US20160367651A1 - T cell inducing vaccine containing an interepitope sequence that promotes antigen presentation - Google Patents

T cell inducing vaccine containing an interepitope sequence that promotes antigen presentation Download PDF

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US20160367651A1
US20160367651A1 US15/026,841 US201415026841A US2016367651A1 US 20160367651 A1 US20160367651 A1 US 20160367651A1 US 201415026841 A US201415026841 A US 201415026841A US 2016367651 A1 US2016367651 A1 US 2016367651A1
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vaccine
consecutive
cells
sequence
long chain
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Hiroshi Shiku
Naozumi Harada
Daisuke Muraoka
Kazunari Akiyoshi
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Mie University NUC
Kyoto University NUC
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Kyoto University NUC
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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
    • 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
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/55583—Polysaccharides
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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

Definitions

  • the present invention relates to a T cell inducing vaccine containing an interepitope sequence that promotes antigen presentation.
  • CD8 + killer T cells CD8 + cytotoxic T cells
  • CD4 + helper T cells are important regulatory cells that enhance the functions of CD8 + killer T cells and antigen-presenting cells
  • professional antigen-presenting cells such as dendritic cells and macrophages, stimulate T cells by presenting antigens thereto and activate T cells via costimulatory molecules, such as CD80, CD86, and cytokines, etc.
  • costimulatory molecules such as CD80, CD86, and cytokines, etc.
  • Tumor cell derived proteins after being phagocytosed by antigen-presenting cells, are cleaved into peptides of various lengths by proteasomes, proteases, and peptidases within the cells.
  • peptides of 8-10 amino acids are loaded as antigen epitope peptides onto major histocompatibility complex (MHC) class I molecules and can be presented on the surfaces of the antigen-presenting cells.
  • MHC major histocompatibility complex
  • CD8 + killer T cells use T cell receptors (TCRs) to specifically recognize the MHC class I/antigenic peptide complexes and become activated.
  • TCRs T cell receptors
  • the activated CD8 + killer T cells detect MHC class I/antigenic peptide complexes that are also present on tumor cells and destroy the tumor cells using effector molecules, such as granzymes and perforin.
  • CD4 + helper T cells The function of CD4 + helper T cells is important for sufficient activation of CD8 + killer T cells (Non-Patent Document 2).
  • Antigenic proteins taken up by the antigen-presenting cells are cleaved into various lengths by proteases and peptidases within the cells and among the resulting antigenic peptides, those of 15-20 amino acids form complexes with MHC class II molecules and can be presented on the antigen-presenting cells.
  • CD4 + helper T cells recognize these specifically and are activated.
  • the activated CD4 + helper T cells enhance differentiation, growth, and functions of CD8 + killer T cells via secretion of cytokines, such as interferon (IFN)- ⁇ and interleukin (IL)-2.
  • IFN interferon
  • IL interleukin
  • the CD4 + helper T cells also have a function of activating antigen-presenting cells via a CD40 ligand/CD40 pathway, and the antigen-presenting cells activated by the CD4 + helper T cells are improved in the capability to stimulate CD8 + killer T cells (Non-Patent Document 3). It is well known from before that CD4 + helper T cells also have an action of enhancing antigen-specific IgG antibody production in B cells.
  • a cancer vaccine therapy has been conceived where a tumor specific antigen is repeatedly administered as a vaccine antigen to induce tumor-specific CD8 + killer T cells within a patient's body to suppress the growth, metastasis, and recurrence of cancer.
  • Various forms of the antigen of the cancer vaccines are known, such as synthetic peptides, recombinant proteins, processed cells.
  • the present inventors have previously prepared a cancer vaccine using a full-length recombinant protein of a tumor antigenic protein as the antigen.
  • the full-length protein includes diverse antigenic peptides recognized by CD8 + killer T cells and CD4 + helper T cells and is expected to activate both types of T cells at the same time.
  • short chain peptides mainly, epitope peptides of 8 to 10 residues recognized by CD8 + killer T cells and clinically apply vaccines using these peptides as antigens.
  • presentation to T cells occurs readily because such peptides bind directly to MHC molecules on cell surfaces without undergoing uptake and antigen processing within antigen-presenting cells.
  • short chain peptides can be manufactured by chemical synthesis and has the advantage of being simpler to manufacture than recombinant proteins, which requires the use of genetically modified organisms.
  • Non-Patent Document 5 Exogenous antigenic proteins are phagocytosed by professional antigen-presenting cells, such as dendritic cells and macrophages, that are provided with costimulatory molecules (CD80, CD86, etc.) and are processed within the cells, and antigen presentation to T cells is performed in a mode with appropriate concentration and costimulation.
  • professional antigen-presenting cells such as dendritic cells and macrophages
  • costimulatory molecules CD80, CD86, etc.
  • short peptide antigens bind directly to MHC molecules on cell surfaces and therefore even general somatic cells, which do not have uptake ability (phagocytic ability) and do not express costimulatory molecules, can present the short peptide antigens in a massive, inappropriate mode that lacks costimulation.
  • the T cells that recognize the complexes of the short peptide antigens and MHCs become prone to depletion and apoptosis and this can consequently lead to immunological tolerance to the targeted antigen.
