WO2014007669A1 - Vaccin cellulaire autologue pour traiter des affections cancéreuses et procédé de production - Google Patents

Vaccin cellulaire autologue pour traiter des affections cancéreuses et procédé de production Download PDF

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WO2014007669A1
WO2014007669A1 PCT/RU2012/000537 RU2012000537W WO2014007669A1 WO 2014007669 A1 WO2014007669 A1 WO 2014007669A1 RU 2012000537 W RU2012000537 W RU 2012000537W WO 2014007669 A1 WO2014007669 A1 WO 2014007669A1
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rhil
cells
medium
mdc
activated
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Сергей Анатольевич ВОЛГУШЕВ
Надежда Владимировна ТЕПЛОВА
Лина Викторовна ЕФИМОВА
Владимир Петрович ОГЛОБЛИН
Сергей Витальевич СЕННИКОВ
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Volgushev Sergei Anatolievich
Teplova Nadezhda Vladimirovna
Efimova Lina Viktorovna
Ogloblin Vladimir Petrovich
Sennikov Sergei Vitalievich
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Priority to PCT/RU2012/000537 priority Critical patent/WO2014007669A1/fr
Publication of WO2014007669A1 publication Critical patent/WO2014007669A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C12N5/064—Immunosuppressive dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2312—Interleukin-12 (IL-12)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2318—Interleukin-18 (IL-18)

Definitions

  • the invention relates to the field of medicine, oncology and can be used for the treatment of oncological diseases of various localizations.
  • NK natural killers
  • NK main class 1 histocompatibility complex
  • Tumor cells may differ from normal cells in a reduced expression of HLA-I, and then such cells become targets for NK.
  • the expression level on tumor cells may be normal, such tumors escape from the control of NK (Immunology of malignant growth. Berezhnaya N.M. Chekhun. V.F.
  • LAK lymphokine-activated killers
  • This cytokine IL-2 helps to generate and enhance the cytotoxicity of lymphokine-activated killer cells (hereinafter LAK), participates in the regulation of the immune response and directs it along the T-helper 1 cell development pathway.
  • LAK lymphokine-activated killer cells
  • IL-2 promotes the proliferation and differentiation of T-lymphocytes, stimulates the clonal proliferation of B-lymphocytes and antibody formation, increases the functional activity of phagocytes and NK cells (Immunology of malignant growth. Berezhnaya N.M. Chekhun V.F.Kiev: Naukova Dumka, 2005 , 791 pp., MV Kiselevsky, GV Casanova, SR. Varfolomeeva et al.
  • Vaccination is a way to create active specific immunity using a vaccine containing an immunogenic antigen (Baldueva I. A. “Antineoplastic vaccines.” Practical oncology. Biotherapy of malignant tumors. 2003, N ° 4 (3), P. 66-70; Moiseenko V .M., Baldueva IA, Hanson KP. “Vaccine therapy of malignant tumors.” Oncology. 1999, T. 3, N ° 1, S. 327-332).
  • antigens in tumors are components of tumor cells that are altered in structure relative to normal body cells.
  • DC dendritic cells
  • Interleukin-10 hereinafter IL-10
  • TGF- ⁇ transforming growth factor beta
  • VEGF vascular endothelial growth factor
  • beta-ECGF endothelial cell growth factor
  • IL-1 cytokines IL-1
  • IL-6 IL-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • TNF- ⁇ Tumor necrosis factor
  • LPS lipopolysaccharides
  • differentiated DC is able to effectively activate T cells, after which the latter can carry out an immune response
  • Baldueva IA Antitumor vaccines.” Practical oncology. Biotherapy of malignant tumors. 2003, N ° 4 (3), P. 66-70; Ptushkin VV "Dendritic cells and the role of cytokines in their differentiation and functioning.
  • the method of administering DC is also important.
