutations into certain HLA anchor motifs, thereby enhancing the peptide/HLA binding affinities, while not interfering with the peptide/T-cell receptor (TCR) binding.
  • the synthetic peptide analogue segments are designed by making one or two amino acid substitutions in anchor or auxiliary residues.
  • the mutated JAK2 peptides, from which the synthetic peptides binding to HLA class I molecules are designed, particularly described herein are nonamers or decamers encompassing the anchor or auxiliary residues
  • analogues may be designed having about 70% to about 130% of the amino acids in the mutated JAK2 peptides.
  • the synthetic peptide analogues binding to class I HLA A2 or HLA A3 molecules may have about 8-12 amino acids.
  • the present invention also provides a pharmaceutical composition of a therapeutic amount of the synthetic peptides or analogue segments or a genetic sequence or DNA encoding the same and a pharmaceutical carrier, as is known in the art.
  • the pharmaceutical composition may be formulated with the pharmaceutical carrier for administration by any of the many techniques known to those of skill in the art.
  • the pharmaceutical composition may be administered parenterally, intravenously, subcutaneously, intradermally, intramucosally, topically, orally, or by inhalation. Therefore, it is contemplated that the synthetic peptides or analogue segments or pharmaceutical compositions thereof may be used in the preparation of an immunogenic composition suitable to effect immunization of a subject.
  • the immunogenic composition may comprise a carrier or a suitable adjuvant to boost immune response or a combination thereof, as are known in the art.
  • the immunogenic composition further may comprise a diluent standard in the art as described herein.
  • the immunogenic composition may comprise a vaccine.
  • a carrier may comprise one or more proteins or peptides.
  • carriers are well known and may be, although not limited to keyhole limpet hemocyanin, an albumin, such as human serum albumin or a polyamino acid.
  • a carrier may comprise a live antigen-presenting cell, such as a dendritic cell, which presents the synthetic peptides described herein.
  • a suitable adjuvant may be incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, alum, QS21, BCG BCG, montinide, and GMCSF.
  • These compositions further may comprise a physiologically acceptable diluent, e.g., water, phosphate buffered saline or saline.
  • a genetic sequence encoding a synthetic peptide or an analogue segment thereof may be delivered as naked DNA to an individual via appropriate methods known in the art.
  • the genetic sequence may be introduced or inserted into a suitable vector, such as for example, but not limited to, attenuated viral or bacterial vectors, as are standard in the art.
  • the naked DNA or vectors comprising the genetic sequence or DNA may be transduced into an antigen-presenting cell, e.g., a dendritic cell.
  • the genetic sequence, DNA, vector or transduced antigen-presenting cell may be introduced into an individual in need of the treatment or into a healthy donor whereupon the DNA encoding the genetic sequence expresses the synthetic peptide to elicit a cytotoxic T-cell response.
  • Donor T-cells may then be infused into a patient in need thereof.
  • the pharmaceutical or immunogenic compositions may be used to treat a neoplastic disease or a neoplastic disorder such as a myeloproliferative disease or disorder, including chronic and atypical myeloproliferative disorders and acute, chronic and atypical leukemias.
  • a neoplastic disease or a neoplastic disorder such as a myeloproliferative disease or disorder, including chronic and atypical myeloproliferative disorders and acute, chronic and atypical leukemias.
  • the pharmaceutical or immunogenic compositions have a therapeutic or immunotherapeutic effect against polycythemia vera (PV), essential thrombocythemia (ET), chronic idiopathic myelofibrosis (IMF), or chronic or acute leukemias, such as, but not limited to, chronic myelogenous leukemia and atypical leukemias, chronic myelomonocytic leukemia (CMML) and chronic neutrophilic leukemia (CNL), and against other atypical myeloproliferative disorders, such as, hypereosinophilic syndrome (HES), systemic mastocytosis (SM) and myelodysplastic syndrome (MDS).
  • PV polycythemia vera
  • ETF chronic idiopathic myelofibrosis
  • chronic or acute leukemias such as, but not limited to, chronic myelogenous leukemia and atypical leukemias, chronic myelomonocytic leukemia (CMML) and chronic neutr
  • the synthetic peptides or synthetic analogue segments thereof or genetic sequences encoding the same or the pharmaceutical or the immunogenic compositions thereof can induce human cytotoxic T cells to produce a heteroclitic immune response against cells presenting the V167F JAK2 mutant peptide, but not the native, non-mutated JAK2 peptide.
