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WO2011024482A1 - Procédé permettant de mesurer la capacité d'induire des lymphocytes t spécifiques de l'antigène - Google Patents

Procédé permettant de mesurer la capacité d'induire des lymphocytes t spécifiques de l'antigène Download PDF

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Publication number
WO2011024482A1
WO2011024482A1 PCT/JP2010/005326 JP2010005326W WO2011024482A1 WO 2011024482 A1 WO2011024482 A1 WO 2011024482A1 JP 2010005326 W JP2010005326 W JP 2010005326W WO 2011024482 A1 WO2011024482 A1 WO 2011024482A1
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seq
antigen
ctls
mannose
cells
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Japanese (ja)
Inventor
俊彦 鳥越
良彦 廣橋
将人 水内
昇志 佐藤
哲 鈴木
佳隆 清水
雄一郎 佐藤
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BIOMEDCORE Inc
Sapporo Medical University
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BIOMEDCORE Inc
Sapporo Medical University
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Priority to JP2011528659A priority Critical patent/JP5833443B2/ja
Publication of WO2011024482A1 publication Critical patent/WO2011024482A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells

Definitions

  • the present invention relates to a method for measuring the antigen-specific T cell inducing ability of human peripheral blood mononuclear cells (hereinafter referred to as “PBMC” or “PBMCs”), and an antigen capable of inducing antigen-specific T cells from PBMC. And a method for producing antigen-specific T cells from lymphocytes outside the patient's body.
  • PBMC peripheral blood mononuclear cells
  • the present invention also relates to an antigenic peptide useful as a vaccine for treating or preventing a disease based on infection with human papillomavirus type 16.
  • CTL cytotoxic T cells
  • CD4 positive T cells CD4 positive T cells
  • the conventional method after obtaining PBMCs, they are separated into a dendritic cell and a CD8 positive T lymphocyte cell fraction, the dendritic cell fraction is proliferated, and a target antigen peptide is pulsed there (dendritic cell).
  • the antigen-presented dendritic cells are mixed with the CD8-positive T lymphocyte fraction to induce CTLs that activate the CD8-positive T lymphocyte cells (second step). Stage). Usually, it took 1-2 weeks for the first stage and another 2-3 weeks for the second stage (Non-patent Document 1).
  • Non-Patent Document 2 An improvement of the mixed lymphocyte-peptide culture (MLPC) method that induces CTL by directly adding an antigen to PBMC has also been reported (Non-Patent Document 2), but also when using this method, the stimulation step (first stage) 2 weeks or more (Non-Patent Document 2, page 4899, 5th line from the bottom of the left column to 7th line of the right column).
  • MLPC mixed lymphocyte-peptide culture
  • Antigenic peptides with a small number of target CTL cells have low inducing ability, and it takes 1 to 2 months or more while re-stimulating antigen-specific CTL induction, confirming that induction efficiency is low It had been.
  • TIL tumor infiltrating lymphocytes
  • Non-Patent Document 3 and Non-Patent Document 4 contacting PBMCs with IL-2, and then antigen-specific groups are subjected to high-throughput quantitative PCR (HT A method of amplification after selection by (qPCR) has been reported (Patent Document 1) and the like.
  • Cervical cancer is the second most common gynecological malignancy in the world. Cervical cancer and premalignant cervical intraepithelial neoplasia are caused by persistent infection with human papillomavirus (hereinafter referred to as “HPV”). HPV is classified into a low-risk type and a high-risk type, and it is known that high-risk HPV causes cervical intraepithelial neoplasia (hereinafter referred to as “CIN”) and cervical cancer.
  • the International Agency for Research on Cancer (IARC) has identified HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 as high-risk HPV (carcinogenic HPV) and 82 are classified.
  • HPV serotypes There are regional differences in the distribution of HPV serotypes, and 21 serotypes of high-risk HPV have been detected in cervical cancer patients in Asia, particularly in Japan. Also worldwide, HPV 16 and 18 are the most common serotypes seen in more than 70% of cases.
  • HPV has a circular double-stranded DNA genome with a size of about 7.9 kb, which encodes 8 genes.
  • L1 and L2 are structural proteins
  • E1 has a viral DNA replication function
  • E2 regulates viral transcription
  • E4 function is unknown
  • E5, E6 and E7 are transformed. It has the properties of E5 is also known to stimulate cell proliferation (Non-Patent Document 5).
  • E6 and E7 viral oncogenes are known to interact with each other to form cervical cancer.
  • E5, E6 and E7 are expressed only in CIN3 and cervical cancer, and are considered to be usable as an ideal therapeutic vaccine (Non-patent Document 6).
  • HPV type 16 HPV16
  • HPV16 prophylactic HPV vaccines are commercially available that prevent persistent HPV infection and related diseases by inducing neutralizing antibodies against HPV16 and HPV18 outer membrane proteins.
  • prophylactic HPV vaccines are commercially available that prevent persistent HPV infection and related diseases by inducing neutralizing antibodies against HPV16 and HPV18 outer membrane proteins.
  • these vaccines are effective against already affected HPV16 and HPV18 genital lesions. It is estimated that HPV infection will kill 5 million cervical cancer patients over the next 20 years, and the need for therapeutic vaccines is still high.
  • Mannose-coated liposomes are known to have IL-12 production-inducing ability, cellular immunity-inducing ability, and CD8 + CTLs-inducing ability (Patent Document 2, Patent Document 3, Non-Patent Document 7, and Non-Patent Document 8). This indicates that the mannose-coated liposome functions as a cellular immune adjuvant, and antitumor activity using ovalbumin as an antigen has already been reported (see Patent Document 3 and Non-Patent Document 9).
  • the present inventors have encapsulated an antigenic substance in liposomes coated with mannose to stimulate antigen-presenting cells, thereby causing problems of antigen presentation time and low induction efficiency, which have been problems in the past. It was found that antigen-specific CTL can be induced stably in a short time. The present inventors have found that antigen-specific CTL can be induced in a stable and short time by encapsulating an antigenic substance in a mannose-coated liposome and stimulating antigen-presenting cells. . According to the method of the present invention, CTL can be induced and detected at a very high level (at least 10 times the level of the conventional method) in a short time of less than two weeks.
  • an antigenic substance is encapsulated and added to liposomes coated with mannose for antigen exposure in a CTL measurement system, for example, a surprising number of 10 times or more of the conventional method in 7 to 10 days.
  • CTL can be induced and detected.
  • antigen-specific CTL can be efficiently prepared in vitro by using the above-described method for inducing antigen-specific CTL in vitro. Completed.
  • CTLs specific for HPV16 can be induced by using mannose-coated liposomes encapsulating the E6E7E8 fusion gene of HPV16.
  • CTLs derived from lymphocytes are activated by T2-A2402 cells pulsed with a peptide encoding an amino acid sequence derived from E6 or E7 of HPV16, so that amino acids derived from E6 or E7 of HPV16 It has been found that a peptide encoding the sequence is recognized as an antigen.
  • the present invention is a method for measuring the antigen-specific CTLs inducing ability of lymphocytes derived from mammals, that is, ex vivo, lymphocytes are stimulated with an antigen encapsulated in mannose-coated liposomes, and the antigen-specific CTLs are proliferated.
  • the present invention also relates to a method for measuring the ability of the lymphocytes to induce CTLs against the antigen by measuring the presence or number of the generated CTLs.
  • the present invention also provides a method for screening for an antigen capable of inducing antigen-specific CTLs in a relatively short time.
  • the present invention stimulates lymphocytes with an antigen encapsulated with mannose-coated liposomes, induces the antigen-specific CTLs, and measures the presence or number of the obtained antigen-specific CTLs.
  • the present invention relates to a screening method comprising measuring a CTLs inducing ability of a sphere to the antigen and selecting an antigen having a high inducing ability of CTLs.
  • the present invention provides a method for preparing an antigen-specific T cell for use in a method for preventing and treating cancer, infectious diseases, etc. using a cellular immunity system.
  • the present invention encapsulates PBMC in mannose-coated liposomes ex vivo based on the ability to induce CTLs specific to a desired antigen stably in a relatively short time in vitro.
  • the present invention relates to a method for producing antigen-specific CTLs by stimulating with a selected antigen.