  • Non-Patent Document 5 a polypeptide having several dozen residues such that include two or more T cell recognition epitope peptides. Unlike a short chain peptide, a long chain peptide antigen cannot bind directly in intact form to an MHC molecule.
  • long chain peptide antigens undergo uptake and intracellular processing by professional antigen-presenting cells with phagocytic ability, such as dendritic cells and macrophages, and the T cell epitope peptides included in the long chain peptide antigens form complexes with MHC molecules only thereafter and are thus presented to T cells in a mode with appropriate concentration and costimulation.
  • Long chain peptide antigens do not function as vaccine antigens with general somatic cells lacking antigen phagocytic ability and therefore, unlike short chain peptide vaccines, do not give rise to inappropriate antigen presentation to T cells.
  • chemical synthetic methods can be used to manufacture long chain peptide antigens and therefore, as with short peptide antigens, the advantage of being comparatively easy to manufacture is also provided.
  • Long chain peptide antigens manufactured by chemical synthesis also have a major advantage in that it is possible to freely design the sequence.
  • a long chain peptide antigen is designed so that two or more T cell epitopes are included within a single peptide, and these T cell epitopes may be derived from a single cancer antigenic protein or may be derived from a plurality of cancer antigenic proteins. Also, the T cell epitopes may be restrictive to a single MHC or may be restrictive to a plurality of MHCs. It is also possible to design so that a long chain peptide antigen includes an epitope recognized by a CD8 + killer T cell and an epitope recognized by a CD4 + helper T cell at the same time.
  • Long chain peptide antigens can thus serve as high performance vaccine antigens that can induce diverse T cells.
  • the epitopes must be cut out as epitope peptides of lengths and sequences enabling binding with MHC molecules by sequences between the respective epitopes on the long chain peptide antigen being cleaved appropriately by proteasomes, proteases, and peptidases in an antigen-presenting cell based on the mechanism of antigen presentation reactions.
  • MHC class I binding epitope peptides recognized by CD8 + killer T cells the terminuses of the epitope peptide binding groove on an MHC class I molecule are in a closed state and only epitope peptides, strictly restricted to 8 to 10 residues, can bind to the MHC class I molecule. It is thus especially important with MHC class I binding epitope peptides that peptides of appropriate lengths are produced in antigen-presenting cells.
  • the lengths and sequences of the epitope peptides that bind to MHC molecules are determined by complex cleavage reactions involving intracellular proteasomes and various proteases and peptidases.
  • proteasomes present in the cytoplasm first perform rough cleavage of the antigenic protein or long chain peptide antigen.
  • the terminuses of the resulting peptide fragments are cleaved by other proteases and peptidases based on certain substrate sequence specificities and trimmed to appropriate lengths (trimming reactions).
  • Non-Patent Document 7 the substrate sequence specificities of proteasomes have not been revealed in detail and it is difficult to predict peptide sequences that can be cleaved readily by proteasomes.
  • the epitope peptide sequences included in a certain long chain peptide or protein must be cut out appropriately from the long chain peptide or protein by intracellular proteasomes, proteases, and peptidases.
  • the sequences between the epitopes it is necessary for the sequences between the epitopes to aptly include recognition sites for the proteasomes, proteases, and peptidases.
  • the present invention has been made in view of the circumstances described above, and an object thereof is to provide, in a long chain peptide antigen containing a plurality of epitope peptides, an interepitope sequence that effectively achieves antigen presentation of the respective epitope peptides.
  • each interepitope sequence is one selected from a group consisting of two to ten consecutive tyrosines, two to ten consecutive threonines, two to ten consecutive alanines, two to ten consecutive histidines, two to ten consecutive glutamines, and two to ten consecutive asparagines and it is especially preferable for the sequence to be tyrosines, glutamines, or asparagines.
  • the number of consecutive tyrosines, consecutive threonines, consecutive histidines, consecutive glutamines, or consecutive asparagines is preferably four to eight, more preferably four to six, and especially six.
  • the long chain peptide antigen is cleaved inside a body by enzymes within a living body so that the respective epitopes can perform antigen presentation and the respective epitopes thus exhibit antigen presenting abilities effectively. Also, with a vaccine using along chain peptide antigen having an interepitope sequence constituted of consecutive tyrosines, uptake into antigen-presenting cells is also improved.
  • the vaccine is preferably one selected from the group consisting of anticancer vaccines (including dendritic cell vaccines), antibacterial vaccines, and antiviral vaccines.
  • the vaccine is preferably at least one selected from the group consisting of peptide vaccines, DNA vaccines, mRNA vaccines, and dendritic cell vaccines.
  • a dendritic cell vaccine a peptide antigen or mRNA is added.
  • the vaccine is a peptide vaccine
  • it is preferably arranged as a vaccine in combination with a hydrophobized polysaccharide, especially, cholesterol-modified pullulan (CHP) as a delivery system.
  • a hydrophobized polysaccharide especially, cholesterol-modified pullulan (CHP)
  • the plurality of epitopes within the vaccine can perform antigen presentation effectively and therefore a vaccine having a high effect can be provided.
  • FIG. 1 The influences of differences in interepitope sequence of long chain peptide vaccines, containing a plurality of CD8 + T cell epitopes, on specific CD8 + T cell induction by the vaccines were examined.
