WO2013060328A1 - IMMUNOTHÉRAPIE BASÉE SUR IkB - Google Patents

IMMUNOTHÉRAPIE BASÉE SUR IkB Download PDF

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WO2013060328A1
WO2013060328A1 PCT/DK2012/050387 DK2012050387W WO2013060328A1 WO 2013060328 A1 WO2013060328 A1 WO 2013060328A1 DK 2012050387 W DK2012050387 W DK 2012050387W WO 2013060328 A1 WO2013060328 A1 WO 2013060328A1
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vaccine composition
ikb
hla
cells
composition according
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PCT/DK2012/050387
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Mads Hald Andersen
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Herlev Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464454—Enzymes
    • A61K39/464462—Kinases, e.g. Raf or Src
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464491—Melan-A/MART
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the field of prophylaxis and therapy of clinical conditions including cancer, autoimmune diseases and infectious diseases.
  • vaccine compositions comprising IkB or peptide fragments thereof that are capable of eliciting immune responses useful in treatment of cancer, autoimmune diseases or infectious diseases.
  • the immune system has the capacity to recognize and destroy neoplastic cells
  • CTLA-4 is a key negative regulatorof T-cell responses, which can restrict the antitumor immune response.
  • the anti-CTLA-4 antibody ipilimumab was approved by the FDA as well as EMEA for the treatment of melanoma after showing effect in clinical phase III studies.
  • Another central mechanism counteracting tumor-specific immunity and preventing effective anticancer immunotherapy requires a specific environment in which tolerogenic dendritic cells (DC) play an essential role deviating the immune response away from effective immunity.
  • DC dendritic cells
  • NFkB nuclear factor-kB
  • the problem of cancer immunosuppression is solved by the present invention which is based on the surprising finding by the inventors of spontaneous cytotoxic immune responses against IkB expressing cells in cancer patients. These findings open the way for novel therapeutic and diagnostic approaches which may be generally applicable in the control of cancer diseases.
  • the findings are not restricted to cancer but are also useful in other clinical conditions characterized by constitutive activation of NF-kB correlated with a decrease in stability of IkB proteins.
  • the present invention targets the cancer disease by killing the IkB expressing cancer cells directly and by killing the IkB expressing regulatory cells. This is done by enabling the T cells to recognize the IkB expressing cells. Likewise, when the clinical condition is an infection, T cells are enabled to kill IkB expressing APCs / DCs.
  • the expression of the immune suppressing enzyme IkB in cancer cells and APCs is positive in conjunction with the application of the method of the present invention, which targets these IkB expressing cells.
  • T-cell response against IkB were elicited in cancer patients.
  • the presence of an in vivo T-cell response specific for IkB demonstrates that cancer patients are capable of generating T-cell responses to IkB in vivo in response to the presence of IkB peptides.
  • the two conditions for generating a T-cell response are met: the T cells are present in the cancer patient and they have the ability to expand, which are shown in the application as filed. It follows from the general knowledge in the field of immunology that providing additional IkB protein or IkB peptides will lead to generation of IkB specific T-cell responses.
  • the T-cells recognizes a complex ligand, comprising an antigenic peptide bound to a protein called the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • Class I HLA molecules sample peptides from protein-degradation inside the cell and present these at the cell surface to T cells. Hence, this enables T-cells to scan for cellular alterations.
  • the ELISPOT method used in Examples 1 and 3 of the present application is a very sensitive assay that demonstrates the presence of in vivo immune responses and not of naive T-cells.
  • Peptide-MHC-tetramers have been successfully used to identify and study T cells specific for tumor-associated antigens (TAA) that develop endogenously or after vaccination in patients. Tetramers have also been used to isolate and expand TAA-specific T cells for adoptive cellular immunotherapy.
  • TAA tumor-associated antigens
  • the present application demonstrates the presence of IkB-tetramer specific T-cells (see Example 3) which also demonstrates an ongoing IkB response in vivo.
  • the present invention regards a vaccine composition
  • a vaccine composition comprising IkB of SEQ ID NO: 1 or a functional homologue thereof at least 70% identical thereto or an immunogenically active peptide fragment comprising a consecutive sequence of said IkB or said functional homologue thereof or a nucleic acid encoding said IkB, said functional homologue thereof or said peptide fragment; and an adjuvant for use as a medicament.
  • the synergistic effect of a combination of immunotherapies based on the above disclosed vaccine is provided for in the aspect of the invention which regards a kit-of- parts comprising the vaccine composition and a further immunostimulating
  • a method of treating a clinical condition such as a cancer or infection by any of the means described above falls within the scope of the present invention; the means including administering to an individual suffering from the clinical condition an effective amount of the vaccine composition as disclosed above or a kit-of-parts comprising the aforementioned vaccine together with another immunostimulating composition and/or chemotherapeutic agent. It is thus also an object of the present invention to use IkB or an immunogenically active peptide fragment thereof comprising a consecutive sequence of said IkB or a functional homologue thereof or the vaccine composition of above in the manufacture of a medicament for the treatment or prevention of a cancer disease.
  • PBMC T-cell responses against ⁇ -derived peptides as measured by IFN- ⁇ ELISPOT.
  • PBMC from cancer patients were analyzed. All individuals were HLA-A2 + . Eleven synthetic 9mer and seven 10mer ⁇ -derived peptides were examined.
  • PBMC were stimulated once in vitro with peptide before being plated at 6x10 5 cells per well either without or with the relevant IKB peptide. The average number of IKB -specific spots (after subtraction of spots without added peptide) was calculated per 6x10 5 PBMC for each patient (black circle).
  • ELISPOT (a), Examples of ELISPOT response against ⁇ 10 in PBMC from two cancer patients, (b), The average number of ⁇ -specific spots (after subtraction of spots without added peptide) was calculated per 2 x 10 5 PBMC for each patient (white triangle). PBMC from 17 healthy individuals and 22 malignant melanoma patients
  • T cells were stimulated once with peptide before being plated at 2x10 5 cells per well in duplicates either without or with the ⁇ 10 peptide.
  • ⁇ -specific T-cells (a), GrB ELISPOT response either without or with IKB10 in PBMC from three cancer patients, (b), Lysis by a T-cell bulk culture of T2-cells pulsed with ⁇ 10 peptide (black circles) or an irrelevant peptide (black squares) (HIV-1 pol 476 -484) at different effector to target ratios as measured by 51 Cr- release assay, (c), Lysis of T2-cells pulsed with ⁇ 10 peptide or an irrelevant peptide (HIV-1 pol 476 . 4 8 4 ) , the HLA-A2 + cancer cell line FM3 without or with IFN-g pre-treatment by a T-cell clone at an effector to target ratio at 3:1 as measured by 51 Cr-release assay.
  • IFN- ⁇ producing T-cell responses against ⁇ -derived peptides as measured by IFN-g ELISPOT.
  • PBMC were stimulated once in vitro with peptide before being plated at 6x10 5 cells per well in duplicates either without or with the relevant ⁇ peptide. The average number of ⁇ -specific spots (after subtraction of spots without added peptide) was calculated per 6x10 5 PBMC for each patient (black circle).
  • ELISPOT (A) ELISA analysis of UV-sensitive ligand (KILGFVFJV) exchanged with various peptides: CMV/HLA-A2 (pp65 pos495-503; NLVPMVATV), HIV-1/HLA-A2 (pol476-484; ILKEPVHGV) and ⁇ 10 ( ⁇ 188-196; HLASIHGYL), No-UV (not exposed to UV light) and No peptide (without rescue peptide).
  • B Examples of ELISPOT responses against ⁇ 10 and HIV pol476-484 in PBMC from two melanoma patients (MM 1 and MM3).
  • C) The average number of ⁇ ⁇ -specific spots (after subtraction of spots with irrelevant added HIV peptide) was calculated per 2 x 10 5 PBMC for each patient. PBMC from 17 Healthy Donors (HD, black circles), 22
  • Cytotoxic functionality of ⁇ ⁇ -specific T-cells (A), Examples of GrB releasing cells in response to ⁇ 10 as well as HIV peptide among PBMC from three melanoma patients (MM3, MM 1 and MM5) as measured by GrB ELISPOT. (B), Lysis of T2-cells pulsed with IKB10 peptide or an irrelevant peptide (HIV-1 pol 476 -484) by two T-cell clones expanded as single cells after isolation from the ⁇ ⁇ -specific bulk culture using H LA- A2/lKB10-tetramer-coupled microbeads.
  • ⁇ ⁇ -specific T-cells (A), Lysis by a T-cell bulk culture of T2- cells pulsed with ⁇ 10 peptide (black circles) or an irrelevant peptide (black squares) (HIV-1 pol 476 . 4 8 4 ) at different effector to target ratios as measured by 51 Cr-release assay. (B) Lysis by a ⁇ ⁇ -clone of T2 cells as well as autologous DC either pulsed with IKB10 peptide or irrelevant HIV peptide (C), Lysis of T2-cells pulsed with ⁇ 10 peptide or an irrelevant peptide (HIV-1 pol 476 . 4 8 4 ), the H LA-A2 + melanoma cell line FM3 without or with I FN- ⁇ pre-treatment by the ⁇ ⁇ -clone as measured by 51 Cr-release assay.
  • A Lysis by a T-cell bulk culture of T2- cells pulsed with ⁇ 10 peptide (black circles) or an irrelevant peptid
  • Adjuvant Any substance whose admixture with IkB or an immunologically active peptide fragment thereof upon administration to an individual increases the immune response to IkB or said peptide fragment thereof.
  • said individual is a human being and preferably said immune response is a T-cell response.
  • Antibody Immunoglobulin molecules and active portions of immunoglobulin molecules. Antibodies are for example intact immunoglobulin molecules or fragments thereof retaining the immunologic activity.
  • Antigen Any substance that can bind to a clonally distributed immune receptor (T-cell or B-cell receptor). Usually a peptide, polypeptide or a multimeric polypeptide. Antigens are preferably capable of eliciting an immune response.
  • APC Antigen-presenting cell.
  • An APC is a cell that displays foreign antigen complexed with MHC on its surface. T-cells may recognize this complex using their T-cell receptor (TCR).
  • TCR T-cell receptor
  • APCs fall into two categories: professional, (of which there are three types: Dendritic cells, macrophages and B-cells) or non-professional (does not constitutively express the Major histocompatibility complex proteins required for interaction with naive T cells; these are expressed only upon stimulation of the non-professional APC by certain cytokines such as IFN- ⁇ ).
  • Boost To boost by a booster shot or dose is to give an additional dose of an immunizing agent, such as a vaccine, given at a time after the initial dose to sustain the immune response elicited by the previous dose of the same agent.
  • an immunizing agent such as a vaccine
  • cancer any pre-neoplastic or neoplastic disease, benign or malignant, where "neoplastic” refers to an abnormal proliferation of cells.
  • Carrier Entity or compound to which antigens are coupled to aid in the induction of an immune response.
  • Chimeric protein A genetically engineered protein that is encoded by a nucleotide sequence made by a splicing together of two or more complete or partial genes or a series of (non)random nucleic acids.
  • Clinical condition A condition that requires medical attention, herein especially conditions associated with the expression of IkB. Examples of such conditions include cancers, infectious diseases or autoimmune diseases.
  • CTL Cytotoxic T lymphocyte. A sub group of T-cells expressing CD8 along with the T- cell receptor and therefore able to respond to antigens presented by class I molecules. Cytokine: Growth or differentiation modulator, used non-determinative herein, and should not limit the interpretation of the present invention and claims. In addition to the cytokines, adhesion or accessory molecules, or any combination thereof, may be employed alone or in combination with the cytokines.
  • Delivery vehicle An entity whereby a nucleotide sequence or polypeptide or both can be transported from at least one media to another.
  • DC Dendritic cell.
  • DCs are immune cells and form part of the mammalian immune system. Their main function is to process antigen material and present it on the surface to other cells of the immune system, thus functioning as antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • Fragment is used to indicate a non-full length part of a nucleic acid or polypeptide. Thus, a fragment is itself also a nucleic acid or polypeptide, respectively.
  • a functional homologue may be any polypeptide that exhibits at least some sequence identity with a wild type polypeptide and has retained at least one aspect of the wild type polypeptide's functionality.
  • a functional homologue of IkB has the capability to induce a T-cell immune response to cells expressing IkB.
  • Individual Generally any species or subspecies of bird, mammal, fish, amphibian, or reptile, preferably a mammal, most preferably a human being.