  • the present invention relates to an antigenic substance for use in the prevention or treatment of a disease based on HPV16 infection, specifically, HPV16E6E7E8 fusion gene, and HPV16E6 8-19, HPV16E6 49 -57, HPV16E6 66-74, HPV16E6 82-90, HPV16E6 87-95, HPV16E6 98-106, HPV16E7 10-20, HPV16E7 51-60, and HPV16E7 A peptide having amino acid sequences 83-93 is provided.
  • the present invention also provides CTLs for use in the prevention or treatment of diseases based on HPV16 infection.
  • the present invention provides antigenic substances or CTLs for use in the prevention or treatment of HPV16-infected patients having the HLA-A24 gene.
  • the present invention is a medical composition for preventing or treating a disease based on HPV16 infection, comprising the antigenic substance or the CTLs as an active ingredient.
  • the present invention relates to a method for preventing or treating a disease based on HPV16 infection, comprising administering the antigenic substance or the CTLs to a patient.
  • the present invention relates to the following (1) to (41).
  • (1) A method for measuring the ability of lymphocytes to induce antigen-specific CTLs ex vivo, Preparing lymphocytes from mammals; Contacting the lymphocyte with a mannose-coated liposome encapsulating an antigenic substance; and Detecting CTLs specific for said antigen.
  • (2) The method according to (1), further comprising growing the cells in the presence of interleukin 2.
  • (3) The method according to (1) or (2), wherein the mannose-coated liposome is a mannotriose-coated liposome.
  • (4) The method according to any one of (1) to (3), wherein the mannose-coated liposome encapsulating the antigen and the lymphocyte are contacted for 5 to 30 days.
  • the mammal-derived lymphocytes are human peripheral blood-derived lymphocytes.
  • the antigenic substance is at least one substance selected from the following: Survivin 2B peptide, Human papillomavirus type 16 E5, E6 and E7 fusion genes, Cytomegalovirus pp65-derived peptide, MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID NO: 16), CYSLYGTTL (SEQ ID NO: 17), QYNKPLCDL (SEQ ID NO: 18), IVLHLEPQNEI (SEQ ID NO: 19), H (SEQ ID NO: 20) or a peptide comprising an amino acid sequence consisting of LMGTLGIVCPI (SEQ ID NO: 21), MFQDPQ
  • a method for screening an antigenic substance having a high ability to induce antigen-specific CTLs ex vivo Preparing lymphocytes from mammals; Contacting the lymphocyte with a mannose-coated liposome encapsulating a test substance; Detecting CTLs specific for the test substance or antigen; and A method comprising selecting a test substance having a large amount of detected antigen-specific CTLs as an antigenic substance having a high ability of inducing antigen-specific CTLs to the mammal.
  • Method. (16) The production method according to any one of (12) to (15), wherein the mammal-derived lymphocyte is a human peripheral blood-derived lymphocyte.
  • the antigenic substance is at least one substance selected from the following: Survivin 2B peptide, Human papillomavirus type 16 E5, E6 and E7 fusion genes, Cytomegalovirus pp65-derived peptide, MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID NO: 16), CYSLYGTTL (SEQ ID NO: 17), QYNKPLCDL (SEQ ID NO: 18), IVLHLEPQNEI (SEQ ID NO: 19), H (SEQ ID NO: 20) or a peptide comprising an amino acid sequence consisting of LMGTLGIVCPI (SEQ ID NO: 21), MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID
  • a medical composition for treating or preventing an infectious disease or cancer comprising the antigen-specific CTLs produced by the method according to any one of (12) to (17).
  • the antigenic substance is at least one substance selected from the following: Survivin 2B peptide, Human papillomavirus type 16 E5, E6 and E7 fusion genes, Cytomegalovirus pp65-derived peptide, MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID NO: 16), CYSLYGTTL (SEQ ID NO: 17), QYNKPLCDL (SEQ ID NO: 18), IVLHLEPQNEI (SEQ ID NO: 19), H (SEQ ID NO: 20) or a peptide comprising an amino acid sequence consisting of LMGTLGIVCPI (SEQ ID NO: 21), MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO:
  • a medical composition for treating or preventing an infectious disease or cancer comprising the antigen-specific CTLs produced by the method according to any one of (19) to (21).
  • a method for treating or preventing an infectious disease or cancer A method comprising preparing a mannose-coated liposome encapsulating an antigenic substance, and administering the mannose-coated liposome encapsulating the antigenic substance to a subject.
  • the antigenic substance is at least one substance selected from the following: Survivin 2B peptide, Human papillomavirus type 16 E5, E6 and E7 fusion genes, Cytomegalovirus pp65-derived peptide, MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID NO: 16), CYSLYGTTL (SEQ ID NO: 17), QYNKPLCDL (SEQ ID NO: 18), IVLHLEPQNEI (SEQ ID NO: 19), H (SEQ ID NO: 20) or a peptide comprising an amino acid sequence consisting of LMGTLGIVCPI (SEQ ID NO: 21), MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO:
  • a method for treating or preventing an infectious disease or cancer comprising: (I) A step of selecting an antigenic substance having a high ability of inducing antigen-specific CTLs ex vivo, comprising the following steps (a) to (d): (A) preparing lymphocytes from mammals; (B) contacting the lymphocyte with a mannose-coated liposome encapsulating a test substance; (C) detecting CTLs specific for the test substance or antigen; and (D) selecting a test substance having a large amount of detected antigen-specific CTLs as an antigenic substance having a high ability of inducing antigen-specific CTLs to the mammal; and (Ii) A method comprising administering an antigenic substance selected in (i) to a subject.
  • a method for treating or preventing an infectious disease or cancer comprising the following steps: (A) preparing lymphocytes from mammals; (B) contacting the lymphocyte with a mannose-coated liposome encapsulating a test substance; (C) detecting CTLs specific for the test substance or antigen; and (D) selecting a test substance having a large amount of detected antigen-specific CTLs as an antigenic substance having a high ability of inducing antigen-specific CTLs to the mammal; (Ii) preparing a mannose-coated liposome encapsulating the antigenic substance selected in (i), and (Iii) A method comprising administering a mannose-coated liposome encapsulating the antigenic substance prepared in (ii) to a subject.
  • a method for treating or preventing an infectious disease or cancer (I) A step of obtaining antigen-specific CTLs ex vivo comprising the following steps (a) to (d): (A) preparing lymphocytes from mammals; (B) contacting the lymphocyte with a mannose-coated liposome encapsulating an antigenic substance; and (C) selecting CTLs specific for the antigen; and (Ii) A method comprising administering to the subject the antigen-specific CTLs selected in (i) above.
  • a method for treating or preventing an infectious disease or cancer comprising the following steps (a) to (d): (A) preparing lymphocytes from mammals; (B) contacting the lymphocyte with a mannose-coated liposome encapsulating an antigenic substance; and (C) selecting CTLs specific for the antigen; and (Ii) A method comprising administering the antigen-specific CTLs selected in the above (i) to the mammal itself that provided the lymphocytes used in the preparation of the antigen-specific CTLs.
  • MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID NO: 16), CYSLYGTTL (SEQ ID NO: 17), QYNKPLCDL (SEQ ID NO: 18), IVLHLEPQNEI (SEQ ID NO: 19) ), HYNIVTFCCK (SEQ ID NO: 20), or a peptide comprising an amino acid sequence consisting of LMGTLGIVCPI (SEQ ID NO: 21).
  • MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID NO: 16), CYSLYGTTL (SEQ ID NO: 17), QYNKPLCDL (SEQ ID NO: 18), IVLHLEPQNEI (SEQ ID NO: 19) ), HYNIVTFCCK (SEQ ID NO: 20), or a nucleic acid comprising a DNA encoding an amino acid sequence consisting of LMGTLGIVCPI (SEQ ID NO: 21).
  • a nucleic acid comprising an HPV16E5E6E7 fusion gene (SEQ ID NO: 21).
  • MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID NO: 16), CYSLYGTTL (SEQ ID NO: 17), QYNKPLCDL (SEQ ID NO :) presented by HLA-A24 18) CTLs that recognize at least one of antigens having an amino acid sequence consisting of IVLHLEPQNEI (SEQ ID NO: 19), HYNIVTFCCK (SEQ ID NO: 20), or LMGTLGIVCPI (SEQ ID NO: 21).