  • Contacting human immune cells with at least the analogue segment that is or comprises the synthetic peptides activates the immune cells to induce formation and proliferation of human cytotoxic T cells that will recognize or react against a cell presenting the synthetic peptide.
  • Such cytotoxic T cells cross react with human cells presenting the mutant JAK2 peptide from which the analogue segment is derived thereby producing a heteroclitic response.
  • a synthetic peptide or synthetic analog segment or genetic sequences encoding the same or the pharmaceutical or the immunogenic compositions thereof that binds to HLA DRB molecules may be used to induce a CD4+ T cell response.
  • a CD4+ T cell response could be a heteroclitic response or could be adjunctive to or enhance the heteroclitic CD8+ T cell response generated against cells presenting the V167F JAK2 mutant peptide.
  • synthetic peptide analogues binding to class II HLA DRBl molecules may have about 14-25 amino acids.
  • the synthetic peptides or analogue segments thereof described herein may be used to activate T-cells ex vivo or in vivo.
  • the synthetic peptides or analogue segments thereof or DNA encoding the same may be administered to a patient or to a healthy donor to induce cytotoxic T-cells. If administered to a healthy donor these cytotoxic T-cells are obtained from the donor and infused into an individual in need of them, such as an individual with an active cancer, in remission from a cancer or at risk for developing a cancer.
  • the T cells are obtained from a patient or from a healthy donor and are incubated in the presence of antigen presenting cells and a synthetic peptide or at least an analogue segment thereof to activate the T-cells.
  • the activated T-cells subsequently are infused back into the patient where they will recognize and/or destroy cells presenting the V617F mutant JAK2 peptide.
  • human immune cells may be incubated with the synthetic peptide or at least an analogue segment thereof whereupon the activated immune cells are infused back into the patient to induce T-cell production against both the activated cells and cell presenting the native peptide.
  • immune cells may be peripheral blood mononuclear monocytic cells, bone marrow cells, dendritic cells, or macrophages.
  • the synthetic peptide or at least an analogue segment thereof or pharmaceutical or immunological compositions thereof induces an immune response in a subject, preferably, although not limited to, a CD4+/HLA DRB class II response and/or a CD8+ HLA A class I immune response.
  • the synthetic peptides or at least an analogue segment thereof may be used in a method of immunizing a subject against a neoplastic disease or disorder presenting HLA molecules, e.g., a myeloproliferative or myelodysplastic disease or disorder, including, but not limited to, chronic and acute leukemias, such as chronic myelogenous leukemia.
  • immunizing or immunization of a subject encompasses full and partial immunization whereby the subject becomes fully immune to the condition or partially immune to the condition.
  • the subject may be a mammal, preferably a human.
  • the subject may have a neoplastic disease or disorder, preferably a myeloproliferative or myelodysplastic disease or disorder, including chronic and acute leukemias, more preferably a chronic myeloproliferative disease or disorder, which may be active or in remission, prior to immunization.
  • a neoplastic disease or disorder preferably a myeloproliferative or myelodysplastic disease or disorder, including chronic and acute leukemias, more preferably a chronic myeloproliferative disease or disorder, which may be active or in remission, prior to immunization.
  • the subject may be immunized prior to such development.
  • risk factors such as environmental risk factors or personal risk factors, such as family history, genetic makeup or behavior, to make a determination of risk in the subject.
  • compositions and immunogenic compositions may be administered one or more times to achieve a therapeutic or an immunogenic effect. It is well within the skill of an artisan to determine dosage or whether a suitable dosage comprises a single administered dose or multiple administered doses. As is well known in the art, a specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity, e.g., progression or remission, of the particular disease undergoing therapy. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • Synthetic peptides Peptides were selected from the larger population of all JAK2 peptides spanning 10 amino acids upstream and 10 amino acids downstream of the V617F mutation (amino acids 607-627) by scanning for peptides found in myeloproliferative disorders with a potential binding capacity for HLA-A0201 (about 40% of the Caucasian population) and HLA-A0301 (about 20% of the Caucasian population) using the computer algorithms. Analog sequences with positive scores were selected first, and the corresponding native sequences were included for comparison.