  • Long chain peptide antigens MEN all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized. The sequence between the respective epitopes was set to one of six consecutive tyrosines (Y 6 ), glycines (G 6 ), prolines (P 6 ), or threonines (T 6 ).
  • Each long chain peptide antigen was complexed with cholesterol-modified pullulan (CHP), which is a type of delivery system, and administered as a vaccine to a mouse.
  • CpG oligo DNA was coadministered as an adjuvant.
  • Spleen cells were collected one week after the final administration and the frequencies of CD8 + T cells specific to the respective epitope sequences were measured by an intracellular cytokine staining method.
  • FIG. 2 The influences of differences in interepitope sequence of long chain peptide vaccines, containing a plurality of CD8 + T cell epitopes, on therapeutic effects of the vaccines were examined.
  • Long chain peptide antigens MEN all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized. The sequence between the respective epitopes was set to one of six consecutive tyrosines (Y 6 ), glycines (G 6 ), or prolines (P 6 ). Each long chain peptide antigen was complexed with CHP and administered in a single dose as a vaccine to a mouse.
  • a short chain peptide vaccine constituted of just the mERK2 9m peptide, was mixed with Freund's incomplete adjuvant and administered.
  • CpG oligo DNA was coadministered as an adjuvant.
  • a mouse fibrosarcoma cell line CMS5a expressing mERK2 as a tumor antigen and presenting the CD8 + T cell epitope mERK2 9m derived from the same antigen, was implanted subcutaneously and its growth was recorded over time.
  • FIG. 3 The influences of differences in interepitope sequence of long chain peptide vaccines, containing a plurality of CD8 + T cell epitopes, on specific CD8 + T cell induction by the vaccines were examined.
  • Long chain peptide antigens NME all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized.
  • the antigens differ from the MEN in FIG. 1 in the order of the three types of epitopes.
  • the sequence between the respective epitopes was set to one of six consecutive tyrosines (Y 6 ), glycines (G 6 ), or prolines (P 6 ).
  • Vaccines containing the respective long chain peptide antigens were administered to mice in the same manner as in FIG. 1 and the frequencies of CD8 + T cells specific to the respective epitope sequences were measured by the intracellular cytokine staining method.
  • FIG. 4 Whether or not the usefulness of an interepitope sequence, constituted of consecutive tyrosines, is influenced by preceding and subsequent epitope sequences was examined.
  • Long chain peptide antigens MEN, ENM, and NME all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized.
  • MEN, ENM, and MEN differ in the order of the three types of epitopes.
  • the sequence between the respective epitopes was set to six consecutive tyrosines (Y 6 ).
  • Vaccines containing the respective long chain peptide antigens were administered to mice in the same manner as in FIG. 1 and the frequencies of CD8 + T cells specific to the respective epitope sequences were measured by the intracellular cytokine staining method.
  • FIG. 5 The influences of the difference between a native sequence and a consecutive tyrosine sequence as the interepitope sequence on specific CD8 + T cell induction and specific CD4 + T cell induction by vaccines were examined.
  • the native amino acid sequence of NY-ESO-1 was retained with ESO1 LP (native type) and the sequence of six consecutive tyrosines (Y 6 ) was used with ESO1 LP (Y 6 ).
  • Vaccines containing the respective long chain peptide antigens were administered to mice in the same manner as in FIG. 1 and the frequencies of CD8 + T cells and CD4 + T cells specific to the respective epitope sequences were measured by the intracellular cytokine staining method.
  • FIG. 6 For interepitope sequences constituted of consecutive tyrosines, the relationship between the number of tyrosines and specific T cell induction by vaccines were examined.
  • Long chain peptide antigens MEN all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized. The sequence between the respective epitopes was set to one to six consecutive tyrosines.
  • Vaccines containing the respective long chain peptide antigens were administered to mice in the same manner as in FIG. 1 and the frequencies of CD8 + T cells specific to the respective epitope sequences were measured by the intracellular cytokine staining method.
  • FIG. 7 For interepitope sequences constituted of consecutive tyrosines, the relationship between the number of tyrosines and specific T cell induction by vaccines were examined.
  • Long chain peptide antigens MEN all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized. The sequence between the respective epitopes was set to five to ten consecutive tyrosines.
  • Vaccines containing the respective long chain peptide antigens were administered to mice in the same manner as in FIG. 1 and the frequencies of CD8 + T cells specific to the respective epitope sequences were measured by the intracellular cytokine staining method.
  • FIG. 8 The influences of differences in interepitope sequence of long chain peptide vaccines on uptake of the vaccines into antigen-presenting cells were examined.
  • Long chain peptide antigens MEN all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized. The sequence between the respective epitopes was set to one of six consecutive tyrosines (Y 6 ), glycines (G 6 ), or prolines (P 6 ).
  • Each long chain peptide antigen, labeled with the fluorescent dye FAM was complexed with CHP and administered in vitro to mouse dendritic cells and mouse macrophages. After 60 minutes, the fluorescence uptakes into the respective cells were measured by flow cytometry with the P5 fraction in the figure being deemed to correspond to the dendritic cells and the P 6 fraction in the figure being deemed to correspond to the macrophages.