  • infection relates to any kind of clinical condition giving rise to an immune response and therefore includes infections, chronic infections, autoimmune conditions and allergic inflammations.
  • nucleic acids, polypeptides, and antibodies disclosed herein 'isolated' refers to these having been identified and separated and/or recovered from a component of their natural, typically cellular, environment.
  • Nucleic acids, polypeptides, and antibodies of the invention are preferably isolated, and vaccines and other compositions of the invention preferably comprise isolated nucleic acids, polypeptides or isolated antibodies.
  • MHC Major histocompatibility complex, two main subclasses of MHC, Class I and Class II exist.
  • Nucleic acid construct A genetically engineered nucleic acid. Typically comprising several elements such as genes or fragments of same, promoters, enhancers, terminators, polyA tails, linkers, polylinkers, operative linkers, multiple cloning sites (MCS), markers, STOP codons, other regulatory elements, internal ribosomal entry sites (IRES) or others.
  • Pathogen a specific causative agent of disease, especially a biological agent such as a virus, bacteria, prion or parasite that can cause disease to its host, also referred to as an infectious agent.
  • Peripheral blood cells are the cellular components of blood, consisting of red blood cells, white blood cells, and platelets, which are found within the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow.
  • PBMC Peripheral Blood Mononuclear Cell
  • compositions also termed excipients, or stabilizers are non-toxic to the cell or individual being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins;
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, arginine or lysine
  • chelating agents such as EDTA
  • sugar alcohols such as mannitol or sorbitol
  • salt- forming counterions such as sodium
  • nonionic surfactants such as TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM.
  • Plurality At least two.
  • Promoter A binding site in a DNA chain at which RNA polymerase binds to initiate transcription of messenger RNA by one or more nearby structural genes.
  • Treg Regulatory T cells / T lymphocytes
  • Vaccine A substance or composition capable of inducing an immune response in an animal. Vaccines are also referred to as "vaccine compositions” or as “immunogenic compositions” in the present text. Said immune response is according to the present invention preferably a T-cell response. A vaccine of the present invention may be given as or prophylactic and/or therapeutic medicament.
  • a 'variant' of a given reference nucleic acid or polypeptide refers to a nucleic acid or polypeptide that displays a certain degree of sequence identity to said reference nucleic acid or polypeptide but is not identical to said reference nucleic acid or polypeptide.
  • IkB according to the present invention is an inhibitor of NF-kappaB (NF-kB).
  • I KBS inhibitory proteins
  • I KBS are a family of related proteins that have an N-terminal regulatory domain, followed by six or more ankyrin repeats and a PEST domain near their C terminus.
  • the I KB family consists of ⁇ , ⁇ , ⁇ , and ⁇ .
  • the most prevalent IkB in humans is IkBa. After activation by a large number of inducers, the IkB proteins become phosphorylated, ubiquitylated and subsequently degraded by the proteasome. The degradation of IkB allows NF-kB proteins to translocate to the nucleus and bind their cognate DNA binding sites to regulate the transcription of a large number of genes.
  • the elevated levels of NF-kB correlates with a decreases half-life of IkBs and accordingly IkBs and peptide fragments thereof is present at high levels in cancer cells, as well as in regulatory T-cells. Accordingly the vaccine compositions according to the present invention are useful for prophylaxis and/or treatment of clinical conditions characterized by the presence of undesired cells expressing high levels of NF-kB and IkB.
  • a vaccine composition comprising IkB or an immunologically active polypeptide fragment hereof for use as a medicament for the treatment of a clinical condition.
  • Said clinical condition may be cancer and it is a further aspect of the present invention to prevent, reduce the risk from, or treat cancer.
  • Another aspect relates to the use of the vaccine composition of the present invention in combination with other medicaments such as
  • immunotherapeutic medicaments and/or chemotherapeutic agents are used in combination with other medicaments such as immunotherapeutic medicaments and/or antibiotics and/or anti-viral agents.
  • Another embodiment of the present invention relates to the use of a vaccine composition as herein disclosed for the treatment of autoimmune diseases and further to the use of said vaccine in combination with other medicaments such as immunosuppressive drugs.
  • the vaccine compositions according to the present invention comprise IkB or an immunogenically active peptide fragment thereof for use in treatment of a clinical condition in an individual in need thereof.
  • said IkB is IkB of the species of said individual.
  • the individual in need thereof is a specific kind of mammal
  • said IkB is preferably IkB of said specific kind of mammal.
  • the vaccine compositions comprises human IkBa of SEQ ID NO: 1 or an immunigenically active peptide fragment thereof.
  • the wild-type human IkBa i.e. the naturally occurring non-mutated version of the polypeptide is identified in SEQ ID NO: 1.
  • the vaccine compositions of the invention comprise a functional homologue of IkB or an immunologically active peptide fragment thereof as defined herein below.
  • the present invention thus relates to vaccine compositions comprising an adjuvant and: IkB of SEQ ID NO: 1 ; or
  • An immunologically active peptide fragment IkB of SEQ ID NO:1 or A functional homologue of IkB of SEQ ID NO: 1 at least 70% identical thereto; or
  • a functional homologue of an immunogenically active peptide fragment of IkB of SEQ ID NO: 1 which is a peptide fragment of IkB of SEQ ID NO: 1 , wherein at the most two amino acids have been substituted, or A nucleic acid encoding any of i) to iv)
  • peptide fragment of is used herein to define any non-full length (as compared to SEQ ID NO: 1) string of amino acid residues that are directly derived from or synthesized to be identical with a consecutive sting of amino acids of SEQ ID NO: 1.
  • a functional homologue can be defined as a full length or fragment of IkB that differs in sequence from the wild-type IkB, such as wild-type human IkB of SEQ ID NO: 1 , but is still capable of inducing an immune response against IkB expressing cells such as cancer cells and DCs.
  • the IkB expressed in these cells may be wild type or endogenously mutated (such as a congenital mutant or a mutation induced during cell division or other).
  • a functional homologue may be a mutated version or an alternative splice variant of the wild-type IkB.
  • functional homologues of IkB are defined as described herein below.
  • a functional homologue may be, but is not limited to, a recombinant version of full length or fragmented IkB with one or more mutations and/or one or more sequence deletions and/or additions introduced ex vivo.
  • a functional homologue of IkB may be any protein/polypeptide that exhibits at least some sequence identity with SEQ ID NO: 1 and has the capability to induce an immune response to cells expressing IkB.
  • a functional homologue of IkB preferably share at least 70% sequence identity to IkB of SEQ ID NO: 1 , and accordingly, functional homologue preferably have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85 % sequence identity, for example at least 90 % sequence identity, such as at least 91 % sequence identity, for example at least 91 % sequence identity, such as at least 92 % sequence identity, for example at least 93 % sequence identity, such as at least 94 % sequence identity, for example at least 95 % sequence identity, such as at least 96 % sequence identity, for example at least 97% sequence identity, such as at least 98 % sequence identity, for example 99% sequence identity with the sequence of human IkB of SEQ ID NO: 1 and has the capability of inducing an immune response to cells expressing IkB.
  • Sequence identity are determined over the entire reference sequence and thus sequence identity to SEQ ID NO: 1 is determined over the entire length of SEQ ID NO: 1. Sequence identity can be calculated using a number of well-known algorithms and applying a number of different gap penalties. The sequence identity is calculated relative to full-length SEQ ID NO: 1. Any sequence alignment tool, such as but not limited to FASTA, BLAST, or LALIGN may be used for searching homologues and calculating sequence identity. Moreover, sequence alignments may be performed using a range of penalties for gap opening and extension. For example, the BLAST algorithm may be used with a gap opening penalty in the range 5-12, preferably 8, and a gap extension penalty in the range 1-2, preferably 1.
  • Functional equivalents may further comprise chemical modifications such as ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids (amino acids) such as ornithine, which do not normally occur in human proteins, however it is preferred that the functional equivalent does not contain chemical modifications.
  • chemical modifications such as ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids (amino acids) such as ornithine, which do not normally occur in human proteins, however it is preferred that the functional equivalent does not contain chemical modifications.
  • any changes made to the sequence of amino acid residues compared to that of IkB of SEQ ID NO: 1 are preferably conservative substitutions.
  • a person skilled in the art will know how to make and assess 'conservative' amino acid substitutions, by which one amino acid is substituted for another with one or more shared chemical and/or physical characteristics. Conservative amino acid substitutions are less likely to affect the functionality of the protein.
  • Amino acids may be grouped according to shared characteristics.
  • a conservative amino acid substitution is a substitution of one amino acid within a predetermined group of amino acids for another amino acid within the same group, wherein the amino acids within a predetermined group exhibit similar or substantially similar characteristics.
  • the immunogenically active peptide fragment of IkB to be used with the invention may have any desired length.
  • the immunogenically active peptide fragment of the invention consists of 50 amino acid residues or less, for example of at the most 45 amino acid residues, such as at the most 40 amino acid residues, for example at the most 35 amino acid residues, such as at the most 30 amino acid residues, for example at the most 25 amino acid residues, such as 18 to 25
  • amino acids of IkB as identified in SEQ ID NO: 1 or a functional homologue thereof are consecutive amino acids of IkB as identified in SEQ ID NO: 1 or a functional homologue thereof; the functional homologue being one wherein at the most three amino acids have been substituted, such as two amino acids, such as one amino acid has been substituted for another amino acid, preferably by conservative substitution.
  • the immunogenically active peptide fragment of the invention consists of the most 25 amino acid residues, such as at the most 24 amino acid residues, such as at the most 23 amino acid residues, such as at the most 22 amino acid residues, such as at the most 21 amino acid residues, such as at the most 20 amino acid residues, for example at the most 19 amino acid residues, such as at the most 18 amino acid residues, for example at the most 17 amino acid residues, such as at the most 16 amino acid residues, for example at the most 15 amino acid residues, such as at the most 14 amino acid residues, for example at the most 13 amino acid residues, such as at the most 12 amino acid residues, for example at the most 11 amino acid residues, such as 8 to 10 consecutive amino acids from IkB of SEQ ID no 1 or a functional homologue thereof; the functional homologue being one wherein at the most two amino acids, such as one amino acid has been substituted, preferably by conservative substitution.
  • the peptide comprises at the most 10 consecutive amino acids from IkB of S
  • amino acid residues from IkB as identified in SEQ ID NO: 1 or a functional homologue thereof are consecutive amino acid residues from IkB as identified in SEQ ID NO: 1 or a functional homologue thereof; the functional homologue being one wherein at the most two amino acids, such as one amino acid has been substituted with another amino acid, preferably by conservative substitution.
  • the peptides of the invention are nonapeptides (peptides comprising 9 amino acid residues), and some decapeptides (comprising 10 residues).
  • the immunogenically active peptide fragment comprises a peptide selected from the group consisting of the peptides listed in Table 1 , more preferably a peptide selected from the group consisting of SEQ ID NO: 11 and 19.
  • said immunogenically active peptide fragment consist of at the most 25 amino acid residues, such as at the most 24 amino acid residues, such as at the most 23 amino acid residues, such as at the most 22 amino acid residues, such as at the most 21 amino acid residues, such as at the most 20 amino acid residues, for example at the most 19 amino acid residues, such as at the most 18 amino acid residues, for example at the most 17 amino acid residues, such as at the most 16 amino acid residues, for example at the most 15 amino acid residues, such as at the most 14 amino acid residues, for example at the most 13 amino acid residues, such as at the most 12 amino acid residues, for example at the most 11 amino acid residues, such as of 10 amino acids, for example of 9 amino acids and
  • said immunogenically active peptide fragment is selected from the group consisting peptides listed in Table 1 , and more preferably selected from the group consisting of SEQ ID NO: 1 1 and 19.
  • peptides of the invention comprise (or more preferably consist of) between 4 and 120, preferably between 8 and 100, more preferably between 10 and 75, yet more preferably between 12 and 60, even more preferably between 15 and 40, such as between 18 and 25 contiguous amino acids of IkB of SEQ ID NO: 1 , wherein at the most three amino acids compared to the IkB sequence of SEQ ID NO: 1 have been substituted, deleted or added, such as two amino acids have been substituted, deleted or added, or one amino acid has been substituted, deleted or added.
  • the vaccine composition comprises a polypeptide consisting of a consecutive sequence of IkB of SEQ ID NO: 1 in the range of 8 to 50 amino acids, preferably in the range of 8 to 10 or 20 to 25 amino acids, wherein at the most three amino acid has been substituted, and where the substitution preferably is conservative.