  • peptides used in this study were purchased and synthesized by Genemed Synthesis Inc, (San Francisco, CA) using fluorenylmethoxycarbonyl chemistry and solid phase synthesis and purified by high-pressure liquid chromatography. The quality of the peptides was assessed by high-performance liquid chromatography analysis, and the expected molecular weight was observed using matrix-assisted laser desorption mass spectrometry. Peptides were sterile and 70-90% pure. The peptides were dissolved in DMSO and diluted in phosphate- buffered saline (PBS; pH 7.4) or saline at 5mg/ml and were stored at -80 0 C.
  • PBS phosphate- buffered saline
  • Irrelevant control peptides used for in vitro experiments were: A0201 class I peptides HIV pol (ILKEPVHGV; SEQ ID NO: 48), CML F (YLKALQRPY; SEQ ID NO: 48) or HBV (FLPSDYFPSV; SEQ ID NO: 50); A0301 class I peptides HIV or CML; and RAS (TEYKLWVGAPGVGKSALTIQ; SEQ ID NO: 51) or CML b2a2 (VHSIPLTINKEEALQRPVASDFE; SEQ ID NO: 52) for Class II.
  • Cell lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS), penicillin, streptomycin, 2mM glutamine and 2-mercaptoethanol at 37°C/5% CO 2 .
  • FCS fetal calf serum
  • T2 an A0201+, TAP-deficient human cell line that cannot present endogenous peptides to the surface of the cells was obtained from the ATCC.
  • 293T cells were transfected with letivirus plasmids including: the packaging construct, envelope-coding plasmid and the transfer vector containing GFP and encoding either the WT JAK2, the mutated V617F JAK2 or an empty vector.
  • 293T cells were incubated overnight and viral supernatants were collected and concentrated to 1-5 10 3 titer/ml.
  • 5 x 10 5 SKLY-16, an A0201+ human B cell lymphoma obtained from the ATCC were then infected with 1 ml of lentivirus along with 8 ug/ml of Polybrene. Cells were grown, and infection was repeated at 6-12 hour intervals. Target cells were then sorted by FACs for GFP expression.
  • T2 stabilization assay 1 x 10 6 cells T2 cells/ml were incubated overnight at 37°C in FCS-free RPMI medium supplemented with 10 ⁇ g/ml human ⁇ 2-microglobulin ( ⁇ 2m; Sigma, St Louis, MO) in the absence (negative control) or presence of either control HBV peptide or JAK2 test peptides at various final concentrations ranging from 100-1 ⁇ g/ml. Cells were then incubated with 5 ug/ml of brefeldin-A (Sigma) for 2 hours at 37°C.
  • T2 cells were then washed twice with FACS buffer, and incubated for 30 min at 4 0 C with a saturating amount of FITC labeled anti-human HLA-A2 antibody, (BB7.2. BD PharmingenTM). Fluorescence was assayed on a CytomicsTM FC 500, (Beckman Coulter) and analyzed using the FlowJo program. Each concentration of peptide (1, 10, 50 and 100 mg/ml) was assayed in triplicate wells, and each binding assay was repeated three times. The mean intensity of fluorescence (MIF) for each peptide is calculated by dividing the mean fluorescence of T2 + test peptide by T2 without peptide.
  • MIF mean intensity of fluorescence
  • PBMCs peripheral blood mononuclear cells
  • DCs Monocyte derived dendritic cells
  • the adherent cells were cultured for 7 days in RPM 1640/1-5% autologous plasma, 500 U/mL recombinant human IL-4 (R&D Systems), and 1000 U/mL recombinant human GMCSF (Immunex, Seattle).
  • IL4+ stimulation assays only, 10 ug/ml peptide was added to immature DCs on day 5.
  • maturation cytokine cocktail was added (IL4, GMCSF, 400 IU/ml ILl-beta (R&D Systems), 1000 IU/ml IL-6 (R&D Systems), 10 ng/ml TNF-alpha (R&D Systems) and 1 ug/ml PGE2 (Sigma)).