  • FIG. 9 The influences of differences in interepitope sequence of long chain peptide vaccines on uptake of the vaccines into antigen-presenting cells were examined.
  • the same FAM-labeled long chain peptide antigens as those in FIG. 8 were complexed with CHP and administered subcutaneously to mice. After 16 hours, cells were collected from a regional lymph node of the administration site, and the fluorescence uptakes into dendritic cells and mouse macrophages were measured by flow cytometry with the P4 fraction in the figure being deemed to correspond to the dendritic cells and the P5 fraction in the figure being deemed to correspond to the macrophages.
  • FIG. 10 The influences of differences in interepitope sequence of long chain peptide vaccines, containing a plurality of CD8 + T cell epitopes, on specific CD8 + T cell induction by the vaccines were examined.
  • Long chain peptide antigens NMW all containing three types of human CD8 + T cell epitope sequences (NY p157:HLA-A0201 restrictive, MA4 p143:HLA-A2402 restrictive, and WT1: HLA-A2402 restrictive p235), were synthesized. The sequences between the respective epitopes were set to those of six consecutive amino acids shown in the figure.
  • Each long chain peptide antigen was complexed with cholesterol-modified pullulan (CHP), which is a type of delivery system, and administered in vitro as a vaccine to an immortalized human B cell line (LCL).
  • CHP cholesterol-modified pullulan
  • LCL cholesterol-modified pullulan
  • co-culturing with CD8 + T cell clone 1G4 cells specific to NY p157 was performed and the activation of the 1G4 cells due to antigen presentation was measured by an IFN- ⁇ ELISPOT method.
  • LCL administered with an NY p157 short chain peptide was used as antigen-presenting cells
  • LCL without antigen added was used as antigen-presenting cells.
  • FIG. 11 The influences of differences in interepitope sequence of RNA vaccines, containing a plurality of CD8 + T cell epitopes, on specific CD8 + T cell induction by the vaccines were examined.
  • mRNAs encoding long chain peptide antigens NMW all containing three types of human CD8 + T cell epitope sequences (NY p157:HLA-A0201 restrictive, MA4p143:HLA-A2402 restrictive, and WT1: HLA-A2402 restrictive p235), were synthesized. The sequences between the respective epitopes were set to those of six consecutive amino acids shown in the figure.
  • Each mRNA was introduced in vitro as a vaccine into LCL by an electroporation method.
  • Synthetic long chain peptides were purchased from Bio-Synthesis Inc. The sequences of the synthetic long chain peptides were as follows.
  • Synthetic short chain peptides were purchased from Sigma Genosys. The amino acid sequences of the peptides were as follows.
  • RNA vaccines used to synthesize the RNA vaccines were purchased from Operon Biotechnologies, Inc. The sequences of the cDNAs were as follows.
  • CHP Cholesterol-modified pullulan
  • CHP-80T Cholesterol-modified pullulan
  • CpG oligo DNA was purchased from Hokkaido System Science Co., Ltd.
  • FITC-labeled anti-CD4 monoclonal antibody (clone RM4-5), PerCP-Cy5.5-labeled anti-CD8 monoclonal antibody (clone 53-6.7), and APC-labeled anti-IFN- ⁇ antibody (clone XMG1.2) were purchased from eBiosciece Inc. or BD Biosciences.
  • Anti-human IFN- ⁇ antibody and biotinylated anti-human IFN- ⁇ antibody was purchased from Mabtech AB.
  • Each long chain peptide was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL.
  • CHP was dissolved in 6M urea-containing phosphate buffered saline (PBS) at a concentration of 10 mg/mL.
  • 1 mL (10 mg) of the long chain peptide solution and 20 mL (200 mg) of the CHP solution were mixed and left to stand at room temperature overnight in a dark place.
  • the liquid mixture was transferred into a dialysis membrane (molecular weight cutoff: 3,500; Thermo Fisher Scientific, Inc.) and dialyzed for 2 hours to overnight at 4° C.
  • Dialysis was then performed for 2 hours to overnight at 4° C. against 0.06M urea-containing PBS of a volume ratio of not less than 100 times as the dialysis outer solution. Dialysis was performed again for 2 hours to overnight at 4° C. against PBS of a volume ratio of not less than 100 times as the dialysis outer solution.
  • the dialyzed inner solution was collected, filtered through a filtration sterilization filter of 0.45 ⁇ m or 0.22 ⁇ m pore size, and thereafter the UV absorption at 280 nm was measured to determine the final concentration of the long chain peptide from its molecular extinction coefficient.
  • Each CHP/long chain peptide complex as the vaccine and the CpG oligo DNA as the adjuvant were administered at the same time to a mouse. Administration was performed by subcutaneous injection on the back of the mouse. As the dose, the CHP/long chain peptide complex was administered at 0.05 to 0.1 mg equivalent of long chain peptide per administration. The CpG oligo DNA was administered at 0.05 mg per administration.
  • spleen cells were separated by the following procedure from each vaccine-administered mouse. The spleen was isolated from the mouse and removed of blood by rinsing with RPMI1640 medium. After the spleen was triturated using a glass slide, the released cells were collected in RPMI1640 medium.