  • the peptide according to (28) or the nucleic acid according to (29) or (30) is contained as an antigenic substance, or the CTLs according to (33) or (34) is contained as an active ingredient
  • the medical composition according to (35) wherein the antigenic substance is encapsulated in mannose-coated liposome.
  • a method for treating or preventing an infectious disease or cancer, the peptide according to (28), the nucleic acid according to (29) or (30), or the method according to (33) or (34) Administering a lipotoxic T cell to a subject.
  • (40) The method according to (35), wherein the antigenic substance is encapsulated in mannose-coated liposome.
  • (41) The method according to (39) or (40), wherein the subject has the HLA-A24 gene.
  • CTLs are cells that destroy infected cells among T lymphocyte cells, and are also called CD8 + T cells or killer T cells. is there.
  • CTLs are preferably T cells (antigen-specific CTLs) that are specifically activated to a target antigen or cause apoptosis specifically to cells that present the target antigen.
  • antigen-specific CTLs examples include survivin 2B peptide, human papillomavirus type 16-derived peptide (MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID NO: 16), CYSLYGTTL ( SEQ ID NO: 17), QYNKPLCDL (SEQ ID NO: 18), IVLHLEPQNEI (SEQ ID NO: 19), HYNIVTFCCK (SEQ ID NO: 20) or a peptide containing an amino acid sequence consisting of LMGTLGIVCPI (SEQ ID NO: 21)), or cytomegalovirus pp65 peptide as an antigen CTLs that are specifically recognized can be mentioned.
  • MFQDPQERPRKL human papillomavirus type 16-derived peptide
  • VYDFAFFRDL SEQ ID NO: 14
  • lymph-derived lymphocyte is not particularly limited as long as it is a lymphocyte collected from a mammal.
  • PBMC peripheral blood lymphocyte
  • TIL intratumoral infiltrating lymphocyte
  • affiliation Includes lymph node lymphocytes and lymphocytes in thoracoascites.
  • pets such as dogs, cats, rabbits, squirrels, hamsters, economical animals such as cattle, horses, sheep, pigs, goats, or lymphocytes collected from humans, more preferably humans. It is a lymphocyte.
  • blood for collecting lymphocytes may be collected from anywhere, preferably peripheral blood lymphocytes collected from peripheral blood.
  • the lymphocytes derived from mammals are not limited in the degree of purification as long as they include lymphocytes, and may be in a state separated and purified as lymphocytes. It may be in a state containing blood components and impurities.
  • the mammal-derived lymphocytes of the present invention include at least T cells such as lymphoid stem cells or naive T cells or precursors thereof, and antigen-presenting cells such as macrophages or dendritic cells.
  • the mammal-derived lymphocytes of the present invention are preferably pets such as dogs, cats, rabbits, squirrels and hamsters, economical animals such as cattle, horses, sheep, pigs, goats, or human peripheral blood lymphocytes. And more preferably human peripheral blood lymphocytes.
  • the “antigenic substance” is not particularly limited as long as it is a substance capable of inducing CTLs specific to the target antigen.
  • Antigenic substances include, for example, substances that are specifically expressed in cells in which cell death is to be induced by CTLs, substances that are improved in expression, or substances that are present by infecting the cells (preferably Non-self substance) or a part thereof can be used, and may be derived from microorganisms such as bacteria and fungi, viruses, and cancer cells.
  • the antigenic substance may be a substance that has already been reported as a preventive or therapeutic vaccine, for example, influenza vaccine, HIV vaccine, cholera vaccine, hepatitis B vaccine, tetanus vaccine, rabies vaccine, survivin It may be a vaccine such as a cancer vaccine containing 2B, or a part or modification thereof.
  • the antigenic substance used in the method of the present invention does not need to be one kind, and may contain two or more different antigenic substances individually, or a combination of two or more kinds of antigenic substances. There may be.
  • antigenic substances include survivin 2B peptide, human papillomavirus type 16-derived peptide (MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15), EYRHYCYSL (SEQ ID NO: 16), CYSLYGTTL (SEQ ID NO: 16) No.
  • antigenic peptide of the present invention a DNA encoding the peptides or may include the HPVE5E6E7 fusion gene.
  • MFQDPQERPRKL SEQ ID NO: 13
  • VYDFAFFRDL SEQ ID NO: 14
  • PYAVCDKCL SEQ ID NO: 15
  • EYRHYCYSL SEQ ID NO: 15
  • a peptide comprising an amino acid sequence consisting of CYSLYGTTTL SEQ ID NO: 17
  • QYNKPLCDL SEQ ID NO: 18
  • IVLHLEPQNEI SEQ ID NO: 19
  • HYNIVTFCCK SEQ ID NO: 20
  • LMGTLGIVCPI SEQ ID NO: 21
  • the “mannose-coated liposome” refers to a liposome having at least one mannose bound to the surface.
  • the mannose may be, for example, D-mannose.
  • the number of mannoses bonded to the surface is preferably 2 to 11, for example, mannobiose, mannotriose, mannotetraose, mannopentaose, mannohexaose or mannopentaose.
  • mannose having two or more sugars can be bound by ⁇ 1 ⁇ 2 bond, ⁇ 1 ⁇ 3 bond, ⁇ 1 ⁇ 4 bond, ⁇ 1 ⁇ 6 bond, and ⁇ 1 ⁇ 4 bond.
  • mannopentaose and mannohendekaose can take the following structures.
  • the mannose-coated liposome may be a mixture of mannose-coated liposomes of various particle shapes, but is preferably a liposome exhibiting a desired average particle size or particle size distribution, for example, an average particle size of 100 to 1000 nm, 150 to Liposomes in the range of 700 nm, 200-600 nm, 450-550 nm.
  • the mannose-coated liposome of the present invention may have a polydispersity index (PDI) of 0.3 or less.
  • the mannose-coated liposome of the present invention is preferably a mannobiose-coated liposome, a mannotriose-coated liposome, a mannotetraose-coated liposome, a mannopentaose-coated liposome, a mannohexaose-coated liposome, or a mannopentaose-coated liposome. More preferably, it is a mannotriose-coated liposome.
  • the “liposome” means an endoplasmic reticulum formed of a lipid bilayer including an aqueous medium inside, and the lipid bilayer may be a monolayer or a multilayer.
  • the lipid constituting the liposome is not particularly limited as long as it has a hydrophilic group and a hydrophobic group and can form an endoplasmic reticulum.
  • cholesterol (Chol), 3 ⁇ - [N- (dimethylaminoethane) Carbamoyl] sterols such as cholesterol (DC-Chol), N- (trimethylammonioethyl) carbamoylcholesterol (TC-Chol); dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE) Phosphatidylethanolamines such as: dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholines such as distearoylphosphatidylcholine (DSPC); dipalmitoylphosphatidylserine (DPPS), distea Phosphatidylserines such as yl phosphatidylserine (DSPS); dipalmitoyl phosphatidic acid (DPPA), and a combination of distearoyl lysophosphatidic acid (DSPA) phosphatidic acids and
  • the “medical composition” refers to a composition that, when administered to a subject, exerts an effect of treating or preventing the disease of the subject.
  • having a therapeutic or preventive effect includes not only a complete therapeutic or preventive effect, but also an effect of reducing the severity of the disease or reducing the morbidity.
  • the medical composition is a vaccine, more preferably a vaccine for treating or preventing cancer or infectious diseases.
  • the vaccine includes, as additives, preservatives such as sodium edetate, polymyxin B, neomycin or thimerosal, adjuvants such as aluminum salts or light liquid paraffin, emulsifiers such as sorbitan monooleate, and surfactants such as polysorbate 80, An inactivating agent such as formaldehyde may be contained.
  • the medical composition is a cell composition containing antigen-specific CTLs as an active ingredient, and more preferably in the body of a subject for the treatment or prevention of cancer or infectious diseases. A cell composition for administration.