  • CD3+ T lymphocytes were isolated from the same donors using negative selection by depletion with an anti-CDl lb, anti-CD56 and CD19 MoAb (Miltenyi, CA), and stimulated at a 10:1 effecto ⁇ target (E:T) ratio with the monocyte-derived DCs.
  • the mature DCs expressed dendritic cell-associated antigens, such as CD80, CD83, CD86, and HLA class I and class II on their cell surfaces (data not shown).
  • CD3+ cells were stimulated for 7 days in the presence of RPMI 1640/5% autologous plasma, 10 ug/ml JAK2 synthetic peptides, 1 ug/ml b2 microglobulin (Sigma, St. Louis) and 10 ng/ml IL-15 (R&D Systems).
  • CD3+ cells were re-stimulated with either a 5:1 E:T ratio of freshly isolated CD14+ cells, or a 50:1 E:T ratio of monocyte derived DCs.
  • cells were re- stimulated after another 6-7 days, in the same manner.
  • gamma-IFN secretion of these cells was examined by ELISPOT.
  • T cells were again stimulated for 7 days with autologous CD 14+ cells and used to test cytotoxicity in a standard 51 Cr-release assay.
  • Gamma interferon ELISPOT HA-Multiscreen plates (Millipore, Burlington,
  • MA mouse-anti-human IFN-gamma antibody (10 ⁇ g/ml; clone 1-DlK, Mabtech, Sweden) in PBS, incubated overnight at 4 0 C, washed with PBS to remove unbound antibody and blocked with RPM/10% autologous plasma.
  • Peptide stimulated CD3+ T cells were challenged with either autologous CD 14+ (5:1 E:T ratio) or T2 cells (10:1 E:T ratio) in the presence of lO ⁇ g/ml B2-microglobulin (Sigma, St. Louis) and 50 ug/ml of various test peptides.
  • Negative control wells contained APCs with or without T cells or T cells with peptides alone.
  • Positive control wells contained T cells + APC+ 10 ug/ml PHA (Sigma). AU conditions were done in triplicate. After incubation for 20 h at 37 0 C, plates were extensively washed with PBS/0.05% Tween and 100 ⁇ l/well biotinylated detection antibody against human IFN-g (2 ⁇ g/ml; clone 7-B6-1, Mabtech, Sweden) was added. Plates were incubated for an additional 2 h at 37 0 C and spot development was performed as described (Herr W). Spot numbers were automatically determined with the use of a computer-assisted video image analyzer with KS ELISPOT 4.0 software (Carl Zeiss Vision, Germany).
  • Chromium 51 cytotoxicity assay The presence of specific CTLs was measured in a standard 4 hour 51 Chromium release assay as described. Briefly, target cells are pulsed with 10 ug/ml of synthetic peptides overnight at 37 0 C, after which they are labeled with 300 ⁇ Ci Of Na 2 51 CrO 4 (NEN Life Science Products, Inc., Boston, MA). After extensive washing, target cells are incubated with T cells at an E:T ratio ranging from 100:1 to 10:1. AU conditions were performed in triplicate. Plates were incubated for 4 hours at 37 0 C in 5% CO 2 . Supernatant fluids were harvested and radioactivity was measured in a gamma counter. Percent specific lysis was determined from the following formula: 100 x [(experimental release minus spontaneous release)/(maximum release minus spontaneous release)]. Maximum release was determined by lysis of radiolabeled targets in 2.5% Triton X- 100.
  • Tables 1 and 2 show the amino acid sequences and binding predictions of native and mutated JAK2 peptides and synthetic analogues of mutated JAK2 peptide to human class I molecules HLA A2 and HLA A3, particularly A0201 and A0301.
  • the amino acid residues in bold represent modifications from the native and mutated JAK2 sequence, particularly the sequence from about position 609 to about position 625 of SEQ ID NO: 1 (603KLSHKHLVLNYGVCVCGDENILVQEFV).
  • Two A0201 specific peptides containing the V617F mutation were identified based on the presence of preferred primary anchor motifs at position two and nine (Table 1).