  • mice spleen cells were added at 5 ⁇ 10 6 cells/0.5 mL per well to a 24-well culture plate (Nunc). NY p81, MAGE p265, or mERK2 9m as the short chain peptide for CD8 + T cell stimulation or ESO1 LP (native type) or ESO1 LP (Y 6 ) CD4 + T cell stimulation was added at a concentration of 10 ⁇ M and culturing under 37° C. and 5% CO 2 was performed for 6 hours. Thereafter, GoldiPlug (BD Biosciences), diluted 10-fold with 10% FBS-containing RPMI1640 medium, was added at 50 ⁇ L per well and culturing under 37° C. and 5% CO 2 was performed for 6 hours.
  • GoldiPlug (BD Biosciences)
  • the cells were collected and transferred to a 96-well round bottom microplate (Nunc). After centrifuging (1200 rpm, 1 minute, 4° C.) and removing the supernatant, the cells were suspended in 50 ⁇ L of staining buffer (PBS containing 0.5% bovine serum albumin) per well. The FITC-labeled anti-CD8 antibody or the FITC-labeled anti-CD4 antibody was added and after mixing, the cells were left to stand for 15 minutes in a dark place at 4° C. After rinsing the cells twice with 200 ⁇ L of the staining buffer, 100 ⁇ L of Cytofix/Cytoperm buffer (BD Biosciences) were added and mixed gently.
  • staining buffer PBS containing 0.5% bovine serum albumin
  • rinsing with 100 ⁇ L of Perm/Wash buffer was performed twice. 50 ⁇ L of Perm/Wash buffer with the respective types of anti-cytokine antibodies added were added to the cells and after suspending gently, the cells were left to stand for 15 minutes in a dark place at room temperature. After rinsing twice with 100 ⁇ L of Perm/Wash buffer, the cells were re-suspended in 200 ⁇ L of the staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). The cells were analyzed by a flow cytometer (FACS Canto II, BD Biosciences) using the included analysis software (FACSDiva).
  • a subcloned CMS5a cell line obtained from a CMS5 cell line isolated from fibrosarcoma induced by administering 3-methylcholanthrene to a BALB/c mouse, expresses mutant ERK2 (mERK2) as a tumor antigen and presents a CD8 + T cell epitope derived from the mERK2.
  • the CMS5a cell line cultured in a T75 flask (Nunc) was detached using PBS containing 0.5% trypsin and collected in RPMI1640 medium containing 10% FBS.
  • a test of uptake of long chain peptide antigens by antigen-presenting cells in individual animals was performed as follows. Each long chain peptide was fluorescence-labeled, complexed with a CHP nanogel, administered subcutaneously to BALB/c mice. 16 hours after administration, cells were collected from lymph nodes and after staining with the anti-CD11c antibody and the anti-F4/80 antibody, the uptake of the fluorescence-labeled long chain peptide antigens by CD11c + cells (dendritic cells) and F4/80 + cells (macrophages) was analyzed by flow cytometry.
  • Cryostored LCL was rinsed with RPMI medium and suspended at 1.25 ⁇ 10 6 /mL in X-VIVO15 medium. This was dispensed in 0.4 mL aliquots into polypropylene tubes, and 0.1 mL of a vaccine solution (0.1 mg/mL as peptide) was added to each tube. The cells were cultured for 24 hours at 37° C. in the presence of 5% CO 2 and then used as antigen-presenting cells.
  • mRNA was introduced by an electroporation method (300V, 700 ⁇ s) using ECM830 into LCL that was rinsed and suspended in the same manner as in (9). The cells were cultured for 24 hours at 37° C. in the presence of 5% CO 2 and then used as antigen-presenting cells.
  • the cryostored CD8 + T cell clones were thawed, rinsed, adjusted to 5 ⁇ 10 5 /mL with RPMI medium, and thereafter added in 0.1 mL aliquots to each well. After culturing for 24 hours at 37° C. in the presence of 5% CO 2 , the liquid was discarded and the plate was rinsed well with phosphate buffered saline containing 0.05% Tween 20 (PBS-T). A biotin-labeled IFN- ⁇ antibody for detection was diluted to an appropriate concentration and dispensed in 0.1 mL aliquots into each well.
  • PBS-T phosphate buffered saline containing 0.05% Tween 20
  • the plate was rinsed well with PBS-T, and an alkaline phosphatase-labeled streptavidin diluted to an appropriate concentration was added in 0.1 mL aliquots. After incubating for 1 hour at room temperature, the plate was rinsed well with PBS-T. A coloring solution was added in 0.1 mL aliquots and allowed to react for 5 minutes to 30 minutes at room temperature. When the formation of spots was observed, the reaction was stopped by rinsing with water.
  • cDNAs encoding the intended long chain peptide antigens were purchased as synthetic genes from Operon Biotechnologies, Inc. Each of these was cloned into the multiple cloning site of a pcDNA3.1 vector.
  • the priming site on the T7 promoter contained in the pcDNA3.1 was used to synthesize mRNA by a conventional method using MEGAscript (registered trademark) T7 Transcription Kit, made by Life Technologies, Inc., etc.