  • MHC class I molecules are recognized by CD8 T-cells, which are the principal effector cells of the adaptive immune response.
  • MHC class II molecules are mainly expressed on the surface of antigen presenting cells (APCs), the most important of which appears to be the dendritic cells.
  • APCs stimulate naive T-cells, as well as other cells in the immune system. They stimulate both CD8 T-cells and CD4 T-cells.
  • novel MHC Class l-restricted peptide fragments consisting of 8-10 amino acids from IkB of SEQ ID NO 1 or a functional homologue thereof, wherein at the most two amino acids of SEQ ID NO 1 have been substituted, which are characterized by having at least one of several features, one of which is the ability to bind to the Class I HLA molecule to which it is restricted at an affinity as measured by the amount of the peptide that is capable of half maximal recovery of the Class I HLA molecule (C 50 value) which is at the most 50 ⁇ as determined by the assembly binding assay as described herein.
  • This assembly assay is based on stabilization of the HLA molecule after loading of peptide to the peptide transporter deficient cell line T2.
  • the peptides of this embodiment comprises (or more preferably consists of) at the most 200, preferably at the most 100, more preferably at the most 50, yet more preferably at the most 25, even more preferably at the most 20, yet even more preferably at the most 15, such as at the most 10, for example in the range of 8 to 10 contiguous amino acids of IkB of SEQ ID NO 1or a functional homologue thereof wherein at the most two amino acids of SEQ ID NO 1 have been substituted.
  • the peptide fragment of the invention in one having a C 50 value, which is at the most 30 ⁇ , such as a C 50 value, which is at the most 20 ⁇ including C 50 values of at the most 10 ⁇ , at the most 5 ⁇ and at the most 2 ⁇ .
  • novel MHC Class ll-restricted peptide fragments of IkB of SEQ ID NO 1 or a functional homologue thereof, wherein at the most two amino acids of SEQ ID NO 1 have been substituted (also referred to herein as "peptides"), which are characterized by having at least one of several features described herein below.
  • the peptides of this embodiment comprises (or more preferably consists of) between 4 and 120, preferably between 8 and 100, more preferably between 10 and 75, yet more preferably between 12 and 60, even more preferably between 15 and 40, such as between 18 and 25 contiguous amino acids of IkB of SEQ ID NO 1 of SEQ ID NO 1 or a functional homologue thereof, wherein at the most two amino acids of SEQ ID NO 1 have been substituted,
  • novel MHC Class l-restricted peptide fragments of 8-10 amino acids or novel MHC Class ll-restricted peptide fragments of 18-25 amino acids of IkB of SEQ ID NO 1 or a functional homologue thereof, wherein at the most two amino acids of SEQ ID NO 1 have been substituted which are characterized by having at least one of several features described herein below, one of which is the ability to bind to the Class I or Class II HLA molecule to which it is restricted.
  • peptide fragments which is an MHC Class l-restricted peptide or an MHC class ll-restricted peptide having at least one of the following characteristics: (i) capable of eliciting INF- ⁇ -producing cells in a PBMC population of a cancer patient at a frequency of at least 25 per 2x10 5 PBMCs as determined by an ELISPOT assay, and/or
  • More preferred peptides according to the present invention are peptides capable of raising a specific T-cell response as determined by an ELISPOT assay, for example the ELISPOT assay described in Example 1 herein below. Some peptides although they do not bind MHC class I or class II with high affinity, may still give rise to a T-cell response as determined by ELISPOT. Other peptides capable of binding MHC class I or class II with high affinity also give rise to a T-cell response as determined by ELISPOT. Both kinds of peptides are preferred peptides according to the invention.
  • preferred peptides according to the present invention are peptides capable of raising a specific T-cell response as measured by an ELISPOT assay, wherein more than 25 peptide specific spots per 2x10 8 cells, more preferably per 2x10 7 , even more preferably per 2x10 6 , yet more preferably per 2x10 5 cells are measured.
  • preferred peptides according to the present invention are peptides capable of raising a specific T-cell response of more than 25 peptide specific spots per 2x10 8 PBMC, more preferably per 2x10 7 , even more preferably per 2x10 6 , yet more preferably per 2x10 5 PBMC, when measured by the ELISPOT assay described in Example 1 including stimulation once with peptide in vitro.
  • Most preferred peptides according to the present invention are peptides that are capable of eliciting a cellular immune response in an individual suffering from a clinical condition characterized by the expression of IkB, the clinical condition preferably being a cancer, an autoimmune disease or an infectious disease, and most preferably a cancer.
  • the HLA system represents the human major histocompatibility (MHC) system.
  • MHC human major histocompatibility
  • the MHC codes for three different types of molecules, i.e. Class I, II and III molecules, which determine the more general characteristics of the MHC.
  • Class I molecules are so-called HLA-A, HLA-B and HLA-C molecules that are presented on the surface of most nucleated cells and thrombocytes.
  • the peptides of the present invention are characterized by their ability to bind to (being restricted by) a particular MHC Class I HLA molecule.
  • the peptide is one which is restricted by a MHC Class I HLA-A molecule including HLA-A1 , HLA-A2, HLA-A3, HLA-A9, HLA-A 10, HLA-A11 , HLA-Aw19, HLA-A23(9), HLA-A24(9), HLA-A25(10), HLA-A26(10), HLA-A28, HLA-A29(w19), HLA-A30(w19), HLA-A31 (w19), HLA-A32(w19), HLA-Aw33(w19), HLA-Aw34(10), HLA-Aw36, HLA-Aw43, HLA- Aw66(10), HLA-Aw68(28), HLA-A69(28).
  • the peptide of the invention is restricted a MHC Class I HLA species selected from the group consisting of HLA-A1 , HLA-A2, HLA-A3, HLA-A11 and HLA- A24. In specific embodiment, the peptide of the invention is restricted a MHC Class I HLA species HLA-A2 or HLA-A3.
  • the peptide of the invention is a peptide, which is restricted by a MHC Class I HLA-B molecule including any of the following: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA-B15, HLA-B16, HLA-B17, HLA- B18, HLA-B21 , HLA-Bw22, HLA-B27, HLA-B35, HLA-B37, HLA-B38, HLA-B39, HLA- B40, HLA-Bw41 , HLA-Bw42, HLA-B44, HLA-B45, HLA-Bw46 and HLA-Bw47.
  • MHC Class I HLA-B molecule including any of the following: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA-B15, HLA-B16, HLA-B17
  • the MHC Class I HLA-B species to which the peptide of the invention is capable of binding is selected from HLA-B7, HLA-B35, HLA- B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51.
  • the peptide of the invention is a peptide, which is restricted by a MHC Class I HLA-C molecule including but not limited to any of the following: HLA-Cw1 , HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw5, HLA-Cw6, HLA-Cw7 and HLA-Cw1.
  • the peptide of the invention is a peptide, which is restricted by a MHC Class II HLA molecule including but not limited to any of the following: HLA-DPA-1 , HLA-DPB-1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB and all alleles in these groups and HLA-DM, H LA-DO.
  • a MHC Class II HLA molecule including but not limited to any of the following: HLA-DPA-1 , HLA-DPB-1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB and all alleles in these groups and HLA-DM, H LA-DO.
  • peptides potentially having the ability to bind to a particular HLA molecule can be made by the alignment of known sequences that bind to a given particular HLA molecule to thereby reveal the predominance of a few related amino acids at particular positions in the peptides. Such predominant amino acid residues are also referred to herein as “anchor residues” or “anchor residue motifs”.
  • anchor residues or “anchor residue motifs”.
  • peptides can be derived from IkB, which are likely to bind to a specific HLA molecule. Representative examples of such analyses for a range of HLA molecules are given in the below table:
  • anchor residue is R or A.
  • nonapeptides potentially having the ability to bind to HLA-A3 would have one of the following sequences: Xaa-L-Y-Xaa-Xaa-Xaa-Xaa-Xaa-K, Xaa-L- Y-Xaa-Xaa-Xaa-Xaa-Y; Xaa-L-Y-Xaa-Xaa-Xaa-Xaa-Xaa-F or Xaa-V-Y-Xaa-Xaa- Xaa-Xaa-Xaa-K (Xaa indicating any amino acid residue).
  • sequences potentially having the ability to bind to any other HLA molecule can be designed. It will be appreciated that the person of ordinary skill in the art will be able to identify further "anchor residue motifs" for a given HLA molecule.
  • the peptide of the invention may have a sequence which is a consecutive sequence of the native sequence of the IkB of SEQ ID NO: 1.
  • peptides having a higher affinity to any given HLA molecule may be derived from such a native sequence by modifying the sequence by substituting, deleting or adding at least one amino acid residue, whereby anchor residue motifs in respect of the given HLA molecule are identified.
  • polypeptides of the invention include peptides, the sequences of which comprise, for each of the specific HLA alleles listed in the table, any of the amino acid residues as indicated in the table.
  • the peptides of the invention may be any of the above-mentioned peptides comprising contiguous sequences from IkB, wherein in the range of 1 to 10, preferably in the range of 1 to 5, more preferably in the range of 1 to 3, even more preferably in the range of 1 to 2, yet more preferably 1 amino acid has been exchanged for another amino acid, preferably in a manner so that the peptide comprises one or more, preferably all anchor residues of a given HLA-A specific peptide as indicated in Table 2 above.
  • HLA species include, to which preferred peptides of the present invention are restricted include: a MHC Class I HLA species selected from the group consisting of HLA-A1 , HLA-A2, HLA-A3, HLA-A11 and HLA-A24, more preferably the peptide is restricted by HLA-A3 or HLA-A2.
  • a preferred HLA species includes MHC Class I HLA-B species selected from the group consisting of HLA-B7, HLA -B35, HLA -B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51.
  • An approach to identifying polypeptides of the invention includes the following steps: selecting a particular HLA molecule, e.g. one occurring at a high rate in a given population, carrying out an alignment analysis as described above to identify "anchor residue motifs" in the IkB protein, isolating or constructing peptides of a suitable size that comprise one or more of the identified anchor residues and testing the resulting peptides for the capability of the peptides to elicit INF- ⁇ -producing cells in a PBMC population of a cancer patient at a frequency of at least 25 per 2x10 5 PBMCs as determined by an ELISPOT assay as described in Example 1.
  • IkB derived peptides longer than 8 to 10 amino acid residues are provided.
  • Polypeptides longer than 8 to 10 amino acids are processed by the proteasome to a shorter length for binding to HLA molecules.
  • the "long" polypeptide / protein / protein fragment / variant of IkB is processed into a series of smaller peptides in the cytosol by the proteasome.
  • the vaccine composition of the present invention comprises one or more of the following: IkB full length polypeptide (SEQ ID NO: 1), polypeptide fragments here from, functional homologues of full length IkB of SEQ ID NO: 1 and immunigenically active peptide fragments of IkB wherein one or two amino acids have been substituted, added or deleted.
  • the vaccine composition comprises any of the sequences listed in the sequence listing of the present disclosure.
  • the vaccine composition comprises the peptides lkB10 (SEQ ID NO: 11) and/or lkB18 (SEQ ID NO: 19).
  • a significant feature of the peptide of the invention is its capability to recognize or elicit INF- ⁇ -producing responder T cells, i.e. cytotoxic T cells (CTLs) that specifically recognize the particular peptide in a PBMC population, on an APC or tumor / neoplastic cells of an individual suffering from a cancer and/or an infection (target cells).
  • CTLs cytotoxic T cells
  • This activity is readily determined by subjecting PBMCs, APCs or tumor cells from an individual to an ELISPOT assay. Prior to the assay, it may be advantageous to stimulate the cells to be assayed by contacting the cells with the peptide to be tested.
  • the peptide is capable of eliciting or recognizing INF- ⁇ -producing T cells at a frequency of at least 25 per 2x10 5 PBMCs as determined by an ELISPOT assay as used herein. More preferably the frequency is at least 30 per 2x10 5 PBMCs.
  • the ELISPOT assay represents a strong tool to monitor IkB specific T-cell responses.
  • a major implication of the findings herein is that the peptides of the invention are expressed and complexed with HLA molecules on cancer cells and/or IkB expressing APCs. This renders these cancer cells susceptible to destruction by CTLs and emphasizes the potential usefulness of IkB immunization to fight cancer and infections.
  • the immunigenically active peptide fragment of the invention is capable of inducing IkB specific T-cells capable of killing cells, such as cancer cells expressing IkB.