  • subcutaneously administered DCs have greater immunogenicity and the ability to migrate, in contrast to DCs administered intravenously (Korostelev SA “Antineoplastic vaccines.” Modern oncology. 2003, ° 4 (3), C. 9-15; Moskaleva E. Yu., Severin S.E. “Prospects for the development of antitumor vaccines using human dendritic cells. Immunology. 2002, N ° 1, S. 8-15).
  • LAK autologous lymphokinactivated killers
  • a method of immunotherapy of malignant brain tumors comprising locoregional administration in the tumor bed after its removal of LAK in combination with IL-2 and cytotoxic lymphocytes (CTL) stimulated by a tumor antigen in the presence of IL-2, in which sequentially:
  • IFN- ⁇ leukinferon interferon-alpha
  • monocytes are isolated from peripheral blood, which are cultured with GM-CSF and IFN-a for 24 with maturation of DC in the presence of monocyte conditioned medium for 24 hours and incubation of DC in the presence of antigenic tumor material for 1 hours for their loading with a tumor antigen, while a tumor cell lysate can be used as antigenic material;
  • a vaccine therapy course of DC loaded with tumor antigen is given as subcutaneous injections administered at 2-week intervals in combination with subcutaneous injections of recombinant IL-2.
  • the T-cell population is activated with a population of antigen-presenting DCs to thereby provide an activated T-cell population and the effect of these antigen-presenting DCs on TNF-a.
  • the antigen is obtained from a protein or carbohydrate located on the surface of the transformed cell, using a peptide obtained from a protein selected from the group consisting of HIV Gag, HIV Env, C-EJAB- [beta] -2 HER2 / neu, PEM / MUC -1, Int-2, Hst, BRCA-1, BRCA-2, truncated EGFRvIII, MUC-1, p53, ras, RK, Myc, Myb, OB-1, OB-2, BCR / ABL, GIP, GSP, RET, ROS, FIS, SRC, TRC, WTI, DCC, NFl, FAP, MEN-1, ERB-Bl MART-1, gp-100, PSA, HBVc, HBVs, HPV E6, HPV E7, idiotypic immunoglobulin, tyrosinase, MAGE-1, trp-1, and mycobacterial antigen.
  • the differentially presented protein is selected from the group consisting of HIV Gag, HIV Env, HER-2, MART-1, gp-100, PSA, HBVc, HBVs, tyrosinases, MAGE-1, trp-1, mycobacterial antigens, and CEA.
  • the method may further comprise contacting the DC with a protein differentially present on the cell, for example, a transformed cell selected from the group consisting of: a cancer cell, a bacterial cell, a parasitically infected cell, and a virus infected cell.
  • the method may further include transducing said DC with a nucleic acid vector that differentially encodes a protein present on a cell selected from the group consisting of a cancer cell, a bacterial cell, a parasitically infected cell and a virus infected cell.
  • the patient When vaccinated, the patient is administered the above activated T cells and the above antigen presenting DC.
  • antigen-presenting DCs stimulate the patient's NK cell activity.
  • the T cell may be a helper of T cells
  • DC may be an antigenic protein comprising a peptide sequence derived from a peptide that appears on the surface of a cancer cell.
  • the method described above has insufficient clinical efficacy due to the lack of LAK.
  • the use of a peptide sequence as an antigen reduces the specificity of this vaccine.
  • a known method of obtaining and using a combined cell transplant used for the prevention and treatment of cancer of various localization obtained on the basis of LAK and DC, containing lymphocytes activated by IL-2, and mature DC (mature DC, mDC) obtained by incubating immature DC (immature DC, iDC) with a tumor lysate, in which:
  • MNCs mononuclear cells
  • - MNCs are cultured by incubation in RPMI 1640 culture medium or in DMEM medium with 5% human patient serum or donor AB serum for 2-4 hours;
  • MNCs divide MNCs into fractions of monocytes / macrophages adhered to the substrate (for example, to the wall of a culture bottle), and lymphocytes that do not adhere to the substrate;
  • iDC CD83 low CD86 low
  • CD80 low GM-CSF and IL-4 are added to the attached monocyte / macrophage cells
  • proinflammatory factors IL- ⁇ , TNF-a, IL-6 are added to stimulate DC maturation, all in concentration 10 mg / ml;
  • - iDC is incubated with tumor lysate for 1.0 day and get mature DC (mDC) pulsed with antigens, CB83 hi h , CD86 hi h , CD80 hi h ,
  • IL-2 at a concentration of 1000 IU / ml is added to lymphocytes that do not adhere to the substrate.