  • FIG. 1 shows that with vaccines having a long chain peptide, which contains a plurality of T cell epitopes, as an antigen, differences in interepitope sequence influence the success or failure of specific T cell induction by the respective epitopes.
  • the long chain peptide antigens MEN all containing the three types of mouse CD8 + T cell epitope sequences, MA p265, NY p81, and mERK2 9m, which are derived from the human tumor antigens MAGE-A4 and NY-ESO-1 and the mouse tumor antigen, mutant ERK2 (mERK2), were synthesized.
  • the sequence between the three types of epitopes was set to one of six consecutive tyrosines (Y 6 ), glycines (G 6 ), prolines (P 6 ), or threonines (T 6 ).
  • Each long chain peptide antigen was complexed with cholesterol-modified pullulan (CHP), which is a type of delivery system, and administered as a vaccine to a mouse.
  • CHP cholesterol-modified pullulan
  • specific CD8 + T cells corresponding to all three types of epitopes were clearly induced.
  • the interepitope sequence strongly influences T cell induction by the preceding and subsequent epitopes and that consecutive tyrosines or threonines is preferable as the interepitope sequence.
  • the influences of differences in interepitope sequence of long chain peptide antigens on antitumor effects of vaccines were examined using a mouse tumor implant model ( FIG. 2 ).
  • the long chain peptide antigens MEN all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized, and the sequence between the respective epitopes was set to one of six consecutive tyrosines (Y 6 ) glycines (G 6 ), or prolines (P 6 ).
  • Each long chain peptide antigen was complexed with CHP and administered in a single dose as a vaccine to a mouse.
  • a short chain peptide vaccine constituted of just the mERK2 9m peptide
  • the mouse fibrosarcoma cell line CMS5a presenting the CD8 + T cell epitope mERK2 9m derived from the mERK2 antigen
  • the vaccine using the long chain peptide antigen MEN (Y 6 ) the growth of the tumor was suppressed significantly (p ⁇ 0.05).
  • the vaccine using MEN (G 6 ) or MEN (P 6 ) or the mERK2 9m short chain peptide vaccine significant suppression of tumor growth was not observed.
  • the usefulness of the consecutive tyrosine sequence as an interepitope sequence when the order of epitopes on the long chain peptide antigen differs from that in the case of FIG. 1 , that is, when the epitope sequences preceding and subsequent the interepitope sequence differ was examined.
  • the long chain peptide antigens NME all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized. The NME differ from the MEN in FIG. 1 in the order of the three types of epitopes.
  • the sequence between the respective epitopes was set to one of six consecutive tyrosines (Y 6 ), glycines (G 6 ), or prolines (P 6 ).
  • Y 6 tyrosines
  • G 6 glycines
  • P 6 prolines
  • the long chain peptide antigens MEN, ENM, and NME which are changed in the order of the three types of epitope sequences (MA p265, NY p81, and mERK2 9m) but with which the interepitope sequence is fixed at six consecutive tyrosines (Y 6 ), were prepared.
  • vaccines containing the respective long chain peptide antigens were administered to mice, specific CD8 + T cells corresponding to all three types of epitopes were clearly induced with all of the long chain peptide antigens ( FIG. 4 ).
  • the native amino acid sequence of NY-ESO-1 was retained with ESO1 LP (native type) and the sequence of six consecutive tyrosines (Y 6 ) was used with ESO1 LP (Y 6 ).
  • MEN long chain peptide antigens MEN, all containing three types of mouse CD8 + T cell epitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized. The sequence between the respective epitopes was set to one to six ( FIG. 6 ) or five to ten ( FIG. 7 ) consecutive tyrosines. Vaccines containing the respective long chain peptide antigens were administered to mice in the same manner as in FIG. 1 and the frequencies of the induced CD8 + T cells were measured. In the case of comparing one to six tyrosines ( FIG.
  • Non-Patent Document 7 Long chain peptide antigens MEN, with the interepitope sequence being set to one of six consecutive tyrosines (Y 6 ), glycines (G 6 ), or prolines (P 6 ), were synthesized.
  • FAM fluorescent dye
  • the sequence between the three types of epitopes was set to that in which six of one of alanine (A), glutamic acid (E), glycine (G), histidine (H), asparagine (N), proline (P), glutamine (Q), serine (S), or tyrosine (Y) are made consecutive.
  • a long chain peptide containing an interepitope sequence constituted of an amino acid besides the above was difficult to synthesize or difficult to complex with CHP.
  • Immortalized human B cell lines (LCL) administered with vaccines prepared by complexing the respective long chain peptide antigens with CHP were used as antigen-presenting cells to evaluate the antigen presenting activity with respect to NY p157 specific CD8 + T cell clone 1G4 cells by the IFN- ⁇ ELISPOT method.
  • the activation of 1G4 cells was clearly confirmed with the long chain peptide vaccine adopting the Y 6 interepitope sequence, the activation of 1G4 cells was not clearly confirmed with the vaccine adopting the G 6 or the P 6 interepitope sequence.
  • the sequence between the three types of epitopes was set to that in which six of one of glycine (G), proline (P), threonine (T), or tyrosine (Y) are made consecutive.