  • said peptide fragment is capable of inducing IkB specific T-cells capable of lysing at least 5% cancer cells, such as FM86 cells after co-incubation in vitro.
  • the protein from which the peptide can be derived can be any IkB polypeptide from any animal species in which the protein is expressed.
  • the starting protein is from a mammalian species including a rodent species, rabbit and a primate species such as humans.
  • the peptide of the invention is derived by any appropriate chemical or enzymatic treatment of the protein starting material that results in a peptide of a suitable size as indicated above, or it can be synthesized by any conventional peptide synthesis procedures with which the person of ordinary skills in the art is familiar.
  • the IkB polypeptide is human IkB and more preferably human IkB of SEQ ID NO:1.
  • the individual to be treated with the vaccine composition of the present invention is an individual suffering from a clinical condition.
  • the individual is preferably of a
  • the individual may be of any age, young or old, and may be either male or female.
  • the clinical condition from which the individual suffers may be a neoplastic disease such as a cancer, or an infectious disease such as an intracellular infection or a viral infection or an autoimmune disease.
  • An embodiment of the present invention provides a vaccine for the treatment, reduction of risk of, stabilization of or prevention of a cancer.
  • the present invention provides a vaccine for the treatment, reduction of risk of, stabilization of or prevention of a disease stemming from an infection, such as an intracellular infection or viral infection.
  • the invention provides vaccine compositions for treatment, reduction of risk of, stabilization of or prevention of an autoimmune disease.
  • the vaccine composition of the present invention may be used to prevent, reduce the risk from or treat a clinical condition.
  • the clinical condition is associated with or characterized by constitutive activation of NFkB and the expression of IkB.
  • IkB may be IkB as identified in SEQ ID NO: 1 or a homolog sharing at least 70% identity with SEQ ID NO: 1. It is understood hereby that the expression level of IkB (the expression being expression of hnRNA, mRNA, precursor protein, fully processed protein and so on) is the same or higher than in an individual not suffering from said clinical condition.
  • the clinical condition is cancer.
  • Cancer malignant neoplasm
  • a group of cells display the traits of uncontrolled growth (growth and division beyond the normal limits), invasion
  • cancer as used herein is meant to encompass any cancer, neoplastic and
  • a non-limiting group of cancers given as examples of cancers that may be treated, managed and/or prevented by administration of the vaccine of the present invention include: colon carcinoma, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangeosarcoma, lymphangeoendothelia sarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystandeocarcinoma, medullary carcinoma, bronchogenic carcinoma
  • the vaccine composition according to the invention vaccine composition is capable of eliciting a clinical response in subject, wherein the clinical response may be characterized by a stable disease, in a preferred embodiment the clinical response may be characterized by a partial response or preferably the clinical response may be characterized by complete remission of a cancer.
  • the cancer is selected from the group of; melanoma, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, hematologic cancers (such as leukemias), colon and renal cell cancers, more preferably from the group consisting of melanoma, renal cell cancer and breast cancer.
  • the vaccine composition is capable of eliciting a clinical response in an individual.
  • the clinical response may be
  • the clinical response may be characterized by a partial response or preferably the clinical response may be characterized by complete remission of a cancer or infections.
  • the clinical response may be determined as described herein below.
  • the vaccine composition is capable of eliciting a clinical response in subject, wherein the clinical response is characterized by a decrease in the sum of the longest diameter of the largest target lesion.
  • the decrease may be determined as described herein below.
  • Target lesions should be selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate repeated measurements (either by imaging techniques or clinically).
  • a sum of the longest diameter (LD) for all target lesions will be calculated and reported as the baseline sum LD.
  • the baseline sum LD will be used as reference by which to characterize the objective tumor.
  • Partial Response At least a 30% decrease in the sum of the LD of target lesions, taking as reference the baseline sum LD Progressive Disease (PD): At least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions
  • Stable Disease Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started
  • SD Incomplete Response/ Stable Disease
  • the vaccine composition comprising any of the herein mentioned proteins and/or polypeptides is capable of eliciting a clinical response in subject, wherein the clinical response is characterized by a decrease in the sum of the longest diameter of the largest target lesion
  • the vaccine composition of the invention is capable of eliciting an immune response against a cancer expressing IkB of SEQ ID NO: 1 or a functional homologue thereof having at least 70% identity to IkB.
  • the vaccine composition of the invention is capable of eliciting the production in a vaccinated individual of effector T- cells having a cytotoxic effect against the cancer cells, IkB expressing APCs and/or inducing infiltration of antigen specific T-cells in tumor stroma in a subject.
  • the peptides of the invention are capable of eliciting cytolytic immune responses in situ, i.e. in solid tumor tissues.
  • This may for example be demonstrated by providing HLA-peptide complexes, e.g. being multimerized and being provided with a detectable label, and using such complexes for immunohistochemistry stainings to detect in a tumor tissue CTLs that are reactive with the immunogenically active peptide fragments of the invention.
  • a further significant feature of the peptide of the invention is that it is capable of in situ detection in a tumor tissue of CTLs that are reactive with the epitope peptide.
  • the peptides of the invention in addition to their capacity to bind to HLA molecules resulting in the presentation of complexes of HLA and peptides on cell surfaces, which complexes in turn act as epitopes or targets for cytolytic T cells, may elicit other types of immune responses, such as B-cell responses resulting in the production of antibodies against the complexes and/or a Delayed Type Hypersensitivity (DTH) reaction.
  • DTH Delayed Type Hypersensitivity
  • the latter type of immune response is defined as a redness and palpable induration at the site of injection of the peptide of the invention.
  • It is an object of the present invention to provide a vaccine composition comprising IkB of SEQ ID NO: 1 or a functional homologue thereof having at least 70% identity to SEQ ID NO: 1 or an immunogenically active peptide fragment comprising a consecutive sequence of said IkB or said functional homologue thereof or a nucleic acid encoding said IkB or said peptide fragment; and an adjuvant, for the prevention of, reduction of risk from or treatment of cancer.
  • the invention relates to vaccine compositions comprising IkB or an immunigenically active peptide fragment thereof for treatment of cancer
  • a further conventional cancer treatment such as chemotherapy, radiotherapy, treatment with immunostimulating substances, gene therapy, treatment with antibodies and treatment using dendritic cells.
  • NFkB-based immunotherapy as disclosed by the present invention with either of these NFkB suppressants may be used for treatment of cancer.
  • IkB-based immunotherapy as disclosed by the present invention may be combined with cytotoxic chemotherapy and or another anti-cancer immunotherapeutic treatment may be used for treatment of cancer.
  • second active ingredients are also referred to herein as "second active ingredients”.
  • NFkB suppressants that are of relevance in regards to administration (sequentially or simultaneously) with the vaccine composition of the present invention include, but are not limited to: Herceptin, Tykerb, Erbitux, Iressa, Vectibix, Humira, Remicade, Revlimid, Enbrel, Sutent, Sprycel, Avastin, Nexavar, Gleevec and Tasigna.
  • vaccine composition of the present invention include, but are not limited to: all-trans retinoic acid, Actimide, Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabine, Cisplatin,
  • Vinblastine Vincristine
  • Vindesine Vinorelbine.
  • chemotherapeutic agent for use in the combination of the present agent may, itself, be a combination of different chemotherapeutic agents. Suitable combinations include FOLFOX and IFL.
  • FOLFOX is a combination which includes 5-fluorouracil (5-FU), leucovorin, and oxaliplatin.
  • IFL treatment includes irinotecan, 5-FU, and leucovorin.
  • Another second active ingredient may be a kinase inhibitor, for separate, simultaneous or combined use in the treatment of tumors.
  • Suitable kinase inhibitors include those which have been shown to possess anti-tumor activity (such as gefitinib (Iressa) and erlotinib (Tarceva) and these could be used in combination with the peptides.
  • the receptor tyrosine kinase inhibitors such as Sunitinib malate and Sorafenib which have been shown to be effective in the treatment of renal cell carcinoma are also suitable to be used as second active ingredients.
  • second active ingredients are immunostimulating substances e.g. cytokines and antibodies.
  • cytokines may be selected from the group consisting of, but not limited to: GM-CSF, type I IFN, interleukin 21 , interleukin 2, interleukin 12 and interleukin 15.
  • the antibody is preferably an immunostimulating antibody such as anti-CD40 or anti-CTLA-4 antibodies.
  • the immunostimulatory substance may also be a substance capable of depletion of immune inhibitory cells (e.g. regulatory T-cells) or factors, said substance may for example be E3 ubiquitin ligases.
  • E3 ubiquitin ligases have emerged as key molecular regulators of immune cell function, and each may be involved in the regulation of immune responses during infection by targeting specific inhibitory molecules for proteolytic destruction.
  • HECT and RING E3 proteins have now also been linked to the induction and maintenance of immune self-tolerance: c-Cbl, Cbl-b, GRAIL, Itch and Nedd4 each negatively regulate T cell growth factor production and proliferation.
  • the vaccine composition of the present invention comprising IkB, any of the functional homologues thereof described herein above or any of the immunigenically active peptide fragments thereof described herein above, is administered in combination with a second active ingredient, such as an
  • the immunostimulatory substance is preferably an interleukin such as IL-21 or IL-2 or a chemotherapeutic agent. Infectious diseases and autoimmune diseases
  • NFkB is found to be constitutively active in many inflammation and autoimmune diseases, resulting in expression of IkB.
  • the present invention also relates to vaccine compositions comprising IkB or any of the immunogenically active peptide fragments thereof described herein above for the treatment of a clinical condition, wherein the clinical condition may be an infection or the clinical condition may be an autoimmune disease.
  • infection as used herein relates to any kind of clinical condition giving rise to an immune response, such as an inflammation, and therefore includes infectious diseases, chronic infections and autoimmune conditions.
  • infections such as infectious diseases, chronic infections and autoimmune conditions are all clinical conditions of relevance for the present invention, and are dealt with in turn hereunder.
  • the infection or the autoimmune disease may be a disease associated with inflammation.
  • Inflammation is the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. Inflammation can be classified as either acute or chronic.
  • Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes from the blood into the injured tissues.
  • a cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue.
  • Prolonged inflammation known as chronic inflammation, leads to a progressive shift in the type of cells which are present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.
  • IkB is expressed by cells of the immune system such as the APCs and therefore infections and inflammations are clinical conditions that may be treated, prevented, or from which the risk may be reduced by the administration of the vaccine composition of the present invention.
  • the vaccine composition preferably comprises IkB or any of the immunogenically active peptides fragments thereof described herein above.
  • disorders associated with inflammation include, but are not limited to: Autoimmune diseases, Chronic inflammations, Infectious diseases, Inflammatory bowel diseases, Arthritis, Sepsis, Gastritis, Asthma, Neurodegenerative diseases, Heart disease, Septic shock,
  • Schizophrenia Human cerebral infarction, Alzheimer and virus infections.
  • the clinical condition is a chronic inflammation in which a constitutive active NFkB is observed.
  • the autoimmune disease to be treated with the vaccine compositions of the invention may be a chronic inflammation.
  • a chronic inflammation is a pathological condition characterized by concurrent active inflammation, tissue destruction, and attempts at repair.
  • Chronically inflamed tissue is characterized by the infiltration of mononuclear immune cells (monocytes, macrophages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, which include angiogenesis and fibrosis.
  • It is an object of the presenting invention to provide a vaccine composition comprising IkB of SEQ ID NO: 1 or a functional homologue thereof having at least 70% identity to SEQ ID NO: 1 or an immunogenically active peptide fragment comprising a
  • IkB or said functional homologue thereof for example any of the immunogenically active peptide fragments described herein above or a nucleic acid encoding said IkB or said peptide fragment; and an adjuvant, for the prevention of, reduction of risk from or treatment of an autoimmune disease, for example treatment of a chronic inflammation.
  • infectious diseases for example any of the immunogenically active peptide fragments described herein above or a nucleic acid encoding said IkB or said peptide fragment; and an adjuvant, for the prevention of, reduction of risk from or treatment of an autoimmune disease, for example treatment of a chronic inflammation.
  • the vaccine composition of the present invention may be used to prevent, reduce the risk from or treat a clinical condition in which a constitutively active NFkB is observed.
  • the clinical condition is an infectious disease.
  • the infectious disease may be promoted by any infectious agent such as bacteria, virus, parasites and or fungi that are capable of inducing an increased expression of NFkB and in turn IkB in the individual suffering from the infectious disease, preferably, the infectious disease is or is at risk of becoming a chronic disease.