  • J2A1 contained a leucine (L) at position two and had a relatively high predicted binding affinity.
  • J2C1 contained a valine (V) at position nine, but had a much lower predicted binding affinity.
  • Their native counterparts without the V617F mutation, J2A and J2C had very low binding scores.
  • Each mutant peptide was then modified at one amino acid to improve the predicted binding.
  • Two different modifications of the J2A1 peptide were made.
  • the introduction of an aromatic amino acid residue in the first or third position greatly increases the binding affinity. Therefore, in J2.1, the third amino acid was changed to a tryptophan (W).
  • Peptide J2.2 contained a substitution of the valine (V) anchor residue at position nine. These changes unproved the predicted half-life of the peptides up to ten times that of the original V617F mutated sequence, and close to a hundred fold greater than the JAK2 native sequence in the BIMAS algorithm.
  • J3.8 VMFCGDENK 100 ND 45 Three A0301 peptides from the JAK2 V617F mutated sequence J3A1, J3B1 and J3C1 were identified based on the position of preferred primary or secondary anchor residues. Despite the presence of either a hydrophobic leucine (L) at position two, or a tyrosine (Y) at position 3, neither these, nor their native counterparts J3A, J3B or J3C had any predicted affinity to the HLA A0301 molecule. Only once two mutations were introduced, a lysine at position nine or ten and an aromatic at position three or a hydrophobic residue at position two, was there an increase in the predicted affinity of the analog peptides (Table 2). These mutations reflect the necessity of having both anchor residues present for increased binding predictions to HLA A0301.
  • the synthetic peptides J3.1 and J3.2 and the synthetic peptides J3.3 and J3.4 were generated from mutant V617F peptides J3A1 and J3B1, respectively, that demonstrate very low or no binding to HLA A0301 (Table 2).
  • the synthetic peptides bound well to HLA A0301.
  • J3.1 and J3.2 have a tyrosine or phenylalanine (F) substitution in position 3 and a lysine substitution in position 9.
  • J3.3 and J3.4 have a leucine or methionine substitution in position 3 and a lysine substitution in position 9.
  • CD4+ T cells recognize peptides bound to the HLA class II molecule on APC. Once activated, CD4+ cells enhance immunity by licensing dendritic cells thereby sustaining the activation and survival of the cytotoxic T cells. ⁇ Rammensee, 1999 #124 ⁇ (Table 1). The V617F amino acid change in the long CD4+ epitope appears to have an impact on the binding to some of the HLA-DRBl alleles, but not to others.
  • a CD4+ epitope spanning the JAK2 V617F region was identified using a predictive algorithm based on binding motifs through the SYFPEITHI Database as described in Example 1. Long peptides comprising about 14 to about 25 amino acids from mutated V617F JAK2 that have a high predicted affinity to HLA class II molecules, e.g., HLA DRB, were identified. Table 3 lists the binding predictions to various HLA DRB molecules for the JAK2DR WT (SEQ ID NO: 46) V617F mutant (SEQ ID NO: 47) peptide sequences from about position 614 to about position 628 of SEQ ID NO: 1. The amino acid residues in bold represent modifications from the native and mutated JAK2 sequence.
  • V617F (SEQ ID NO: 46)
  • WT (SEQ ID NO: 47) Freq. Caucasian 18.5 17.7 23.6 26.2 17.0 19.9
  • the immunogenicity of MHC class I-restricted peptides requires the capacity to bind and stabilize MHC class I molecules on the live cell surface.
  • the computer prediction models used to identify the potential CD8+ epitopes have only 60-80% predictive accuracy, so direct measurement of the strength of the interaction between the peptides and the HLA-A0201 molecules using a conventional binding and stabilization assay that uses the antigen-transporting deficient (TAP2 negative) HLA-A0201 human T2 cells was used.
  • T2 cells lack TAP function and consequently are defective in properly loading class I molecules with antigenic peptides generated in the cytosol.
  • thermolabile, empty HLA-A0201 molecules stabilizes them and results in an increase in the level of surface HLA-A0201 recognizable by specific anti- HLA A0201 mAb such as BB7.2.
  • the two sets of A0201 peptides (Figs. IA- IB) and two sets of A0301 peptides (Figs. 1C- ID) were analyzed, and their in vitro binding appears to correlate with the predictive binding.