  • LCL with the respective mRNAs introduced therein were used as antigen-presenting cells to evaluate the antigen presenting activity with respect to NY p157 specific CD8 + T cell clone 1G4 cells or MA4 p143 specific CD8 + T cell clone RNT007#45 cells by the IFN- ⁇ ELISPOT method.
  • Activations of 1G4 cells and RNT007#45 were clearly confirmed with the RNA vaccine encoding the long chain peptide adopting Y 6 as the interepitope sequence. From this, it has been revealed that the interepitope sequence of the present invention is useful not only in peptide vaccines but also in RNA vaccines.
  • the usefulness of consecutive tyrosines or threonines as an interepitope sequence is not limited to the long chain peptide vaccines described above and may also be applied to DNA vaccines, mRNA vaccines, or dendritic cell vaccines.
  • a DNA vaccine may be prepared by using artificial gene synthesis techniques to synthesize a cDNA, encoding a long chain peptide antigen having a single methionine at the N-terminus and having a plurality of T cell epitopes linked by consecutive tyrosine sequences or consecutive threonine sequences, and inserting it into a gene expression plasmid vector for mammals.
  • the cDNA of the long chain peptide antigen is synthesized to be in the range of 66 to several kbp according to the number of T cell epitopes to be included.
  • plasmid that which contains pcDNA3, pVAX, or other promoter (CMV promoter, etc.) that operates in mammalian cells
  • polyA derived from bovine growth hormone, etc.
  • a drug resistance gene such as that for kanamycin
  • the plasmid may carry a plurality of long chain peptide antigen cDNAs and the respective antigen cDNAs can be co-expressed by linking with an IRES sequence, etc.
  • the plasmid may carry, at the same time, accessory genes for enhancing tumor immune response, for example, cytokines such as IFN- ⁇ and IL-12, immunostimulatory molecules, such as GITR ligand-Fc, immunosuppression inhibitors, such as PD-L1-Fc.
  • accessory genes for enhancing tumor immune response for example, cytokines such as IFN- ⁇ and IL-12, immunostimulatory molecules, such as GITR ligand-Fc, immunosuppression inhibitors, such as PD-L1-Fc.
  • a plurality of plasmid DNAs that differ in the numbers and types of antigen cDNAs and accessories molecules carried may be administered at the same time.
  • the DNA vaccine that is obtained is repeatedly administered subcutaneously, intradermally, intravenously, intramuscularly, intralymphnodally, epicutaneously, or intratumorally to the living body of an animal, such as a mouse (BALB/c mouse or C57BL/6 mouse, etc.), or a human, etc., at a dose of 1 ⁇ g to 1 mg per individual and an interval of one to four weeks using an administration technique such as a gene gun, needle-free injector, electroporation method, DNA tattooing, delivery system (cationic liposome, polyethylene imine, etc.), hydrodynamic method, transdermal administration method.
  • an administration technique such as a gene gun, needle-free injector, electroporation method, DNA tattooing, delivery system (cationic liposome, polyethylene imine, etc.), hydrodynamic method, transdermal administration method.
  • the specific T cells induced by the T cell epitopes contained in the long chain peptide antigens that are transcribed and translated from the cDNA on the DNA vaccine may be detected by an immunological technique such as an intracellular cytokine staining method, ELISPOT method, MHC tetramer staining method.
  • CMS5a fibrosarcoma, CT26 colorectal cancer, 4T1 breast cancer (hereabove in the case of BALB/c mouse), B16 melanoma, or LLC lung cancer (hereabove in the case of C57BL/6 mouse) incorporating a wild type or model antigen gene may be implanted subcutaneously to observe the inhibitory effect of the DNA vaccine against growth and metastasis of the tumor.
  • Tumor growth may be measured by measuring the size of the tumor or, if tumor cells incorporating a monitor gene such as a luciferase gene, are used, by an in vivo imaging technique, such as IVIS (PerkinElmer Inc.), etc.
  • tumor nodules which, upon intravenous or subcutaneous administration of tumor, occur in the lungs, etc., that are the metastasis destinations, may be visually counted after dissection or be evaluated by an in vivo imaging technique.
  • a biological vector using a virus or microorganism may be used instead of a plasmid vector.
  • a viral vector a retroviral vector, lentiviral vector, adenoviral vector, adeno-associated virus vector, vaccinia virus vector, fowlpox virus vector, alphavirus vector, or Sendai virus vector, etc.
  • a microorganism vector yeast, listeria, salmonella, E. coli , or lactobacillus , etc., may be used.
  • a DNA vaccine using such a biological vector is administered intravenously, subcutaneously, intradermally, intramuscularly, intralymphnodally, supramucosally, or intratumorally to a test animal, such as a mouse, or a human.
  • the arrangement and evaluation methods (immunogenicity and therapeutic effects) of the genes carried on the biological vector are the same as in the example of the plasmid vector described above.
  • An mRNA vaccine encoding consecutive tyrosines or threonines as the interepitope sequence may be implemented in the same manner as a DNA vaccine.
  • Artificial gene synthesis techniques are used to synthesize a cDNA, encoding a long chain peptide antigen having a single methionine at the N-terminus and having a plurality of T cell epitopes linked by consecutive tyrosine sequences or consecutive threonine sequences, and inserting it into a template plasmid DNA for in vitro transfer.