  • a vaccine composition comprising IkB or any of the immunogenically active peptide fragment thereof described herein above for the treatment, amelioration of (lessening of severity) stabilization and/ or prevention of a disease caused by an infectious agent.
  • An infectious diseases may be caused by a virus, and viral diseases against which the vaccine composition of the present invention may be administered in the treatment of include, but are not limited to the following viral diseases: HIV, AIDS, AIDS Related
  • the vaccine composition is administered to individuals suffering from HIV/AIDS or viral infections that may cause cancer.
  • the main viruses associated with human cancers are human papillomavirus, hepatitis B and hepatitis C virus, Epstein-Barr virus, and human T-lymphotropic virus; thus it is an object of the present invention to be administered as the treatment of or as part of the treatment of these viral infections.
  • the infectious disease may be infection with a virus selected from the group consisting of HIV and Hepatitis virus.
  • bacterial infections include, but are not limited to: Anthrax, Bacterial Meningitis, Botulism, Brucellosis, Campylobacteriosis, Cat Scratch Disease, Cholera, Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis, Leprosy (Hansen's Disease), Leptospirosis, Listeriosis, Lyme disease, Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis, Pertussis (Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis, Q fever, Rocky Mountain Spotted Fever (RMSF), Salmonellosis, Scarlet Fever, Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia, Typhoid Fever, Typhus, and Urinary Tract Infections. It is an object of the present invention to provide
  • Parasitic infectious diseases such as, but not limited to: African trypanosomiasis, Amebiasis, Ascariasis, Babesiosis, Chagas Disease, Clonorchiasis, Cryptosporidiosis,
  • Onchocerciasis Pediculosis, Pinworm Infection, Scabies, Schistosomiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis, Trichomoniasis, and Trypanosomiasis; Fungal infectious diseases such as but not limited to:
  • transmissible spongiform encephalopathy Bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Kuru-Fatal Familial Insomnia, and Alpers Syndrome; thus it is an object of the present invention to be administered as the treatment of or as part of the treatment of these parasitic, fungal or prion caused infections.
  • the vaccine compositions of the invention are for treatment of an infectious disease, which is an intracellular infection, preferably in intracellular infection with a pathogen selected from the group consisting of L. monocytogenes and Plasmodium.
  • constitutively active NFkB is observed by the administration of the vaccine composition according to the present invention may be given in conjunction with a further (second) active ingredient either sequentially in any order or simultaneously or in combination with a further treatment such as antibiotic treatment, chemotherapy, treatment with immunostimulating substances, treatment using dendritic cells, antiviral agents anti parasitic agents and so forth.
  • a further treatment such as antibiotic treatment, chemotherapy, treatment with immunostimulating substances, treatment using dendritic cells, antiviral agents anti parasitic agents and so forth.
  • antibiotics refers to substances with antibacterial, anti-fungal, anti-viral and/or anti-parasitical activity; examples of relevance to the present invention include, but are not limited to: Amikacin, Gentamycin, Kanamycin, Neomycin, Netilmicin, Paromomycin, Streptomycin, Tobramycin, Ertapenem,
  • Imipenem Meropenem, Chloramphenicol, Fluoroquinolones, Ciprofloxacin,
  • Cefepime Monobactams, Aztreonam, Nitroimidazoles, Metronidazole, Oxazolidinones, Linezolid, Penicillins, Amoxicillin, Amoxicillin / Clavulanate, Ampicillin, Sulbactam, Bacampicillin, Carbenicillin, Cloxacillin, Dicloxacillin, Methicillin, Mezlocillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Piperacillin / Tazobactam, Ticarcillin, Ticarcillin / Clavulanate, Streptogramins, Quinupristin, Dalfopristin, Sulfonamide / Sulfamethoxazole, Trimethoprim, Tetracyclines, Demeclocycline, Doxycycline,
  • Minocycline, Tetracycline, Azole antifungals Clotrimazole Fluconazole, Itraconazole, Ketoconazole, Miconazole, Voriconazole, Amphotericin B, Nystatin, Echinocandin, Caspofungin, Micafungin, Ciclopirox, Flucytosine, Griseofulvin, and Terbinafine.
  • antivirals such as Vidarabine, Acyclovir, Gancyclovir and Valcyte (valganciclovir), Nucleoside-analog reverse transcriptase inhibitors (NRTI): AZT (Zidovudine), ddl (Didanosine), ddC (Zalcitabine), d4T (Stavudine), 3TC (Lamivudine), Non-nucleoside reverse transcriptase inhibitors (NNRTI): Nevirapine, Delavirdine, Protease Inhibitors: Saquinavir, Ritonavir, Indinavir, Nelfinavir, Ribavirin, Amantadine / Rimantadine, Relenza and Tamiflu, Pleconaril, Interferons
  • the present invention regards a vaccine composition comprising IkB of SEQ ID NO: 1 , any of the functional homologues thereof described herein above or any of the immunigenically active peptide fragments thereof described herein above for the treatment of an infectious disease in combination with at least one antibiotic.
  • the vaccine composition of the present invention is used for the treatment of chronic infections e.g. HIV and therefore is used in combination with any of the above listed antibiotics such as anti-viral agents.
  • Autoimmune diseases arise when an organism fails to recognize its own constituent parts (down to the sub-molecular levels) as self, which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease and is of relevance to the present invention. Autoimmune diseases have also been demonstrated to be linked to an constitutive activation of NFkB and expression of IkB.
  • It is an object of the present invention to provide a vaccine composition comprising IkB of SEQ ID NO: 1 or a functional homologue thereof having at least 70% identity to SEQ ID NO: 1 or any immunogenically active peptide fragments of IkB described herein above or a nucleic acid encoding said IkB or said peptide fragment; and an adjuvant, for the prevention of, reduction of risk from or treatment of autoimmune diseases.
  • Said autoimmune diseases may preferably be selected from the group consisting of the Coeliac disease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Sjogren's syndrome, multiple sclerosis (MS), Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis (RA).
  • IDDM diabetes mellitus type 1
  • SLE systemic lupus erythematosus
  • MS multiple sclerosis
  • Hashimoto's thyroiditis Graves' disease
  • idiopathic thrombocytopenic purpura rheumatoid arthritis
  • RA rheumatoid arthritis
  • Non-immune therapies such as hormone replacement in Hashimoto's thyroiditis or diabetes mellitus Type 1 treatment outcomes of the autoaggressive response. Dietary manipulation limits the severity of celiac disease. Steroidal or NSAID treatment limits inflammatory symptoms of many diseases.
  • IVIG immune globulin
  • CIDP Chronic Inflammatory Demyelinating Polyneuropathy
  • GSS Guillain-Barre syndrome
  • immunomodulatory therapies such as the TNFa antagonist Etanercept, have been shown to be useful in treating RA. These immunotherapies may be associated with increased risk of adverse effects, such as susceptibility to infection.
  • Helminthic therapy has developed based on these observations and involves inoculation of the individual with specific parasitic intestinal nematodes (helminths).
  • helminths There are currently two closely-related treatments available, inoculation with either Necator americanus, commonly known as hookworms, or Trichuris Suis Ova, commonly known as Pig Whipworm Eggs. Research is available that demonstrates this approach is highly effective in treating a variety of autoimmune disorders, including Crohn's, Ulcerative Colitis, Asthma, allergies, Multiple Sclerosis, and chronic inflammatory disorders
  • the vaccine herein disclosed is used in combination with a second active ingredient such as any of the above mentioned drugs and treatments against autoimmune diseases.
  • the present invention regards pharmaceutical compositions capable of treating, reducing the risk of and/or preventing a clinical disorder associated with constitutive expression of NFkB and IkB expression in an individual; in other words the terms vaccine and pharmaceutical composition are used interchangeably herein.
  • the vaccine / pharmaceutical compositions of the present invention may be "traditional" vaccine compositions comprising antigens such as proteins polypeptides and/or nucleic acid molecules. They may also be in the form of compositions comprising cells, such as modified cells originating from the individual and later processed, or to compositions comprising complex molecules such as antibodies or TCRs.
  • a vaccine is a substance or composition capable of inducing an immune response in an individual.
  • the composition may comprise one or more of the following: an "active component" such as an antigen(s) (e.g. protein, polypeptides, peptides, nucleic acids and the like), nucleic acid constructs comprising one or more antigens amongst other elements, cells, (e.g. loaded APC, T cells for adoptive transder aso.), complex molecules (Antibodies, TCRs and MHC complexes and more), carriers, adjuvants and pharmaceutical carriers.
  • an active component such as an antigen(s) (e.g. protein, polypeptides, peptides, nucleic acids and the like), nucleic acid constructs comprising one or more antigens amongst other elements, cells, (e.g. loaded APC, T cells for adoptive transder aso.), complex molecules (Antibodies, TCRs and MHC complexes and more), carriers, adjuvants and pharmaceutical carriers.
  • an antigen(s) e.g. protein, polypeptides, peptides,
  • the vaccine composition of the invention is capable of eliciting an immune response against a cancer, DC or APC expressing IkB of SEQ ID NO: 1 or a functional homologue thereof having at least 70% identity to SEQ ID NO 1 , when administered to an individual suffering from a cancer and/or infection (leading to the constitutive expression of NFkB and expression of IkB).
  • the clinical condition is a cancer.
  • the vaccine composition of the invention is capable of eliciting the production in a vaccinated individual of effector T-cells having a cytotoxic effect against cancer cells, APCs and DCs expressing IkB and/or inducing infiltration of antigen specific T-cells in tumor stroma in a subject.
  • the vaccine composition of the present invention comprises one or more of the following: IkB (SEQ ID NO: 1), immunogenically active peptide fragments here from, functional homologues of full length and partial length IkB, in particular any of the fragments described herein above. More preferably, the vaccine composition comprises any of the sequences listed in the sequence list of the present disclosure. Very preferably, the vaccine composition comprises the peptides lkB10 (SEQ ID NO: 11) and/or lkB18 (SEQ ID NO: 19).
  • antigen in the vaccine composition of the invention will depend on parameters determinable by the person of skill in the art. As it has been mentioned, each of the different peptides of the invention is presented on the cell surfaces by a particular HLA molecule. As such, if a subject to be treated is typed with respect to HLA phenotype, a peptide/peptides are selected that is/are known to bind to that particular HLA molecule. Alternatively, the antigen of interest is selected based on the prevalence of the various HLA phenotypes in a given population.
  • HLA-A2 is the most prevalent phenotype in the Caucasian population, and therefore, a composition containing a peptide binding to HLA-A2 will be active in a large proportion of that population.
  • the antigens / peptides of the present invention may be modified according to the anchor residue motifs presented in Table 2, to enhance binding to particular HLA molecules.
  • the composition of the invention may also contain a combination of two or more IkB derived peptides, each interacting specifically with a different HLA molecule so as to cover a larger proportion of the target population.
  • the pharmaceutical composition may contain a combination of a peptide restricted by a HLA-A molecule and a peptide restricted by a HLA-B molecule, e.g. including those HLA-A and HLA-B molecules that correspond to the prevalence of HLA phenotypes in the target population, such as e.g. HLA-A2 and HLA-B35.
  • the composition may comprise a peptide restricted by an HLA-C molecule.
  • epitopes can be administered in an 'MHC-ready' form, which enables presentation through exogenous loading independently of antigen uptake and processing by host antigen-presenting cells.
  • the peptides of the present invention comprise both peptides in a short 'MHC-ready' form and in a longer form requiring processing by the proteasome thus providing a more complex vaccine composition that can target multiple tumor antigens.
  • the more different HLA groups are targeted by a vaccine the higher likelihood of the vaccine functioning in diverse populations.
  • the present invention regards in a preferred embodiment a vaccine composition
  • a vaccine composition comprising IkB of SEQ ID NO: 1 or a functional homologue thereof having at least 70% identity to SEQ ID NO: 1 or an immunogenically active peptide fragment comprising a consecutive sequence of said IkB or said functional homologue thereof or a nucleic acid encoding said IkB or said peptide fragment; in combination with an adjuvant for use as a medicament.
  • the vaccine composition may be administered to treat, prevent, or reduce the risk associated with a clinical condition in an individual.
  • the invention also relates to highly immunogenic multi-epitope vaccines.
  • such vaccines should be designed so as to facilitate a simultaneous delivery of the best-suited IkB-derived peptides optionally in combination with other suitable peptides and/or adjuvants as described hereinafter.
  • the present invention encompasses such multiepitope vaccines comprising IkB-derived peptides optionally in combination with further proteins or peptides fragments not belonging to or derived from IkB and/or adjuvants as described hereinafter.