  • MHC binding affinity and/or stability of MHC-peptide complexes for class I epitopes does not necessarily correlate with antigenicity.
  • TAP2 deficient cells that express A0301 to test the in vitro binding of the A0301 specific peptides requires in vitro stimulation assays to confirm that the peptides described above are immunogenic and can stimulate CD8+ T cells that recognize the mutant V617F JAK2 kinase and not the native JAK2 kinase.
  • CD3+ cells from seven healthy HLA-A0201 donors were stimulated in vitro two or three times with autologous DCs in the presence of the A0201 JAK2 heteroclitic peptides or the V617F mutated parent peptide. Consistently, J2.2 was able to generate T cells that secreted IFN-gamma when challenged with A0201 target cells pulsed with either J2.2, the immunizing peptide or J2A1, the V617F mutant parent (Figs. 2A-2B). These T cells did not recognize target cells pulsed with the J2A, the native JAK2 sequence.
  • J2.1 and J2.3 were able to generate T cells that recognized target cells pulsed with the immunizing peptide, but they never induced a heteroclitic response that could recognize target cells pulsed with peptide J2A1 or J2A.
  • the V617F mutated peptides J2A1 and J2C1, and peptides J2.4 and J2.5 were not immunogenic, and were unable to generate a significant T cell response.
  • CD3+ cells from four healthy HLA-A0301 donors were stimulated in the same manner in the presence of the A0301 JAK2 heteroclitic peptides.
  • a heteroclitic response was generated by analog peptides J3.3 and J3.6 (Figs. 2D-2E).
  • CD3+ T cells from an A0201 donor were stimulated five times with
  • JAK2 heteroclitic peptide J2.2 as described in Example 1.
  • Stimulated T cells were used as effector cells in a 51 Cr release assay. T cells were incubated at a 60:1 E:T ratio with radiolabeled SKLY (A0201+ JAK2 V617F-) target cells pulsed with various peptides (Fig. 3)
  • CD3+ cells from healthy donors were stimulated with either the native JAK2DR peptide, or the V617F JAK2DR peptide as described in Example 1.
  • Donors expressing HLA DRBl*701/1202; HLA DRBl*407/1302 and HLA DRB1*7O1/13O3 induced a peptide specific response to the WT and mutant JAK2DR peptide. Only in the setting of HLA DRB1*7O1/1 IXX was it possible to discriminate between the WT and mutant JAK2DR peptide ( Figure 4). It is contemplated that the long peptide is a better vaccine candidate since the DR expression varies greatly in the population.

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Abstract

L'invention concerne des peptides synthétiques comprenant au moins des analogues d'un peptide mutant V617F JAK2 qui se lie spécifiquement à des molécules HLA de classe I ou de classe II sur une caractéristique cellulaire d'un trouble myéloprolifératif ou d'une maladie myéloproliférative, chez un patient. L'invention concerne des compositions pharmaceutiques et des compositions immunogéniques comprenant au moins lesdits segments analogues peptidiques ou un ADN codant ceux-ci. L'invention concerne encore des méthodes d'utilisation de ces peptides synthétiques et de ces compositions immunogéniques, lesquelles méthodes étant destinées à induire une réponse immunitaire hétéroclitique ou à traiter un trouble myéloprolifératif ou une maladie myéloproliférative.