  • antigen-specific CTLs contained in the cell composition include survivin 2B peptide, human papillomavirus type 16-derived peptide (preferably MFQDPQERPRKL (SEQ ID NO: 13), VYDFAFRDL (SEQ ID NO: 14), PYAVCDKCL (SEQ ID NO: 15)
  • CTLs that recognize cytomegalovirus-derived peptides preferably, cytomegalovirus pp65 495-503 peptide (SEQ ID NO: 26)
  • said peptide may be mentioned specifically recognizing CTLs).
  • antigen-specific CTLs are prepared by a method comprising preparing lymphocytes derived from mammals and contacting with mannose-coated liposomes in which an antigenic substance (eg, HPVE5E6E7 fusion gene) is encapsulated in the lymphocytes. CTLs included.
  • an antigenic substance eg, HPVE5E6E7 fusion gene
  • the type of cancer to be treated or prevented is not particularly limited.
  • lung cancer head and neck cancer, esophageal cancer, liver cancer, pancreatic cancer, cervical cancer, biliary tract cancer, breast cancer.
  • malignant tumors such as oral cancer, malignant melanoma, malignant lymphoma, and colon cancer.
  • cancer caused by infection.
  • the target cancers include Burkitt lymphoma, nasopharyngeal cancer, and gastric cancer; antigenicity derived from hepatitis B virus and hepatitis C virus
  • liver cancer can be mentioned as a target cancer
  • cervical cancer can be mentioned as a target cancer
  • human when an antigenic substance derived from T cell leukemia virus (HTLV) is used, human adult T cell leukemia and hairy cell leukemia can be exemplified as target cancers.
  • the infectious disease to be treated or prevented is not limited as long as it is a disease based on infection with bacteria, fungi, viruses and the like.
  • the in vitro CTLs induction method using the mannose-coated liposome-encapsulated antigen of the present invention can efficiently induce CTLs in a short time and / or with a small amount of antigen. Therefore, it is possible to measure the ability of inducing CTLs against a candidate antigen of PBMCs in a short time, and early detection of the effectiveness of treatment or prevention using the target antigen. Therefore, since the method of the present invention can induce CTLs stably in a short time, for example, the response based on CTLs in a patient with a candidate peptide antigen can be determined at an early stage. Can be expedited.
  • the ability to induce CTLs for each candidate antigen of the target PBMCs can be measured in a short time. It is possible to select the antigen that is most effective against Furthermore, by using the in vitro CTLs preparation method using the mannose-coated liposome-encapsulated antigen of the present invention, antigen-specific CTLs used in CTL transfer cell therapy (adoptive immunotherapy) can be adjusted in a short time. , Treatment time can be shortened.
  • the mannose-coated liposome-encapsulated HPVE5E6E7 fusion gene antigen of the present invention can efficiently induce CTLs that specifically recognize HPV16.
  • MFQDPQERPRKL SEQ ID NO: 13
  • VYDFAFFRDL SEQ ID NO: 14
  • PYAVCDKCL SEQ ID NO: 15
  • EYRHYCYSL SEQ ID NO: 16
  • CYSLYGTTL SEQ ID NO: 17
  • QYNKPLCDL SEQ ID NO: 18
  • IVLHLEPQNEI SEQ ID NO: 18
  • a peptide comprising an amino acid sequence consisting of HYNIVTFCCK (SEQ ID NO: 20) or LMGTLGIVCPI (SEQ ID NO: 21) has a HLA-A24 gene and is highly immunogenic in patients with diseases based on HPV16 infection.
  • FLAG E5E6E7 indicates 293T cells transduced with pCAGGS-E5E6E7
  • “Mock” indicates 293T cells as a negative control.
  • the arrow in a figure shows HPV16E5E6E7 protein. It is a figure which shows the result of having detected the expression of E5E6E7 protein in PBMCs by Western blot.
  • PBMC alone indicates untransduced PBMCs
  • PBMC + OML-HPV indicates PBMCs transduced with OML-HPV
  • 293T / E5E6E7 transduces pCAGGS-E5E6E7. 293T cells are shown.
  • PBMC + OML-HPV indicates the amount of OML-HPV used ( ⁇ g / mL).
  • the arrow in a figure shows HPV16E5E6E7 protein.
  • ⁇ -actin indicates the expression of beta-actin measured as an internal control. It is a figure which shows the result of the HLA-A24 binding test of HPV16E6 and E7 origin peptide which has a HLA-A24 binding motif.
  • “Peptide” on the vertical axis represents the peptide used, and “peptide ( ⁇ )” represents a control to which no peptide was added. It is a figure which shows the result of the CTL induction ability comparison test of OML-HPV and Lipofectamine 2000.
  • the horizontal axis indicates the number of spots obtained by ELISPOT assay.
  • T2A24 alone on the vertical axis represents a control in which no HPV16-derived synthetic peptide was added, and the others represent HPV16-derived synthetic peptides added in the ELISPOT assay.
  • the horizontal axis indicates the number of spots obtained by ELISPOT assay.
  • T2A24 alone on the vertical axis represents a control in which no HPV16-derived synthetic peptide was added, and the others represent HPV16-derived synthetic peptides added in the ELISPOT assay. It is a figure which shows the result of the cytotoxic activity test of the peptide specific CTLs induced
  • E / T on the horizontal axis indicates the ratio of effector cells to target cells.
  • the left figure shows the induction result of antigen-specific CTL upon stimulation with CMVpp65 epitope peptide antigen alone, and the right figure shows the antigen-specific CTL using CMVpp65 upon stimulation with mannose-coated liposome-encapsulated CMVpp65 epitope peptide antigen. The result of induction is shown.
  • the vertical axis represents CD8 positive fluorescence intensity
  • the horizontal axis represents CMVpp65 / MHC tetramer positive fluorescence intensity.
  • the present invention relates to a method for measuring CTLs inducibility against PBMC antigen ex vivo: (a ′) preparing mammalian-derived PBMC, (b ′) mannose encapsulating said antigen Contacting coated liposomes with the PBMC and (c ′) detecting CTLs specific for the antigen.
  • the present invention relates to a method for measuring CTLs inducibility of PBMC antigens ex vivo: (a ′) preparing mammalian-derived PBMC, (b ′) mannose coating encapsulating said antigens Contacting the liposome with the PBMC, (c ′) detecting CTLs specific for the antigen, and (d ′) growing the cell in the presence of interleukin 2 (IL-2).
  • the step of (a ′) preparing PBMC derived from a mammal can be performed by following the method described in the production method of the present invention.
  • the step of bringing the mannose-coated liposome encapsulating the antigen into contact with the PBMC uses, as the antigen, a desired antigen for measuring the CTLs inducing ability of the test PBMC by the antigen stimulation. Then, it can be carried out by following the method described in the production method of the present invention.
  • the detection of CTLs specific to the antigen used in the present invention is not particularly limited as long as it is a method capable of detecting CTLs specific to the antigen, and for example, chromium release assay (CTL assay) (Matsueda S. et al. Cancer Immunol Immunother, 53, 479-489 (2004)) or the tetramer method (Altman J.D. et al., Science, 274, 94-96 (1996); Lechner F. et al., J. Exp. Med., 191 (9) ), 1499-1512 (2000)).
  • CTL assay chromium release assay
  • tetramer method Altman J.D. et al., Science, 274, 94-96 (1996); Lechner F. et al., J. Exp. Med., 191 (9) ), 1499-1512 (2000).
  • a human when detecting using the tetramer method, is a complex of a target antigen peptide consisting of an ⁇ chain of a membrane-bound protein having a molecular weight of 44 kDa, ⁇ 2 myoglobulin ( ⁇ 2m) having a molecular weight of 11 kDa, and 8-10 amino acids.
  • a tetramer (tetramer) in which leukocyte antigen (HLA) molecules are modified with biotin and the HLA is bound to biotin-labeled streptavidin is used.
  • HLA leukocyte antigen
  • streptavidin a label well known to those skilled in the art can be used.
  • fluorescein isocyanate FITC
  • PE phycoerythrin
  • PerCP periodinin chlorophyll protein
  • carbocyanine that can be used for subsequent FACS analysis.
  • Fluorescent labels such as (Cy3, Cy5, PE-Cy5) can be used.