  • An important factor driving the development of vaccines having a more complex composition is the desire to target multiple tumor antigens e.g. by designing vaccines comprising or encoding a collection of carefully selected CTL and T h cell epitopes.
  • the invention thus in one aspect relates to vaccine compositions comprising both Class I and Class ll-restricted IkB epitopes.
  • composition according to the present invention may be provided as a multiepitope vaccine comprising class I restricted epitope and/or class II restricted epitopes as defined hereinbefore.
  • Nucleic acid based vaccine composition
  • the vaccine composition according to the present invention may comprise a nucleic acid encoding IkB or an immunologically active peptide fragment thereof, in particular any of the fragments described herein above.
  • Said nucleic acid may thus encode any of the above-mentioned proteins and peptide fragments.
  • the nucleic acid may for example be DNA, RNA, LNA, HNA, PNA, preferably the nucleic acid is DNA or RNA.
  • the nucleic acids of the invention may be comprised within any suitable vector, such as an expression vector. Numerous vectors are available and the skilled person will be able to select a useful vector for the specific purpose.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle or artificial chromosome.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures, for example, DNA may be inserted into an appropriate restriction endonuclease site(s) using techniques well known in the art.
  • the vector may furthermore comprise one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • the vector may also comprise additional sequences, such as enhancers, poly-A tails, linkers, polylinkers, operative linkers, multiple cloning sites (MCS), STOP codons, internal ribosomal entry sites (IRES) and host homologous sequences for integration or other defined elements.
  • MCS multiple cloning sites
  • IRS internal ribosomal entry sites
  • the vector is preferably an expression vector, comprising the nucleic acid operably linked to a regulatory nucleic acid sequence directing expression thereof in a suitable cell.
  • said regulatory nucleic acid sequence should in general be capable of directing expression in a mammalian cell, preferably a human cell, more preferably in an antigen presenting cell.
  • the vector is a viral vector.
  • the vector may also be a bacterial vector, such as an attenuated bacterial vector. Attenuated bacterial vectors may be used in order to induce lasting mucosal immune responses at the sites of infection and persistence. Different recombinant bacteria may be used as vectors, for example the bacterial vector may be selected from the group consisting of Salmonella, Lactococcus, and Listeria. In general, induction of immunity to the heterologous antigen HPV16 L1 or E7 could be shown, with strong CTL induction and tumor regression in mice.
  • the vector may furthermore comprise a nucleic acid encoding a T-cell stimulatory polypeptide.
  • an immunogenic response directed against a cancer disease is elicited by administering the peptide of the invention either by loading MHC class I or class II molecules on antigen presenting cells (APCs) from the individual, by isolating PBLs from the individual and incubating the cells with the peptide prior to injecting the cells back into the individual or by isolating precursor APCs from the individual and differentiating the cells into professional APCs using cytokines and antigen before injecting the cells back into the individual.
  • APCs antigen presenting cells
  • antigen presenting cells comprising IkB or any of the immunologically active peptide fragment thereof described herein above or a nucleic acid encoding said IkB or said immunologically active peptide fragment.
  • the antigen presenting cell may be any cell capable of presenting an antigen to a T-cell.
  • Preferred antigen presenting cells are dendritic cells.
  • the dendritic cells (DC) may be prepared and used in therapeutic procedure according to any suitable protocol, for example as described herein below. It will be appreciated by the person skilled in the art that the protocol may be adopted to use with individuals with different HLA type and different diseases.
  • Dendritic cells may be pulsed with 50 ⁇ g/ml HLA-restricted peptide (synthesized at GMP quality) for 1 h at 37°C peptide and 5 x 10 6 cells are administered
  • a method for treating an individual suffering from a clinical condition characterized by the expression of IkB, preferably wherein the clinical condition is cancer or an infection is one wherein the peptide is administered by presenting the peptide to the individual's antigen presenting cells (APCs) ex vivo followed by injecting the thus treated APCs back into the individual.
  • APCs antigen presenting cells
  • Loading the MHC class I molecules means incubating the APCs with the peptide so that the APCs with MHC class I molecules specific for the peptide will bind the peptide and therefore be able to present it to T cells. Subsequently, the APCs are re-injected into the individual.
  • Another alternative way relies on the recent discoveries made in the field of dendritic cell biology. In this case, monocytes (being dendritic cell precursors) are isolated from the individual and differentiated in vitro into professional APC (or dendritic cells) by use of cytokines and antigen. Subsequently, the in vitro generated DCs are pulsed with the peptide and injected into the individual.
  • An important aspect the invention relates to cultivating IkB specific T-cells in vitro and adoptive transfer of these to individuals.
  • Adoptive transfer means that the physician directly transfers the actual components of the immune system that are already capable of producing a specific immune response, into an individual.
  • IkB specific T-cells which may be useful for example for adoptive transfer.
  • Isolated T-cells comprising T-cell receptors capable of binding specifically to IkB peptide/MHC class I or IkB peptide/MHC class II complexes can be adoptively transferred to individuals, said T-cells preferably being T- cells that have been expanded in vitro, wherein the IkB peptide may be any of the IkB peptides mentioned herein above.
  • the invention also relates to methods of treatment comprising administering T-cells comprising T-cell receptors capable of binding specifically to a MHC- restricted IkB peptide complex to an individual, such as a human being suffering from a cancer disease, wherein the IkB derived peptide may be any of the IkB peptides mentioned herein above.
  • the invention furthermore relates to use of T-cells comprising T-cell receptors capable of binding specifically to IkB or peptide fragments thereof for the preparation of a medicament for the treatment of a cancer or infection.
  • Autologous T-cell transfer may be performed essentially as described in Walter et al., (1995).
  • such T-cells could be irradiated before adoptive transfer to control proliferation in the individual. It is possible to genetically engineer the specificity of T cells by TCR gene transfer (Engels et al., 2007). This allows the transfer of T cells bearing IkB peptide specificity into individuals. In general, the use of T cells for adoptive immunotherapy is attractive because it allows the expansion of T cells in a tumor- or virus-free environment, and the analysis of T cell function prior to infusion.
  • TCR gene-modified T cells such as T-cells transformed with an expression construct directing expressing of a heterologous TCR
  • TCR gene-modified T cells has several advantages in comparison to the transfer of T cell lines: (i) the generation of redirected T cells is generally applicable, (ii) High-affinity or very high-affinity TCRs can be selected or created and used to engineer T cells, (iii) High-avidity T cells can be generated using codon optimized or murinized TCRs allowing better surface expression of the stabilized TCRs. Genetic engineering of T cell specificity by T cell receptor (TCR) gene transfer may be performed essentially as described in Morgan et al., (2006).
  • TCR with known anti-tumor reactivity can be genetically introduced into primary human T lymphocytes.
  • Genes encoding TCR alpha and beta chains from a tumor specific CTL clone can be transfected into primary T cells and in this way reprogram T cells with specificity against the tumor antigen.
  • TCR RNA is transfected into PBL by
  • T cells can be provided with at new specificity by TCR gene transfer using retroviral vectors (Morgan et al., 2006).
  • the provirus from the retroviral vector might integrate at random in the genome of the transfected cells and subsequently disturb cell growth. Electroporation of T cells with TCR-coding RNA overcome this disadvantage, since RNA is only transiently present in the transfected cells and can not be integrated in the genome (Schaft et al., 2006). Furthermore, transfection of cells is routinely used in the laboratory.
  • the vaccine composition according to the invention preferably comprises an adjuvant and/or a carrier.
  • an adjuvant and/or a carrier examples of useful adjuvants and carriers are given herein below.
  • useful adjuvants and carriers are given herein below.
  • immunogenically active peptide fragment thereof may in a composition of the present invention be associated with an adjuvant and/or a carrier.
  • Adjuvants are any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response to the IkB or peptide fragment thereof, see further in the below.
  • Carriers are scaffold structures, for example a polypeptide or a polysaccharide, to which the IkB or peptide fragment thereof is capable of being associated and which aids in the presentation of especially the peptides of the present invention.
  • peptides of the invention are relatively small molecules and it may therefore be required in compositions as described herein to combine the peptides with various materials such as adjuvants and/or carriers, to produce vaccines, immunogenic compositions, etc.
  • adjuvants broadly defined, are substances which promote immune responses. A general discussion of adjuvants is provided in Goding, Monoclonal
  • Antibodies Principles & Practice (2nd edition, 1986) at pages 61-63. Goding notes, that when the antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended.
  • carrier molecules include keyhole limpet haemocyanin, bovine serum albumin, ovalbumin and fowl immunoglobulin.
  • saponin extracts have also been suggested to be useful as adjuvants in immunogenic compositions. It has been proposed to use granulocyte- macrophage colony stimulating factor (GM-CSF), a well known cytokine, as an adjuvant (WO 97/28816).
  • GM-CSF granulocyte- macrophage colony stimulating factor
  • a carrier may be present independently of an adjuvant.
  • a carrier can for example be to increase the molecular weight of in particular peptide fragments in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life.
  • a carrier may aid in presenting the IkB polypeptide or said fragment thereof to T-cells.
  • the carrier may be any suitable carrier known to a person skilled in the art, for example a protein or an antigen presenting cell.
  • a carrier protein could be, but is not limited to, keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier For immunization of humans, the carrier must be a physiologically acceptable carrier acceptable to humans and safe. However, tetanus toxoid and/or diptheria toxoid are suitable carriers in one embodiment of the invention. Alternatively, the carrier may be dextrans for example sepharose.
  • the vaccine compositions comprise IkB of SEQ ID NO: 1 , a functional homologue thereof sharing at least 70% sequence identity or any of the immunogenically active peptide fragments described herein above is associated with a carrier such as e.g. a protein of the above or an antigen-presenting cell such as e.g. a dendritic cell (DC).
  • a carrier such as e.g. a protein of the above or an antigen-presenting cell such as e.g. a dendritic cell (DC).
  • the vaccine compositions of the invention in general comprises an adjuvant.
  • Adjuvants could for example be selected from the group consisting of: AIK(S0 4 ) 2 , AINa(S0 4 ) 2 , AINH 4 (S0 4 ), silica, alum, AI(OH) 3 , Ca 3 (P0 4 ) 2 , kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl- nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N- acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 '2'-dipalmitoyl-sn -glycero-3- hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to
  • lipid A lipid A
  • FCA Freund's Complete Adjuvant
  • FCA Freund 's Incomplete Adjuvants
  • Merck Adjuvant 65 polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1 , Interleukin 2, Montanide ISA-51 and QS-21.
  • Preferred adjuvants to be used with the invention include oil/surfactant based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51.
  • Other preferred adjuvants are bacterial DNA based adjuvants, such as adjuvants including CpG oligonucleotide sequences.
  • Yet other preferred adjuvants are viral dsRNA based adjuvants, such as poly l:C.
  • Imidazochinilines are yet another example of preferred adjuvants. The most preferred adjuvants are adjuvants suitable for human use.
  • Montanide adjuvants may be selected from the group consisting of Montanide ISA-51 , Montanide ISA-50, Montanide ISA-70,
  • Montanide ISA-720 more preferably from the group consisting of Montanide ISA-51.
  • Montanide ISA-51 (Seppic, Inc.) is oil/surfactant based adjuvants in which different surfactants are combined with a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. They are prepared for use as an emulsion with an aqueous solution comprising IkB of SEQ ID NO: 1 , any of the functional homologues thereof described herein above or any of the immunogenically active peptide fragments thereof described herein above.
  • the surfactant is mannide oleate.
  • QS-21 Antigenics; Aquila
  • Biopharmaceuticals, Framingham, MA is a highly purified, water-soluble saponin that handles as an aqueous solution.
  • QS-21 and Montanide ISA-51 adjuvants can be provided in sterile, single-use vials.
  • the well-known cytokine GM-CSF is another preferred adjuvant of the present invention.
  • GM-CSF has been used as an adjuvant for a decade and may preferably be GM-CSF as described in WO 97/28816.
  • CTL induction Particles which can bind or Cytosolic processing of protein enclose immunogen and which yielding correct class 1 restricted can fuse with or disrupt cell peptides
  • Carbohydrate adjuvants which As above. May also determine target lectin receptors on type of response if targeting macrophages and DCs selective
  • a vaccine composition according to the present invention may comprise more than one adjuvant.