  • mDC pulsed with antigens is administered subcutaneously, intravenously, interarterially or regionally (in the cavity), and LAK is administered intravenously.
  • the vaccine obtained by the described method has insufficient clinical efficacy due to the absence of specifically activated in vitro T-lymphocytes, and specific cytotoxic cells are not obtained in the described method.
  • a significant drawback of this method is the use of autologous serum of cancer patients or human heterologous serum, since autologous serum of cancer patients may contain dissolved factors that suppress the immune response, and heterologous serum may have an additional non-specific antigenic effect.
  • the simultaneous introduction of antigen and acute phase cytokines into the medium, initiating the maturation of DC can lead to early cell maturation with incomplete antigen presentation.
  • the addition of mercaptoethanol to the medium increases survival and increases the yield of DC.
  • an autologous vaccine for the treatment of cancer consisting of two parts: a part containing T lymphocytes obtained from a non-adherent fraction of the patient’s peripheral blood MNCs, specifically activated by mature DC (mDC) and LAK and activated IL-2, and portions containing mature DC (mDC) pulsed by antigens obtained by incubating immature DC (iDC) of the attached fraction of peripheral MNCs blood of a patient with a tumor lysate.
  • the method of obtaining the specified autologous vaccines includes:
  • the aim of the present invention was to develop an autologous cell vaccine for the effective treatment of oncological diseases of various localization by simultaneously activating specific and nonspecific units of the antitumor immune response in patients with tumors of various localizations, obtained on the basis of autologous mononuclear blood cells of a patient with cancer and containing mature vaccines in one part dendritic cells antigenactivated during maturation (hereinafter AA mDC ⁇ and in another part of the vaccine, T-lymphocytes specifically activated by the indicated mature dendritic cells, antigen-activated during maturation (AA mDC), and lymphokine-activated killers (LAK).
  • AA mDC dendritic cells antigenactivated during maturation
  • LAK lymphokine-activated killers
  • the task was to create a method for producing an autologous cell vaccine containing mature dendritic cells antigenactivated during maturation (AA mDC) in one part of the vaccine and T-lymphocytes specifically activated with the indicated mature dendritic cells antigenactivated in one part of the vaccine maturation process (AA mDC), and lymphokinactivated killer cells (LAK cells).
  • AA mDC mature dendritic cells antigenactivated during maturation
  • LAK cells lymphokinactivated killer cells
  • the task was solved by creating an autologous cell vaccine for the treatment of cancer by simultaneously acting on a specific and nonspecific link of the antitumor immune response containing mature dendritic cells antigenactivated during maturation in one part of the vaccine (AA mDC), and in the other part of the vaccine containing T-lymphocytes specifically activated by these mature dendritic cells antigenactivated during maturation (AA mDC ⁇ and lymphokinactivated killers (LAK).