  • the determination of the ability to induce CTLs can be performed by determining an antigen having a large number of CTLs specific to the target antigen measured by the above method as an antigen having a high ability to induce CTLs.
  • the determination of the ability to induce CTLs can be performed by determining the presence of CTLs specific to the target antigen by the above method as an antigen having the ability to induce CTLs.
  • the present invention relates to a method for measuring CTLs inducibility to an antigen of PBMC ex vivo, (a ′) preparing a PBMC derived from a mammal, and (b ′) encapsulating the antigen. (C ′) detecting the number of CTLs specific to the antigen, and (d ′) contacting an antigen having a large number of CTLs specific to the antigen with CTLs inducing ability.
  • the present invention relates to a method comprising determining a high antigen.
  • the present invention is a method for measuring CTLs-inducing ability against an antigen of PBMC ex vivo: (a ′) preparing PBMC derived from a mammal; (b ′) a mannose-coated liposome encapsulating the antigen; Contacting the PBMC, (c ′) detecting the number of CTLs specific to the antigen, and (d ′) determining an antigen having a low number of CTLs specific to the antigen as an antigen having a low CTLs inducing ability
  • the present invention relates to a method comprising:
  • the method for measuring the ability of inducing CTLs against PBMC antigen in ex vivo according to the present invention is the method of interleukin 2 (IL-2) of the mannose-coated liposome encapsulating the antigen and the PBMC at the same time or after the contact. It may include growing the cells in the presence.
  • IL-2 interleukin 2
  • the method for measuring the ability to induce CTLs against PBMC antigens ex vivo may further include a functional analysis of CTLs, if necessary.
  • the function of CTLs to be measured can be appropriately selected depending on the purpose of the measurement, and examples thereof include interferon ⁇ (IFN ⁇ ) production ability analysis using the ELISPOT method or the like.
  • the method for measuring the ability to induce CTLs against PBMC antigens ex vivo of the present invention can also be used as a diagnostic method for CTLs induction function. That is, the present invention is a method for diagnosing CTLs-induced dysfunction characterized in that it is performed ex vivo, (a ′) preparing PBMC derived from a subject, (b ′) mannose encapsulating the antigen. Contacting the coated liposome with the PBMC, (c ′) detecting CTLs specific to the antigen, and (d ′) if no CTLs specific to the antigen are detected or the number of detections is small, The method comprises determining that the subject is in a state of CTLs-induced dysfunction. When used in the present diagnostic method, it is preferable to use an antigen in which induction of CTLs is recognized in a relatively wide range of subjects and / or to use a plurality of types of antigens.
  • the present invention relates to a method for measuring the antigen-specific CTLs inducing ability of an antigenic substance ex vivo, comprising: (a '') preparing a PBMC derived from a mammal; (b '') Contacting the PBMC with the mannose-coated liposome encapsulating the antigenic substance; (c ′′) detecting CTLs specific to the antigen; and (d ′) detecting CTLs specific to the antigen.
  • a method comprising determining an antigen having an ability to induce CTLs.
  • the present invention relates to a method for measuring the antigen-specific CTLs inducing ability of an antigenic substance ex vivo: (a ′′) preparing PBMC derived from a mammal; (b ′′) Contacting the encapsulated mannose-coated liposome with the PBMC, (c ′′) detecting CTLs specific to the antigen, and (d ′′) an antigen in which the number of CTLs specific to the antigen is not detected.
  • the present invention relates to a method comprising determining an antigen having a low ability to induce CTLs or an antigen having no ability to induce CTLs.
  • the method for measuring the antigen-specific CTLs inducing ability of an antigenic substance ex vivo is performed by contacting the mannose-coated liposome encapsulating the antigen with the PBMC, or after contacting the interleukin 2 (IL -2) may be included in the presence of cells.
  • IL -2 interleukin 2
  • the present invention relates to a method for screening a vaccine antigen having high CTLs-inducing ability ex vivo, comprising (a) preparing a mammal-derived PBMC, and (b) mannose encapsulating a test substance. Contacting the coated liposome with the PBMC, (c) detecting CTLs specific to the test substance, and (d) a test substance having a large amount of detected CTLs having an ability to induce CTLs to the mammal. It relates to a method comprising selecting as a high vaccine antigen. In this method, (a) the step of preparing a mammal-derived PBMC can be performed by following the method described in the production method of the present invention.
  • the step of bringing the mannose-coated liposome encapsulating the antigen into contact with the PBMC comprises, as an antigen, a vaccine antigen to be screened, that is, the ability to induce CTLs of a test PBMC by the antigen stimulation. It can be carried out by following the method described in the production method of the present invention using one or more desired vaccine antigens to be measured. Further, (c) the step of detecting CTLs specific to the test substance can be performed by following the method described in the measurement method of the present invention.
  • a test substance having a large amount of detected CTLs can be determined as a vaccine antigen having a high CTLs-inducing ability for the mammal, and in particular, CTLs specific to the detected antigen. Can be determined as a vaccine antigen having a high ability to induce CTLs against the mammal.
  • a method for screening a vaccine antigen having a high CTLs-inducing ability is performed by contacting interleukin 2 (IL-2) simultaneously with or after contacting the mannose-coated liposome encapsulating the antigen and the PBMC. In the presence of a cell.
  • IL-2 interleukin 2
  • the test substance in which the detected CTLs are present can be determined as a vaccine antigen having a CTLs inducing ability for the mammal.
  • the present invention is an ex vivo screening method for a vaccine antigen having an ability to induce CTLs, comprising (a ′′) preparing a mammal-derived PBMC, and (b ′′) a mannose encapsulating a test substance. Contacting the coated liposome with the PBMC, (c ′′) detecting CTLs specific to the test substance, and (d ′′) detecting a test substance in which CTLs are detected from the CTLs.
  • the present invention relates to a method comprising selecting as a vaccine antigen having inducibility.
  • diagnosis method, and screening method refer to an example that has already been reported to be immunologically effective, or the number of CTLs induced for a general subject or test animal May be performed by setting a threshold level and comparing the number of CTLs derived from PBMC derived from the subject with the threshold level.
  • Statistical significance in a decision is determined by comparing two or more populations and determining confidence intervals and / or p-values (Dowdy and Wearden, Statistics for Research, John Wiery & Sons, NewYord, 1983). .
  • the confidence interval of the present invention may be, for example, 90%, 95%, 98%, 99%, 99.5%, 99.9% or 99.99%.
  • the p value of the present invention is, for example, 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0002, or 0.0001. It may be.
  • the present invention is a method for producing CTLs ex vivo, wherein (a) preparing PBMC derived from a mammal, and (b) contacting the PBMC with a mannose-coated liposome encapsulating an antigen.
  • a method comprising:
  • the present invention provides an ex vivo method for producing CTLs, comprising: (a) preparing a PBMC derived from a mammal; (b) contacting the PBMC with a mannose-coated liposome encapsulating an antigen; And (c) a method comprising growing cells in the presence of interleukin 2 (IL-2).
  • IL-2 interleukin 2
  • the present invention relates to a method for producing CTLs ex vivo, comprising (a) preparing PBMC derived from a mammal, and (b) contacting the PBMC with a mannose-coated liposome encapsulating an antigen. (C) growing cells in the presence of interleukin 2 (IL-2) and (d) selecting CTLs specific for the antigen.
  • Mammal-derived PBMC can be prepared by using a method well known to those skilled in the art as a method for collecting PBMC. For example, it may be prepared as blood collected from a mammal or by separating a PBMC component from the blood, or only PBMC may be collected directly from a mammal by PBMC separation and collection.
  • PBMCs may be used by thawing and culturing those that have been cryopreserved as appropriate, or PBMCs prepared by the above method may be further grown by culturing and used.
  • PBMC is, for example, a serum-free medium such as IL-2 and 10% FCS-containing RPMI1640 medium or AIM (Invitrogen) under conditions of 5% CO 2 and 37 ° C. (10% human serum and IL- 2 or the like may be added).
  • expansion culture can also be performed by adding an anti-CD3 monoclonal antibody to the medium.
  • the “mannose-coated liposome encapsulating an antigenic substance” used in the method of the present invention is a method known as a method for producing an antigen-encapsulated liposome using the lipid constituting the above-mentioned liposome as a raw material, for example, International Publication WO95 / 11704, WO2005 / 087196, WO2006 / 104199, WO2007 / 122756, or the method described in Japanese Patent Application Publication No. 2001-081044.