  • the invention encompasses a therapeutic composition further comprising any adjuvant substance and/or carrier including any of the above or combinations thereof. It is also contemplated that the IkB protein, the functional homologue thereof or any of the immunogenically active peptide fragments thereof, and the adjuvant can be administered separately in any appropriate sequence.
  • the vaccine compositions of the present invention comprise a Montanide adjuvant such as Montanide ISA 51 or Montanide ISA 720 or the GM-CSF adjuvant.
  • the invention encompasses a therapeutic composition further comprising an adjuvant substance including any of the above or combinations thereof. It is also contemplated that the antigen, i.e. the peptide of the invention and the adjuvant can be administered simultaneously or separately in any appropriate sequence.
  • the amount of the IkB or the immunogenically active peptide fragment thereof in the vaccine composition may vary, depending on the particular application. However, a single dose of the IkB or the peptide fragment thereof is preferably anywhere from about 10 ⁇ g to about 5000 ⁇ g, more preferably from about 50 ⁇ g to about 2500 ⁇ g such as about 100 ⁇ g to about 1000 ⁇ g.
  • Modes of administration include intradermal, subcutaneous and intravenous administration, implantation in the form of a time release formulation, etc. Any and all forms of administration known to the art are encompassed herein.
  • any and all conventional dosage forms that are known in the art to be appropriate for formulating injectable immunogenic peptide composition are encompassed, such as lyophilized forms and solutions, suspensions or emulsion forms containing, if required, conventional pharmaceutically acceptable carriers, diluents, preservatives, adjuvants, buffer components, etc.
  • the pharmaceutical compositions may be prepared and administered using any conventional protocol known by a person skilled in the art. In example 2 non-limiting examples of preparation of a vaccine composition according to the invention is given as well as a non-limiting example of administration of such as a vaccine. It will be appreciated by the person skilled in the art that the protocol may be easily adapted to any of the vaccine compositions described herein.
  • the pharmaceutical composition of the invention is useful for treating an individual suffering from a clinical condition characterized by expression of IkB, such as cancer and infections.
  • the immunoprotective effect of the composition of the invention can be determined using several approaches known to those skilled in the art.
  • a successful immune response may also be determined by the occurrence of DTH reactions after immunization and/or the detection of antibodies specifically recognizing the peptide(s) of the vaccine composition.
  • Vaccine compositions according to the invention may be administered to an individual in therapeutically effective amounts.
  • compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. Administration of pharmaceutical compositions is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (directly to the tissue), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • the present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the methods of prophylaxis and treatment with the vaccine composition.
  • the vaccine compositions can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • An effective but non-toxic amount of the vaccine, comprising any of the herein described compounds can be employed as a prophylactic or therapeutic agent.
  • any and all conventional dosage forms that are known in the art to be appropriate for formulating injectable immunogenic peptide composition are encompassed, such as lyophilized forms and solutions, suspensions or emulsion forms containing, if required, conventional pharmaceutically acceptable carriers, diluents, preservatives, adjuvants, buffer components, etc.
  • Preferred modes of administration of the vaccine composition according to the invention include, but are not limited to systemic administration, such as intravenous or subcutaneous administration, intradermal administration, intramuscular administration, intranasal administration, oral administration, rectal administration, vaginal
  • systemic administration such as intravenous or subcutaneous administration, intradermal administration, intramuscular administration, intranasal administration, oral administration, rectal administration, vaginal
  • a vaccine according to the present invention can be administered once, or any number of times such as two, three, four or five times.
  • the administration of the vaccine composition of the present invention can be administered any number of time such as 1 time monthly the first two years, hereafter administered once every three months for at least two years, such as three years, four years or five years.
  • Administering the vaccine more than once has the effect of boosting the resulting immune response.
  • the vaccine can further be boosted by administering the vaccine in a form or body part different from the previous administration.
  • the booster shot is either a homologous or a heterologous booster shot.
  • a homologous booster shot is a where the first and subsequent vaccinations comprise the same constructs and more specifically the same delivery vehicle especially the same viral vector.
  • a heterologous booster shot is where identical constructs are comprised within different viral vectors.
  • the vaccine composition herein provided is used in combination with a second active ingredient.
  • the administration of the vaccine composition and the second active ingredient may be sequential or combined.
  • the vaccine composition may be used in combination with other therapy of relevance for the given clinical condition to be treated.
  • therapy may include surgery, chemotherapy or gene therapy, immunostimulating substances or antibodies; a person skilled in the art is able to determine the appropriate combination treatment for a given scenario.
  • a further medical treatment such as chemotherapy, radiotherapy, treatment with immunostimulating substances, gene therapy, treatment with antibodies and/or antibiotics and treatment using dendritic cells.
  • composition of the invention is a composition for ex vivo or in situ diagnosis of the presence of IkB expressing cells in an individual.
  • the diagnostic procedure is based on the detection of IkB reactive T cells among PBLs or in tumor tissue.
  • a diagnostic kit for ex vivo or in situ diagnosis of the presence in an individual of IkB reactive T cells among PBLs or in tumour tissue comprising one or more peptides of the invention, and a method of detecting in an individual the presence of such reactive T cells, the method comprising contacting a tumour tissue or a blood sample with a complex of a peptide of the invention and a Class I or Class II HLA molecule or a fragment of such molecule and detecting binding of the complex to the tissue or the blood cells.
  • the invention provides a complex of a peptide of the invention and a Class I or Class II HLA molecule or a fragment of such molecule, which is useful as a diagnostic reagent such as it is described herein.
  • Such a complex may be monomeric or multimeric.
  • Another useful diagnostic or prognostic approach is based on generating antibodies in a heterologous animal species, e.g. murine antibodies directed against human IkB, which can then be used, e.g. to diagnose for the presence of cancer cells presenting the peptide.
  • the amount of peptide may be less than that used in the course of in vivo therapy, such as that mentioned above.
  • a preferred dose can range from about 1 ⁇ g to about 750 ⁇ g of peptide. It is also possible to produce monoclonal antibodies based on immunization with a peptide of the invention.
  • the present invention also relates to a molecule, in particular a monoclonal or polyclonal antibody including a fragment hereof, that is capable of binding specifically to a peptide of the invention and to a molecule that is capable of blocking such a binding, e.g. an antibody raised against the monoclonal or polyclonal antibody directed against a peptide of the invention.
  • the invention furthermore relates to isolated T-cell receptors capable of binding specifically to a peptide or a protein of the invention as well as to isolated nucleic acids encoding same.
  • T-cell receptors may for example be cloned from protein or peptide specific T-cells using standard techniques well known to the skilled person.
  • the invention also relates to isolated T-cells comprising T-cell receptors capable of binding specifically to IkB and/or any of the immunogenically active peptide fragments thereof described herein.
  • the isolated T-cells may be CD8 T-cells or CD4 T- cells.
  • the isolated T-cells are preferably T-cells that have been expanded in vitro. Methods of expanding T-cells in vitro are well known to the skilled person. Such T-cells may in particular be useful in the treatment of cancer by adaptive transfer or autologous cell transfer.
  • the invention also relates to pharmaceutical
  • compositions comprising T-cells as well as methods of treatment comprising administering T-cells comprising T-cell receptors capable of binding specifically to IkB or peptide fragments thereof to an individual, in need thereof such as an individual suffering from cancer and/or infections.
  • Autologous cell transfer may be performed essentially as described in Walter et al., (1995).
  • the present invention provides the means for treating, preventing, alleviating or curing a clinical condition characterized by expression of IkB such as cancers and infections preferably a cancer, comprising administering to an individual suffering from the disease an effective amount of a composition as defined herein, a molecule that is capable of binding specifically to a peptide fragment, which may for example be an antibody or a T-cell receptor or the kit-of-parts described herein. Accordingly, it is a further aspect of the invention to provide a method of treating a clinical condition associated with the constitutive expression of NFkB and expression of IkB of SEQ ID NO: 1. Monitoring immunization
  • the pharmaceutical composition of the invention is a vaccine composition. It is therefore of interest, and an aspect of the present invention to monitor the immunization in an individual to whom the vaccine composition of the present invention is administered.
  • the vaccine composition may thus be capable of eliciting an immune response to a cancer and/or infection.
  • the expression "vaccine composition” refers to a composition eliciting at least one type of immune response directed against IkB expressing cells such as cancer cells, APCs or DCs.
  • an immune response may be any of the following: A CTL response where CTLs are generated that are capable of recognizing the HLA/peptide complex presented on cell surfaces resulting in cell lysis, i.e.
  • the vaccine elicits the production in the vaccinated subject of effector T-cells having a cytotoxic effect against the cancer cells; a B-cell response giving rise to the production of anti-cancer antibodies; and/or a DTH type of immune response. It is on object of the present invention to monitor the immunization of an individual by monitoring any of the above reactions subsequent to administering the composition of the present invention to said individual.
  • the invention relates to methods of monitoring immunization, said method comprising the steps of
  • determining whether said blood sample comprises antibodies or T-cells comprising T-cell receptors specifically binding the protein or peptide iv) thereby determining whether an immune response to said protein or peptide has been raised in said individual.
  • the individual is preferably a human being, for example a human being that has been immunized with IkB or a peptide fragment hereof or a nucleic acid encoding said protein or peptide.
  • the invention also relates to a kit-of-parts comprising
  • the invention also relates to a kit-of-parts comprising
  • the second active ingredient is chosen in correspondence with the clinical condition to be treated so that in the case where a cancer is to be treated the second active ingredient is chosen among e.g. chemotherapeutic agents as listed above.
  • the second active ingredient is preferably an antibiotic and/or an anti-viral agent.
  • kit-of-parts are preferably comprised in individual compositions, it is however within the scope of the present invention that the components of the kit-of- parts all are comprised within the same composition.
  • the components of the kit-of- parts may thus be administered simultaneously or sequentially in any order.
  • PBMC Peripheral Blood Mononuclear Cells
  • the ELISPOT assay was used to quantify peptide specific IFN- ⁇ releasing effector cells as described previously (Andersen et al., 2001 , Cancer Res. 61 :869-872).
  • PBMC were stimulated once in vitro with peptide prior to analysis as described (McCutcheon et al., 1997, J Immunol Methods 210: 149-166) to extend the sensitivity of the assay.
  • nitrocellulose bottomed 96-well plates Multiscreen MAIP N45; Millipore
  • the wells were washed, blocked by X-vivo medium and the effector cells (PBMC collected and when indicated stimulated as described above) were added in duplicates at different cell concentrations, with or without 10 ⁇ peptide.
  • the plates were incubated overnight. The following day, medium was discarded and the wells were washed prior to addition of the relevant biotinylated secondary Ab (Mabtech).
  • the plates were incubated at room temperature (RT) for 2 hours, washed, and Avidin-enzyme conjugate (AP-Avidin; Calbiochem/lnvitrogen Life Technologies) was added to each well.
  • RT room temperature
  • PBL from a specific culture were cloned by limiting dilution in the presence of cloning mix containing 10 6 /ml irradiated (20 Gy) lymphocytes from three healthy donors in X- vivo with 5% heat-inactivated human serum, 25 mM HEPES buffer (GibcoBRL, Paisley, UK), and 120 U/ml IL-2 (PeproTech, London, UK).
  • the plates were incubated at 37°C/5%C0 2 . Every 3-4 days 50 ⁇ fresh media were added containing IL-2 to a final concentration of 120U/ml.
  • Growing clones were expanded using IL-2. After expansion the clones were tested for specificity and cytotoxic potential in a standard 51 Cr-release assay. Cytotoxicity assay
  • Target cells were T2 with or without lkB10 (figure 3a) and the HLA-A2+ cancer cell line FM3 or FM86 (figure 3c). In some assays, target cells were treated with 100 U/ml IFN- ⁇ for 2 days.
  • the amino acid sequence of the ⁇ - ⁇ protein was screened for the most probable HLA-A2 nona- and deca-mer peptide epitopes from the main HLA-A2 specific anchor residues (see Table 2). Eighteen ⁇ - ⁇ -derived peptides (entitled ⁇ 1-18) were selected and subsequently synthesized.
  • ELISPOT IFN- ⁇ secretion assay we examined PBMC from cancer patients and healthy individuals for the presence of specific T-cell responses against these ⁇ - ⁇ -derived peptides.
  • PBMC from HLA-A2 + cancer patients were stimulated once with the different peptides in vitro before examination by ELISPOT.
  • ELISPOT responses were detected against ⁇ 18 (SEQ ID NO: 19) and ⁇ 10 (SEQ ID NO: 11) (Fig. 1).