  • the cDNA is prepared to be in the range of 66 to several kbp according to the number of T cell epitopes to be included.
  • plasmid DNA that which contains a promoter (T7 promoter, T3 promoter, SP6 promoter, etc.) recognized by a phage RNA polymerase, polyA, and a drug resistance gene (such as that for kanamycin), that is for example, pGEM or pcDNA3, etc., may be used.
  • a promoter T7 promoter, T3 promoter, SP6 promoter, etc.
  • polyA a drug resistance gene (such as that for kanamycin), that is for example, pGEM or pcDNA3, etc.
  • MEGAscript commercially available in vitro transfer kit
  • polyA is added to the mRNA as necessary using a polyA tailing kit (Life Technologies, Inc.), etc.
  • the mRNA obtained is administered subcutaneously, intradermally, intramuscularly, intralymphnodally, or intratumorally as it is or upon stabilizing with a protamine or liposome, etc., to a test animal, such as a mouse, or a human.
  • the mRNA vaccine may contain a plurality of mRNAs. For example, a plurality of mRNAs that code long chain peptide antigens may be administered upon mixing.
  • An mRNA encoding accessory molecules for enhancing tumor immune response for example, cytokines such as IFN- ⁇ and IL-12, immunostimulatory molecules, such as CD40 ligand and GITR ligand-Fc, immunosuppression inhibitors, such as PD-L1-Fc, may be administered at the same time as the mRNA vaccine.
  • cytokines such as IFN- ⁇ and IL-12
  • immunostimulatory molecules such as CD40 ligand and GITR ligand-Fc
  • immunosuppression inhibitors such as PD-L1-Fc
  • Dendritic cells to be used in a dendritic cell vaccine may be induced to differentiate in vitro from peripheral blood mononuclear cells in the case of humans and bone marrow cells in the case of mice by a conventional method using GM-CSF and IL-4.
  • a long chain peptide antigen described above or an mRNA encoding a long chain peptide antigen described above is added to the cells to prepare a vaccine. If a long chain peptide antigen is used, the efficiency of uptake and expression can be increased by using CHP as a delivery system ( FIG. 8 ). If an mRNA encoding a long chain peptide antigen is used, the efficiency of uptake and expression in dendritic cells can be increased by electroporation method.
  • an mRNA encoding an accessory molecule for enhancing tumor immune response may be added at the same time as described above.
  • the dendritic cells after addition of antigen may be used upon being stimulated and matured by TNF ⁇ , IL-1 ⁇ , IL-6, Flt3 ligand, PGE 2 , CpG oligo DNA, poly IC RNA, etc.
  • the dendritic cell vaccine obtained is administered subcutaneously, intradermally, intralymphnodally, intratumorally, or intravenously to a test animal, such as a mouse, or a human at a dose of 10 6 to 10 8 cells.
  • the evaluation methods are the same as in the example of the DNA vaccine described above.
  • DNA vaccine, mRNA vaccine, or dendritic cell vaccine adopting consecutive tyrosines or threonines as the interepitope sequence may be applied to diseases other than cancer, for example, to infectious diseases.
  • pathogenic viruses such as hepatitis virus, human papilloma virus, adult T-cell leukemia virus, human immunodeficiency virus, herpes virus, influenza virus, Coxsackie virus, rotavirus, RS virus, varicella zoster virus, measles virus, polio virus, norovirus, pathogenic obligate intracellular parasitic microorganisms, such as rickettsia, chlamydia , phytoplasma, Coxiella, Toxoplasma, Leishmania , protozoa, such as Plasmodium, Cryptosporidium , can be cited.
  • a long chain peptide antigen may be designed with which a plurality of T cell epitopes, identified in hepatitis C virus-derived proteins, such as the core protein, NS4, and NS3, are linked with an interepitope sequence constituted of consecutive tyrosines or threonines.
  • Administration conditions of the vaccine containing the long chain peptide antigen and therapeutic effects on hepatitis C virus infection may be examined using a model system, such as an immunodeficient mouse transplanted with human liver tissue.
  • a long chain peptide antigen may be designed using T cell epitopes contained in the human herpesvirus-derived proteins E6 and E7, and administration conditions and therapeutic effects may be examined with a mouse model transplanted with a tumor that expresses E6 or E7.
  • a long chain peptide antigen is designed with which a group of T cell epitopes, contained in merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP), which are expressed on the surface of the mature body of Plasmodium , and liver-specific protein 2 (LISP2), which is expressed in the intracanal air, are linked with a sequence of consecutive tyrosines or a sequence of consecutive threonines.
  • MSP3 merozoite surface protein 3
  • GLURP glutamate rich protein
  • LISP2 liver-specific protein 2
  • a mouse administered with a vaccine containing the long chain peptide antigen is intravenously administered with 10,000 Plasmodium sporozoites and a peripheral blood smear is prepared 4 to 14 days later.
  • Administration conditions and therapeutic effects of the vaccine may be examined by staining with Giemsa and thereafter observing the parasitemia under a microscope.

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JPWO2015050158A1 (ja) 2017-03-09
EP3053592A4 (en) 2017-09-06
EP3053592A1 (en) 2016-08-10
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US20190015490A1 (en) 2019-01-17

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