  • liposomes are obtained by subjecting lipids such as phospholipids to physical or chemical treatments such as the Bangham method (also referred to as a thin film method), the reverse phase evaporation method, the ultrasonic method, the extrusion method, the French press method, the homogenization method. It can be prepared by applying a genization method, an ethanol injection method, a dehydration-rehydration method, or the like.
  • a conjugate of mannose and lipid for example, a conjugate of mannopentaose and DPPE, or a conjugate of mannotriose and DPPE
  • mannose can be introduced onto the liposome surface by mixing a conjugate of mannose and lipid with liposomes and allowing to stand at 4 ° C. to room temperature for 24 to 120 hours.
  • mannose-binding liposomes can be obtained by directly binding liposomes to mannose.
  • Encapsulation of antigens in liposomes involves dissolving or suspending antigens in a water-soluble solvent used in the production of liposomes, or repeatedly freezing and thawing a liposome suspension in which liposomes are suspended in an antigen-containing aqueous solvent. May be implemented.
  • the amount of the antigen to be encapsulated in the liposome is usually 1 to 100 ⁇ g, more preferably 10 to 50 ⁇ g per 1 mg of lipid constituting the liposome.
  • the mannose-coated liposome encapsulating the antigen may be prepared by the above method, and then prepared by centrifugation or the like so that the concentration of the liposome encapsulating the antigen is increased.
  • the contact between the mannose-coated liposome encapsulating the antigenic substance and the PBMC may be any method as long as the PBMC prepared according to the above and the mannose-coated liposome encapsulating the antigen can be contacted in vitro. May be used.
  • the contact is performed at 37 ° C.
  • the contact period may be determined by collecting a part of the contacted PBMC and confirming the induction of CTLs after a certain period from the start of contact. Confirmation of the induction of CTLs can be performed according to a step of detecting CTLs described later.
  • the PBMC may be cultured in the presence of IL-2 during or after the contact between the mannose-coated liposome encapsulating an antigenic substance and the PBMC, and other methods effective for the proliferation of CTLs may be combined appropriately. (Immunological Investigations, 36 (1), 85-104 (20), (2007)).
  • the present invention relates to a mannose-coated liposome encapsulating an antigenic peptide, an antigenic substance (antigenic peptide or HPVE5E6E7 fusion gene), or a pharmaceutical composition containing the peptide or liposome.
  • the antigenic peptide contained in the medical composition of the present invention can be produced by a method well known to those skilled in the art used for amino acid synthesis. For example, it can be produced by chemical synthesis using the Fmoc method or the Boc method. Can do.
  • the C-terminal amino acid of survivin 2B80-88 peptide is immobilized on a polystyrene carrier, and the amino acid protected with 9-fluorenylmethyloxycarbonyl group (Fmoc group) or tert-butoxycarbonyl group (Boc group) is converted to diisopropyl
  • a peptide having a desired amino acid sequence can be obtained by binding by reacting with a condensing agent such as carbodiimide (DIC) and repeating the steps of washing and deprotection.
  • DIC carbodiimide
  • the antigenic peptide of the present invention can also be synthesized using an automatic peptide synthesizer.
  • a peptide synthesizer for example, PSSM-8 (Shimadzu Corporation); model 433A peptide synthesizer (Applied Biosystems, Inc.); ACT396 Apex (Advanced ChemTech Inc.) Etc.
  • the mannose-coated liposome encapsulating an antigenic substance (antigenic peptide or HPVE5E6E7 fusion gene) contained in the medical composition of the present invention is a “mannose encapsulating an antigenic substance” used in the above-described method for producing CTLs in ex vivo. It can be produced in the same manner as the method for producing “coated liposome”.
  • Encapsulation of antigenic substances in liposomes involves dissolving or suspending the antigen in a water-soluble solvent used in the production of liposomes, or repeatedly freezing the liposome suspension in which the liposomes are suspended in an antigen-containing aqueous solvent. It may be carried out by melting.
  • the amount of the antigenic substance to be encapsulated in the liposome is usually 1 to 100 ⁇ g, more preferably 10 to 50 ⁇ g per mg of lipid constituting the liposome.
  • the mannose-coated liposome encapsulating the antigen may be prepared by the above method, and then prepared by centrifugation or the like so that the concentration of the liposome encapsulating the antigen is increased.
  • Examples of the administration site in the prevention method or treatment method using the medical composition of the present invention include oral administration, buccal administration, intranasal administration, intratracheal administration, subcutaneous administration, transdermal administration, intramuscular administration, intravascular ( Intravenous administration and the like.
  • Examples of the preparation include injections, capsules, tablets, syrups, granules, patches, ointments, powders and the like.
  • the antibody of the present invention may be administered alone, or may be administered together with a pharmacologically acceptable single substance (see “Pharmaceutical Additives Encyclopedia” Yakuho Nippo, “Handbook of Pharmaceutical Excipients” APHA Publications). Good.
  • examples of storage containers include ampoules, vials, prefilled syringes, pen-type syringe cartridges, and infusion bags.
  • the administration method of the medical composition of the present invention is not particularly limited as long as a desired therapeutic effect or prophylactic effect can be obtained, and is preferably administered subcutaneously or intramuscularly.
  • various non-invasive administration methods such as injection, oral, nasal, and transdermal administration can be selected.
  • the medical composition of the present invention may be administered temporarily, or may be administered continuously or intermittently.
  • the frequency of administration of the medical composition of the present invention is preferably a single administration, 1 to 4 administrations per week, and more preferably a single administration or once a week administration.
  • intermittent administration such as once every 1 to 3 months can be selected.
  • the medical composition containing the CTLs of the present invention as an active ingredient is administered according to a method generally known as immunotherapy using T cells, which is performed for the treatment or prevention of infection or cancer. can do.
  • the dosage of the medical composition of the present invention is not particularly limited as long as it provides a desired therapeutic effect or preventive effect, and can be appropriately determined depending on symptoms, sex, age, and the like.
  • the dosage in the cancer treatment method or prevention method (for example, recurrence prevention method) using the medical composition of the present invention is, for example, cancer treatment effect or prevention effect, suppression of cancer metastasis, or patient
  • the number of cytotoxic T cells specific for the antigen in the blood can be determined as an index.
  • the dose of a single antigen of the therapeutic agent or prophylactic agent of the present invention is 0.01 ng / kg to 10 mg / kg, more preferably 0.1 ng / kg to 1 mg / kg, still more preferably 0 0.5 ng to 100 ⁇ g / kg, most preferably 1 ng to 10 ⁇ g / kg.
  • phosphate buffered saline PBS
  • PBS phosphate buffered saline
  • the resulting liposome suspension was pressurized with nitrogen gas using an extruder (Northern Lipids Inc.) and passed through a polycarbonate membrane having a pore size of 1 ⁇ m at 25 ° C. 10 times.
  • the liposome suspension that passed through the polycarbonate membrane was again charged into the extruder, pressurized and extruded. This operation was repeated, and liposomes were sized by passing through the polycarbonate membrane a total of 5 times.
  • the same freeze-thaw operation was further repeated 9 times, and freeze-thaw was performed 10 times in total.
  • the freeze-thawed mannose-coated liposome suspension is precipitated by centrifugation at 15,000 rpm for 10 minutes at 4 ° C. After removing the supernatant, it is resuspended in PBS, transferred to a 15 mL polytube, and the final volume is 8 mL. PBS was added.
  • the prepared mannose-coated liposome suspension was subjected to extrusion a total of 10 times using an extruder in which a polycarbonate membrane having a pore size of 1 ⁇ m was set according to the method described in (1) above.
  • the mannose-coated liposome suspension sized by Extrude is added with PBS so that the final volume is 10 mL, and after centrifuging at room temperature and 3500 rpm for 10 minutes, the operation of removing the supernatant is repeated three times to obtain mannose-coated liposome. Unencapsulated survivin 2B peptide contained in the suspension was removed. The resulting precipitate was suspended in PBS to a final volume of 5 mL, and a survivin 2B peptide-encapsulated mannose-coated liposome suspension was prepared. A part of the suspension was collected and diluted with PBS, and then cholesterol quantification was performed. Based on the results of cholesterol quantification, PBS was added so that the cholesterol concentration was 4 mg / mL, and the mixture was stored at 4 ° C.