  • the peptide specificity was tested in standard 51 Cr-release assays against T2 cells pulsed with ⁇ 10 or an irrelevant peptide (the HLA-A2 high affinity binding epitope HIV-1 pol 476 -484 (ILKEPVHGV)).
  • This showed that only T2 cells loaded with ⁇ 10 were recognized and killed by the T-cell bulk culture (Figure 3b).
  • the T-cell bulk culture did, however, only kill the melanoma cell line FM86 weakly (5% lysis at effector to target ratio 30: 1).
  • to increase the frequency of specific T cells clones were established from the bulk culture as single cell cultures by limiting dilution.
  • T2 cells either loaded with ⁇ 10 peptide or irrelevant HIV peptide served as targets.
  • This assay revealed that only T2 cells pulsed with ⁇ 10 was killed by the T cell clone ( Figure 3c).
  • the ⁇ -specific T cell clone was further used to test the capacity to kill the HLA-A2-positive well established immunogenic melanoma cell line FM3.
  • Figure 3c illustrate that an ⁇ -specific T cell clone was able to kill the cancer cell line. Killing of FM3 were further increased by pre-treatment of the cancer cells with I FN- ⁇ .
  • Vaccine composition 500 ⁇ g IkB-peptide (lkB10 or lkB18) in 500 ⁇ _ phosphate buffer is mixed with 500 ⁇ _
  • Montanide adjuvans (Seppic, France) and administered to the patient.
  • 75 ⁇ g Leukine (Sargramostim; GM-CSF - available from Genzyme, United States) for stimuluation of the immune system.
  • Both the vaccine composition and the GM-CSF is administered by subcutan injection.
  • the area of vaccination is in addition treated topically with Aldara (Imiquimod - available from MEDA AB, Sweden) to increase the local immune response.
  • PBMC Peripheral Blood Mononuclear Cells
  • PBMC peripheral blood mononuclear cells
  • Epitopes from ⁇ - ⁇ were predicted using the "Database SYFPEITHIP" in combination with manual examination of the protein sequence for MHC class I anchor residues. Twelve 9mer and six 10mer synthetic peptides were produced: ⁇ 1 ( ⁇ 197.205;
  • LLLKCGADV LLLKCGADV
  • ⁇ 4 ⁇ 20 ⁇ - 2 09
  • LLVSLGADV LLVSLGADV
  • ⁇ 5 ⁇ 12 - 20 ;
  • AM EGPRDG L AM EGPRDG L
  • ⁇ 6 ⁇ 195 . 2 ⁇ 3
  • ⁇ L G I V ⁇ L L V ⁇ 7
  • ⁇ 44 -52 AM EGPRDG L
  • LLGAGCDPEL ⁇ 16 ( ⁇ 25 . 3 ; LLDDRH DSGL), ⁇ 17 ( ⁇ 268 . 277 ;
  • the ELISPOT assay was used to quantify peptide epitope-specific IFN- ⁇ releasing effector cells.
  • PBMC peripheral blood mononuclear cells
  • IFN- ⁇ capture mAb Mabtech
  • the wells were washed, blocked with X-vivo medium and the effector cells were added in duplicates at different cell concentrations, with or without 10 ⁇ peptide.
  • the plates were incubated overnight. The following day, medium was discarded and the wells were washed prior to addition of the relevant biotinylated secondary Ab (Mabtech).
  • the plates were incubated at room temperature (RT) for 2 hours, washed, and Avidin-enzyme conjugate (AP-Avidin; Mabtech) was added to each well. Plates were incubated at RT for 1 hour and the enzyme substrate NBT/BCIP (Invitrogen Life Technologies) was added to each well and incubated at RT for 5-10 min. Upon the emergence of dark purple spots, the reaction was terminated by washing with tap water. The spots were counted using the ImmunoSpot Series 2.0 Analyzer (CTL Analyzers).
  • CTL Analyzers ImmunoSpot Series 2.0 Analyzer
  • IL-7 5ng/ml
  • IL-12 10ng/ml
  • Stimulation of the cultures were carried out every 8 days with ⁇ -loaded irradiated autologous DC (2x) followed by ⁇ ⁇ -loaded irradiated autologous PBL (3x). After each stimulation 120 U/ml IL-2 (PeproTech, London, UK) was added.
  • the established cultures were tested for specificity for ⁇ 10 after 4 th stimulation.
  • PBL from a specific culture were cloned by limiting dilution in the presence of cloning mix containing 10 6 /ml irradiated (25 Gy) lymphocytes from three different healthy donors in X-vivo with 5% heat-inactivated human serum, 25 mM HEPES buffer (GibcoBRL, Paisley, UK), and 120 U/ml IL-2 (PeproTech, London, UK).
  • the plates were incubated at 37°C/5%C0 2 . Every 2-3 days 25 ⁇ fresh media were added containing IL-2 to a final concentration of 120U/ml.
  • Growing clones were expanded using 120 U/ml IL-2. After expansion the clones were tested for specificity and cytotoxic potential in a standard 51 Cr-release assay.
  • PBMC from a melanoma patient were stimulated with ⁇ 10 peptide and 40U/ml IL-2.
  • SEB super antigen Staphylococcal Enterotoxin B
  • ⁇ g/ml SEB (Sigma Aldrich) was added.
  • tetramer staining was performed where cells were stained with HLA-A2/lkB10 tetramer conjugated with APC or PE fluorochromes.
  • tetramer positive cells were isolated using anti-PE microbeads according to manufacture protocol (Miltenyi Biotec). The purified cells were cloned and growing clones tested for specificity in a standard 51 Cr-release assay.
  • Target cells were T2 and the HLA-A2 + melanoma cell line FM3. In some assays, target cells were treated with 100 U/ml IFN- ⁇ for 2 days.
  • Tetramer staining For tetramer staining, ⁇ 10 cells co-cultured with SEB for 14 days were analysed. Tetramers coupled with PE and APC were prepared using MHC-peptide exchange technology. Cells were stained with tetramers in PBS+2%FCS for 15min at 37 °C/5% C02. Subsequently, surface staining was performed with CD3-FITC, CD8-PB, CD4- PECy7 (BD Bioscience) in PBS+2%FCS for 20min at 4°C in the dark. The cells were analysed on FACS Canto, using FACSDIVA software (BD Bioscience).
  • HLA-A2 light and heavy chains were produced in E. Coli and refolded with a UV-sensitive ligand. This conditional ligand was cleaved upon 1 hour of UV light exposure and substituted with the peptide of interest.
  • affinity of the complex was measured as the absorbance.
  • Two strong binder-peptides HLA-A2/C V pp65 pos 4 g 5 - Jos (NLVPMVATV) and HLA-A2/HIV-1 ⁇ !
  • the ⁇ - ⁇ protein sequence was examined for potential HLA-A2-restricted nona- and decamer peptide epitopes by means of the previously described HLA-A2 peptide binding motifs (Andersen et al., 2000). Eighteen ⁇ - ⁇ -derived peptides (entitled ⁇ 1- 18) were selected for the following studies. We scrutinized PBMC from healthy individuals as well as cancer patients for the presence of specific T-cell responses against these ⁇ - ⁇ -derived peptides using the IFN- ⁇ ELISPOT secretion assay. The ELISPOT has previously been utilized for the identification of novel tumor antigens based on spontaneous immunity in cancer patients.
  • HLA-A2 + PBMC from patients with breast cancer, renal cell carcinoma or melanoma were stimulated once with the different peptides in vitro before examination by ELISPOT.
  • IFN- ⁇ secretion was detected against ⁇ 18 and ⁇ 10 ( Figure 4).
  • IKB10 was examined for its ability to bind to HLA-A2 by the comparison with two HLA- A2-restricted, high affinity epitopes, i.e. HIV-1 pol 47 6-484 (ILKEPVHGV) and CMV pp65 pos495 -5 03 (N LVPMVATV) using peptide exchange technology followed by ELISA.
  • ILKEPVHGV HIV-1 pol 47 6-484
  • CMV pp65 pos495 -5 03 N LVPMVATV
  • ⁇ 10 bound HLA-A2 comparable to the high-affinity control epitope (Figure 5A).
  • reactivity against ⁇ 10 was examined in a larger cohort of patients by means of the ELISPOT assay.
  • Figure 5B exemplifies ⁇ -specific T cell responses in two melanoma patients (MM1 and MM3).
  • MM1 and MM3 melanoma patients
  • PBMC from patients hosting responses against the ⁇ 10 peptide were used to examine if ⁇ 10 specific T cells display cytotoxic function.
  • PBMC from three melanoma patients (MM 1 , MM3 and MM5) hosting ⁇ 10 IFN- ⁇ reactive T cells were analyzed for further reactivity against ⁇ 10 using the Granzyme B (GrB) ELISPOT.
  • GrB Granzyme B
  • Responses against ⁇ 10 could be detected in all three patients with a frequency at about 80-250 ⁇ -specific, GrB releasing cells per 10 5 PBMC ( Figure 6A).
  • CTL clones were generated from expansion of isolated, tetramer-positive single cells.
  • the reactivity of the growing T-cell clones was analyzed after a short expansion step using 51 Cr release assays.
  • a total of 138 T-cell clones were expanded and, subsequently, analysed for cytotoxic capacity against T2 cells loaded with either ⁇ 10 or an irrelevant peptide (HIV-1 pol 47 6-484 (ILKEPVHGV)).
  • 37 of the T-cell clones were ⁇ 10- specific.
  • Figure 6B exemplifies the lytic capacity of two of the ⁇ 10 specific clones.
  • T2 cells and autologous DC either loaded with ⁇ 10 peptide or irrelevant HIV peptide served as targets.
  • This assay revealed that T2 cells and DC were only killed by the T cell clone when pulsed with IKB10 ( Figure 7B).
  • the ⁇ ⁇ -specific T-cell clone was further examined for its capacity to kill the HLA-A2-positive, highly immunogenic melanoma cell line FM3.
  • Figure 7C illustrates that the ⁇ -specific T-cell clone was able to kill the melanoma cell line. Killing of FM3 were further increased by pre-treatment of the melanoma cells with IFN- ⁇ ( Figure 7C).
  • IFN- ⁇ Figure 7C
  • PBMC peripheral blood mononuclear cells
  • NLVPMVATV CMV IE1 3 i 6 -324 (VLEETSVML) or Flu matrix p 58 - 6 6 (GILGFVFTL)
  • 40 U/ml IL-2 was added on day 2 and 6.
  • the PBMC were either cultured alone or added autologous IkB-specific T cells (in a PBMC to IkB-specific T cell ratio of 2000: 1) on day 6.
  • the cultures were stimulated with 120 U/ml IL-2.
  • the number of viral-specific T cells in the cultures, either cultured alone or added IkB- specific T cells was compared by MHC-tetramer staining.
  • Tregs, IL-17A producing T cells and the CD4/CD8 cell ratio in the cultures were also compared.
  • PBMC peripheral blood mononuclear cells
  • CD8 + T cells of irrelevant specificity Co-stimulation with IkB peptide
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • CMV Pp65 49 5-503 NLVPMVATV
  • CMV IE1 3 i 6 -324 VLEETSVML
  • EAAGIGILTV EAAGIGILTV
  • IkB peptide an irrelevant peptide
  • IkB peptide HIV-1 pol 476 - 484 (ILKEPVHGV)
  • 40 U/ml IL-2 was added every third day. Every seven days, the cultures were stimulated with a mixture of CMV- or MART-1 peptide plus PD- L1 peptide, or a mixture of CMV or MART-1 peptide plus HIV-1 pol 476 . 4 8 4 peptide, respectively. Cells were stimulated with 10-, 100-, and 1000-fold diluted peptides for the second, third and fourth peptide stimulation, respectively.
  • the number of CMV- or MART-1 -specific T cells in the cultures was compared by MHC-tetramer staining.
  • the number of Tregs, IL-17A producing T cells and the CD4/CD8 cell ratio in the cultures were also compared.

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Abstract

La présente invention concerne le domaine de la prophylaxie et de la thérapie d'états cliniques, notamment le cancer, des maladies auto-immunes et des maladies infectieuses. L'invention concerne en particulier des compositions de vaccins comprenant IkB ou des fragments peptidiques d'IkB qui sont aptes à induire des réponses immunitaires utiles dans le traitement du cancer, de maladies auto-immunes ou de maladies infectieuses.
PCT/DK2012/050387 2011-10-26 2012-10-17 IMMUNOTHÉRAPIE BASÉE SUR IkB WO2013060328A1 (fr)

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