PCT/US2006/040517 2005-10-17 2006-10-17 Analogues peptidiques de liaison synthetiques hla d'une enzyme v617f jak2 mutante et leurs utilisations Ceased WO2007047653A2 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017211371A3 (fr) * 2016-06-10 2018-01-18 Herlev Hospital Compositions vaccinales à base de calr et de jak2
WO2021099906A1 (fr) * 2019-11-18 2021-05-27 Janssen Biotech, Inc. Vaccins basés sur les mutants du gène calr et de la protéine jak2 et leurs utilisations
US11661422B2 (en) 2020-08-27 2023-05-30 Incyte Corporation Tricyclic urea compounds as JAK2 V617F inhibitors
US11691971B2 (en) 2020-06-19 2023-07-04 Incyte Corporation Naphthyridinone compounds as JAK2 V617F inhibitors
WO2023111862A3 (fr) * 2021-12-16 2023-08-03 Janssen Biotech, Inc. Vaccins à base de mutants du gène calr et de la protéine jak2 et leurs utilisations
US11753413B2 (en) 2020-06-19 2023-09-12 Incyte Corporation Substituted pyrrolo[2,1-f][1,2,4]triazine compounds as JAK2 V617F inhibitors
US11767323B2 (en) 2020-07-02 2023-09-26 Incyte Corporation Tricyclic pyridone compounds as JAK2 V617F inhibitors
US11780840B2 (en) 2020-07-02 2023-10-10 Incyte Corporation Tricyclic urea compounds as JAK2 V617F inhibitors
US11919908B2 (en) 2020-12-21 2024-03-05 Incyte Corporation Substituted pyrrolo[2,3-d]pyrimidine compounds as JAK2 V617F inhibitors
US11958861B2 (en) 2021-02-25 2024-04-16 Incyte Corporation Spirocyclic lactams as JAK2 V617F inhibitors
US12084430B2 (en) 2022-03-17 2024-09-10 Incyte Corporation Tricyclic urea compounds as JAK2 V617F inhibitors

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* Cited by examiner, † Cited by third party
Title
JAMIESON C.H.M. ET AL.: 'The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation' PROC. NATL. ACAD. SCI. vol. 103, no. 16, April 2006, pages 6224 - 6229 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017211371A3 (fr) * 2016-06-10 2018-01-18 Herlev Hospital Compositions vaccinales à base de calr et de jak2
WO2021099906A1 (fr) * 2019-11-18 2021-05-27 Janssen Biotech, Inc. Vaccins basés sur les mutants du gène calr et de la protéine jak2 et leurs utilisations
CN114980920A (zh) * 2019-11-18 2022-08-30 詹森生物科技公司 基于突变体calr和jak2的疫苗及其用途
JP2023509571A (ja) * 2019-11-18 2023-03-09 ヤンセン バイオテツク，インコーポレーテツド 変異型ｃａｌｒ及びｊａｋ２に基づくワクチン並びにこれらの使用
JP7630507B2 (ja) 2019-11-18 2025-02-17 ヤンセン バイオテツク，インコーポレーテツド 変異型ｃａｌｒ及びｊａｋ２に基づくワクチン並びにこれらの使用
US12018289B2 (en) 2019-11-18 2024-06-25 Janssen Biotech, Inc. Vaccines based on mutant CALR and JAK2 and their uses
US11691971B2 (en) 2020-06-19 2023-07-04 Incyte Corporation Naphthyridinone compounds as JAK2 V617F inhibitors
US11753413B2 (en) 2020-06-19 2023-09-12 Incyte Corporation Substituted pyrrolo[2,1-f][1,2,4]triazine compounds as JAK2 V617F inhibitors
US11767323B2 (en) 2020-07-02 2023-09-26 Incyte Corporation Tricyclic pyridone compounds as JAK2 V617F inhibitors
US11780840B2 (en) 2020-07-02 2023-10-10 Incyte Corporation Tricyclic urea compounds as JAK2 V617F inhibitors
US12187725B2 (en) 2020-07-02 2025-01-07 Incyte Corporation Tricyclic urea compounds as JAK2 V617F inhibitors
US11661422B2 (en) 2020-08-27 2023-05-30 Incyte Corporation Tricyclic urea compounds as JAK2 V617F inhibitors
US11919908B2 (en) 2020-12-21 2024-03-05 Incyte Corporation Substituted pyrrolo[2,3-d]pyrimidine compounds as JAK2 V617F inhibitors
US11958861B2 (en) 2021-02-25 2024-04-16 Incyte Corporation Spirocyclic lactams as JAK2 V617F inhibitors
WO2023111862A3 (fr) * 2021-12-16 2023-08-03 Janssen Biotech, Inc. Vaccins à base de mutants du gène calr et de la protéine jak2 et leurs utilisations
US12084430B2 (en) 2022-03-17 2024-09-10 Incyte Corporation Tricyclic urea compounds as JAK2 V617F inhibitors

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