  • the collected supernatant was analyzed by HPLC system (Separations Module 2695-2489, Waters), column (186002684, SunFire, C18 5 ⁇ m, 4.6 ⁇ 250 mm, Waters), and guard column (186002684, SunFire, C18 5 ⁇ m, 4.6 ⁇ 20 mm, Waters) with water (0.1% TFA (208-02741, Wako)) and AcCN (0.1% TFA (208-02741, Wako)) as the mobile phase.
  • the amount of survivin 2B peptide was measured by HPLC and detecting a wavelength of 215 nm.
  • Peripheral Blood PBMC was obtained from Lymphoprep (registered trademark) (Nycomed, Oslo, N. (Norway) and separated by conventional density gradient centrifugation. PBMCs were suspended in a cell banker (Mitsubishi Chemical Rulece), stored frozen at ⁇ 80 ° C., and thawed immediately before antigen stimulation.
  • Lymphoprep registered trademark
  • PBMCs were suspended in a cell banker (Mitsubishi Chemical Rulece), stored frozen at ⁇ 80 ° C., and thawed immediately before antigen stimulation.
  • CTLs were induced by both the antigen alone and the mannose-coated liposome-encapsulated antigen.
  • survivin 2B-specific CTLs were induced in the mannose-coated liposome-encapsulated antigen. It was confirmed that From this, it was shown that the ability to induce antigen-specific CTLs can be measured in 9 days after antigen stimulation in ex vivo by encapsulating the antigen peptide in mannose-coated liposomes.
  • Example 3 Preparation of antigen (HPV16 gene) -encapsulated mannose-coated liposome (1) Construction of HPV16E5E6E7 fusion gene expression plasmid Fusion of HPV16E5 (SEQ ID NO: 1), E6 (SEQ ID NO: 3), and E7 (SEQ ID NO: 5) with FLAG-tag The gene was constructed by inserting it into a pcDNA3.1 expression vector (Invitrogen). The E5, E6 and E7 genes were amplified from Caski HPV16 positive cervical cancer cell lines. The primer pairs used for RT-PCR are shown in Table 1.
  • E5 was digested with the restriction enzymes BamHI and EcoRI, E6 with the restriction enzymes EcoRI and EcoRV, and E7 with the restriction enzymes EcoRV and NotI, and then each product was ligated into the pcDNA3.1 plasmid.
  • the cDNA sequence was confirmed using ABI Genetic analyzer PRIM3100 (Perkin Elmer). Subsequently, the FLAG-tag fusion E5E6E7 gene was subcloned into a pCAGGS expression vector (Miyazaki, J et al., Gene, 79: 269-277 (1989)).
  • E5E6E7 protein was detected by anti-FLAG monoclonal antibody.
  • ⁇ -actin was also detected as an internal control.
  • HLA-A24 binding test of antigen peptide (1) Preparation of HLA-A24 binding peptide candidate As a HLA-A24 specific binding motif, tyrosine (Y), phenylalanine (F), methionine (M), or tryptophan (W ) And a motif having leucine (L), isoleucine (I), phenylalanine (F), or methionine (M) at the C-terminal residue has been reported (Kondo, A et al., J. Immunol., 155). : 4307-4312 (1995)).
  • HPV16E6 8-19 MFQDPQERPRKL: SEQ ID NO: 13
  • HPV16E6 49-57 VYDFAFFRDL: SEQ ID NO: 14
  • HPV16E6 66-74 PYAVCDKCL: SEQ ID NO: 15
  • HPV16E6 82-90 EYRHYCYSL: SEQ ID NO: 16
  • HPV16E6 87- 95 CYSLYGTTL: SEQ ID NO: 17
  • HPV16E6 98-106 QYNKPLCDL: SEQ ID NO: 18
  • HPV16E7 10-20 IVLHLEPQNEI: SEQ ID NO: 19
  • HPV16 E7 51-60 HYNIVTFCCK: SEQ ID NO: 20
  • T2 Transporter (TAP) negative B / T hybrid cell line 174CEM.
  • T2 (hereinafter “T2”) cells were stably transformed with a plasmid encoding HLA-A2402 cDNA.
  • Transformed T2-A2402 cells (provided by Aichi Cancer Research Center) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 0.8 ⁇ g / mL G418 (Invitrogen Life Technologies).
  • HLA-A24 binding ability of each prepared peptide was measured using T2-A2402 cells.
  • the ability of the peptide to bind to HLA-A24 was determined by the HLA-A24 stabilization test according to a previously reported method (Nakao M et al., J. Immunol, 164: 2565-2574 (2000)).
  • HIV-env HIV env-derived peptide (hereinafter referred to as “HIV-env”) (RYLRDQQLLGI: SEQ ID NO: 22) (Sigma Genosis, Ishikari, Hokkaido, Japan), and HLA-A24-restricted EBV LMP2-derived peptide (hereinafter “EBV-”) LMP2 ”) (TYGPVFMSL: SEQ ID NO: 23) (Sigma Genosis, Ishikari, Hokkaido, Japan) was used as a positive control for the HLA-A24 binding test.
  • H2-Kd-restricted ovalbumin (OVA) -derived SL-8 peptide (hereinafter referred to as “SL-8”) (SIINFEKL: SEQ ID NO: 24) (Sigma Genosis, Ishikari, Hokkaido, Japan) and HLA-A68 restriction A sex EBV peptide (hereinafter referred to as “EBV-A68”) (FTASTVSV: SEQ ID NO: 25) (Sigma Genosis, Ishikari, Hokkaido, Japan) was used as a negative control for the HLA-A24 binding test. Each peptide was dissolved in DMSO and stored at ⁇ 80 ° C. until use.
  • This test method is based on the discovery that MHC class I molecules are stabilized on the cell surface in the presence of binding peptides.
  • Embodiment 5 Comparison of the ability of PBMC to induce HPV16 recognition CTLs between MCL-HPV and Lipofectamine 2000
  • PBMCs from HPV16 positive cervical cancer patients were isolated by Lymphoprep® (Nycomed, Oslo, Norway) by conventional density gradient centrifugation.
  • PBMCs isolated from HPV16-positive and HLA-A24-positive cervical cancer patients (case # 18) were seeded at 1 ⁇ 10 7 PBMCs / well, and 10% pooled human AB serum was added. The cells were cultured in the containing AIM medium (Life Technologies). On the first day, it was pulsed with 10 ⁇ g / mL of pCAGGS / E5E6E7-encapsulated mannose-coated liposomes prepared in Example 3, or HPV16E5E6E7 and Lipofectamine 2000 (Life Technologies, Rockville, MD).
  • IL-2 human recombinant IL-2 (provided by Takeda Pharmaceutical) was added to each well to a final concentration of 100 IU / mL.
  • medium change was performed using fresh AIM medium (Life Technologies) containing 10% pooled human AB serum supplemented with IL-2 (100 UI / mL) every 3-4 days.
  • HPV16-specific CTLs were detected by an enzyme linked ELISA (hereinafter referred to as “ELISPOT”) assay.
  • ELISPOT enzyme linked ELISA
  • PBMCs stimulated with MCL-HPV showed HPV16E6 66-74 peptide-specific reactivity, but this reactivity was not seen in PBMCs stimulated with Lipofectamine 2000. This observation showed that CTL induction using mannose-coated liposomes was superior and more efficient than Lipofectamine 2000.
  • Example 6 HPL16-recognized CTLs induction by MCL-HPV To confirm the ability of MCL-HPV DNA vaccine to induce CTLs and to identify more antigenic HLA-A24 restricted peptides, CTLs from CIN3 and PBMCs from cervical cancer patients An induction test was performed. In the same manner as in Example 5 (1), a CTL induction test was performed using PBMCs collected from four HLA-A24 positive and HPV16 positive patients and PBMCs collected from six HLA-A2402 positive and HPV16 negative patients. went. The patient profile is shown in Table 3 below. Patient-derived PBMCs were stimulated with MCL-HPV in vitro. Reactive CTLs were detected by performing IFN ⁇ ELISPOT assay after reacting 6 ⁇ 10 4 CTLs with 5 ⁇ 10 4 T2-A2402 cells in the presence or absence of synthetic peptides.
  • Case number 1 is HPV16E6 82-90 peptide
  • case number 6 is all HPV16E6 or E7-derived peptides
  • case number 16 is HPV16E6 49-57 peptide and HPV16E7 83-93 peptide
  • case number 18 is HPV16E6 98-106 peptide
  • the reactivity was shown respectively.
  • the HPV16 negative case did not show any reactivity with any HPV16-derived peptide. This indicates that the peptide is associated with HPV16 infection.
  • HPV16E6 66-74 peptide is a highly immunogenic peptide of HLA-A24 among peptides derived from HPVE6 and E7 proteins, and thus induces anti-cancer immune response in HLA-A24 positive and HPV16 positive patients. It was shown to be excellent and useful.
  • Example 7 51 Cr release assay of MCL-HPV induced CTLs PBMCs from HPV16 positive, HLA-A24 positive cervical cancer patients (Case No. 6) were stimulated with MCL-HPV to generate CTLs specific for HPV16. 14 days after initiation of stimulation with MCL-HPV, cytotoxicity via CTLs was measured by 51 Cr release assay according to a previously reported method (Sato, T et al., Cancer Res, 46: 4384-4389 (1986)). did. T2-A24 cells or K562 cells were used as target cells. K562 cells were used to monitor the activity of natural killer cells and nonspecific cytotoxicity activated by lymphokines.
  • T2-A2402 cells were cultured for 1 hour at room temperature in the presence or absence of 1 ⁇ g / mL HPV16E7 83-93 peptide or HIV-env prior to 51 Cr release assay.
  • Target cells were labeled with 100 ⁇ Ci of 51 Cr for 1 hour at 37 ° C., washed 5 times and resuspended in RPMI 1640 medium. Thereafter, target cells labeled with 51 Cr (2000 cells / well) and effector cells are adjusted so that the ratio of effector cells to target cells (effector cells / target cells: E / T) is 1, 3 or 10.
  • effector cells / target cells: E / T E / T
  • Results are shown in FIG. CTLs from the patient with case number 18 showed cytotoxicity against T2-A24 cells pulsed with HPV16E7 83-93 peptide. On the other hand, the CTLs did not show cytotoxicity against T2-A24 cells pulsed with a negative control peptide (HIV-env) or K562 NK target cells. This indicates that the CTLs have a specific dissolving action on HPV16E7 83-93.
  • Example 8 FIG. Antigen-specific CTLs induction by mannose-coated liposome CMV
  • PBMCs were collected from heparin from HLA-A2-positive healthy human peripheral blood. Plasma was separated from the collected blood. RPMI1640 medium supplemented with penicillin, streptomycin, and L-glutamine containing 5% of plasma obtained by separation was used as a culture medium. PBMCs were separated using Ficoll Paque (GE Healthcare) according to the manufacturer's protocol.
  • 0.1 mL of an antigen solution having a concentration 100 times the final antigen concentration was prepared with purified water.
  • 0.1 mL of the prepared antigen solution was added to mannose-coated liposomes prepared according to Example 1 (1) and lyophilized. After pipetting 5 times and stirring, the mixture was reacted at room temperature for 30 minutes, and 900 ⁇ L of culture medium was added.
  • CTL induction 900 ⁇ L of culture medium containing 2.0 ⁇ 10 6 cells of PBMCs was added to each well of a 12- or 24-well plate.
  • the CTLs induction method using the mannose-coated liposome-encapsulated antigen of the present invention can induce CTLs in a short time. Therefore, it is possible to measure the ability of mammalian PBMC to induce CTLs and to screen for antigenic substances capable of inducing antigen-specific CTLs in a short time.
  • the mannose-coated liposome-encapsulated HPVE5E6E7 fusion gene antigen of the present invention can efficiently induce CTLs that specifically recognize HPV16. Therefore, it is possible to use the mannose-coated liposome-encapsulated HPVE5E6E7 fusion gene antigen as a vaccine and as a medical composition for diseases based on HPV16 infection.
  • cancer or infectious disease can be treated or prevented by inducing CTLs ex vivo using mannose-coated liposome-encapsulated HPVE5E6E7 fusion gene antigen and then returning the CTLs to the patient's body.
  • MFQDPQERPRKL SEQ ID NO: 13
  • VYDFAFFRDL SEQ ID NO: 14
  • PYAVCDKCL SEQ ID NO: 15
  • EYRHYCYSL SEQ ID NO: 16
  • CYSLYGTTL SEQ ID NO: 17
  • QYNKPLCDL SEQ ID NO: 18
  • IVLHLEPQNEI SEQ ID NO: 18
  • a peptide comprising an amino acid sequence consisting of HYNIVTFCCK (SEQ ID NO: 20) or LMGTLGIVCPI (SEQ ID NO: 21) has an HLA-A24 gene and is highly immunogenic in patients with diseases based on HPV16 infection. It can be used as a vaccine for treating or preventing patients. Moreover, cancer or infectious disease can be treated or prevented by inducing CTLs using these antigenic peptides ex vivo and then returning the CTLs to the body of the patient.

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Abstract

La présente invention concerne un procédé de préparation de lymphocytes T spécifiques de l'antigène destinés à être utilisés dans des méthodes de prévention et de traitement du cancer et des maladies infectieuses faisant appel aux systèmes de l'immunité cellulaire; un procédé permettant de mesurer la capacité de lymphocytes issus de mammifères à induire des lymphocytes T cytotoxiques spécifiques de l'antigène; une substance antigénique destinée à être utilisée dans la prévention ou le traitement de maladies associées à une infection par VPH16; et une méthode permettant de prévenir ou de traiter les maladies associées à une infection par VPH16, qui consiste à administrer une substance antigénique ou des lymphocytes T cytotoxiques à un patient.
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US12435309B2 (en) 2009-08-24 2025-10-07 Baylor College Of Medicine Generation of CTL lines with specificity against multiple tumor antigens or multiple viruses
JPWO2012150663A1 (ja) * 2011-05-02 2014-07-28 株式会社バイオメッドコア 改良された糖被覆リポソーム組成物
WO2012150663A1 (fr) * 2011-05-02 2012-11-08 株式会社バイオメッドコア Composition améliorée de liposomes enrobés de sucre
US11155784B2 (en) 2011-12-12 2021-10-26 Baylor College Of Medicine Process of expanding T cells
US10351824B2 (en) 2011-12-12 2019-07-16 Cell Medica Limited Process of expanding T cells
US11963979B2 (en) 2011-12-12 2024-04-23 Allovir, Inc. Process for T cell expansion
US11981923B2 (en) 2012-02-09 2024-05-14 Baylor College Of Medicine Pepmixes to generate multiviral CTLS with broad specificity
US12227764B2 (en) 2012-02-09 2025-02-18 Baylor College Of Medicine Pepmixes to generate multiviral CTLs with broad specificity
US11001830B2 (en) 2015-03-16 2021-05-11 Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft Method of detecting new immunogenic T cell epitopes and isolating new antigen-specific T cell receptors by means of an MHC cell library
JP2018509911A (ja) * 2015-03-16 2018-04-12 マックス−デルブリュック−ツェントルム フューア モレキュラーレ メディツィン イン デア ヘルムホルツ−ゲマインシャフト Ｍｈｃ細胞ライブラリーを用いる、新規の免疫原性ｔ細胞エピトープの検出方法および新規の抗原特異的ｔ細胞受容体の単離方法
US12215313B2 (en) 2015-03-16 2025-02-04 Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft Method of detecting new immunogenic T cell epitopes and isolating new antigen-specific T cell receptors by means of an MHC cell library
US11931408B2 (en) 2015-09-18 2024-03-19 Baylor College Of Medicine Immunogenic antigen identification from a pathogen and correlation to clinical efficacy
WO2017200017A1 (fr) * 2016-05-19 2017-11-23 東洋製罐グループホールディングス株式会社 Procédé de préparation cellulaire et récipient de culture cellulaire

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