  • the problem was also solved by creating a method for producing an autologous cell vaccine for the treatment of cancer by simultaneously acting on a specific and nonspecific link of the antitumor immune response, containing in one part mature dendritic cells antigenactivated during maturation (AA mDC) and containing in another parts of T-lymphocytes specifically activated by these mature dendritic cells antigen-activated during maturation (AA mDC), and lymphokinact Rowan killers (LAK), comprising the steps of:
  • MNCs mononuclear cells
  • step b) culturing the mononuclear cells obtained in step a) in vitro in a base medium with a high content of amino acids and vitamins, which provides cell viability, including lymphoid cells, supplemented with FCS, HEPES, Glutamine, Gentamicin, to obtain populations of monocytes and lymphocytes;
  • step b) separation of the mononuclear cells obtained in step b) into an adherent fraction of monocytes and an adherent fraction of lymphocytes;
  • step d) culturing the monocytes of the adherent fraction obtained in step c) in vitro in a fresh base medium, similar in composition to the medium used in step b), with the addition of 2-Mercaptoethanol, a recombinant analogue of granulocyte-macrophage colony stimulating factor (rhGM-CSF) and growth factor, to obtain immature dendritic cells (iDC), which are capable of actively capturing antigen by phagocytosis and macropinocytosis;
  • rhGM-CSF granulocyte-macrophage colony stimulating factor
  • iDC immature dendritic cells
  • step d) culturing the fraction obtained in step d) containing immature dendritic cells, in a fresh base medium, similar in composition to the medium used in step b), with the addition of 2-Mercaptoethanol, a tumor cell lysate obtained from a fragment of a patient’s autologous tumor, to produce antigen-activated immature dendritic cells (AA AL); f) culturing the fraction obtained in step e) containing antigen-activated immature dendritic cells in vitro in the medium used in step e) with the addition of recombinant human tumor necrosis factor alpha (rhTNF-a) to obtain mature dendritic cells antigenactivated in the process ripening (AA mDC);
  • rhTNF-a recombinant human tumor necrosis factor alpha
  • step c) exposure of the lymphocytes of the non-adherent fraction obtained in step c) in a fresh medium, similar in composition to the medium used in step b), for a time sufficient to maintain the viability of the lymphocyte fraction;
  • step j) co-culturing the fraction obtained in step e) and part of the non-adherent fraction obtained in step i) in vitro in a ratio of 1: 10 in fresh medium, similar in composition to the medium used in step b), and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) and recombinant human Interleukin-12 (rhIL-12) or supplemented with recombinant human Interleukin-12 (rhIL-12) and recombinant human Interleukin-18 (rhIL-18), to produce T-lymphocytes specifically activated by mature dendritic cells antigenic during maturation (AA mDC);
  • Roncoleukin, rhIL-2 Noncoleukin, rhIL-2
  • rhIL-12 recombinant human Interleukin-12
  • rhIL-18 recombinant human Interleukin-18
  • step k) in vitro cultivation of the portion of lymphocytes of the non-adherent fraction not used in step k) obtained in step c) in a medium similar in composition to the medium used in step b) and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) obtaining lymphokinactivated killer cells (LAK cells);
  • Roncoleukin, rhIL-2 recombinant human Interleukin-2
  • LAK cells lymphokinactivated killer cells
  • step l selection from the fraction obtained in step k) of adherent mature dendritic cells antigen-activated during maturation (AA mDC) and washing them to obtain one part of the vaccine;
  • stage k selection from fractions of cells obtained in stage k) and in stage k) that did not adhere specifically activated T-lymphocytes and lymphokine-activated killer cells (LAK cells) and their washing with receiving another part of the vaccine.
  • LAK cells lymphokine-activated killer cells
  • step b) it is advisable to cultivate in step b) for 1.0 hour at a temperature of 37 ° C in an atmosphere of 5% CO in a base medium supplemented with 100 ml of a base medium: FCS -1%, HEPES -5 mm, Glutamine - 2mM, Gentamicin -1000 ⁇ g / ml.
  • step d) it is advisable to cultivate in step d) for 48 hours in a basic medium supplemented with 100 ml of a basic medium: FCS -10%, HEPES -5 mM, Glutamine-2 mM, 2-Mercaptoethanol - 5x10 "5 M, Gentamicin - 1000 ⁇ g / ml, and supplemented with 1 ml of medium containing 1.0 million cells, recombinant human granulocyte-macrophage colony stimulating factor Neupogen (rhGM-CSF) - 50 ng / ml and recombinant human Interleukin -4 (rhIL- 4) - 100 ng / ml.
  • a basic medium FCS -10%, HEPES -5 mM, Glutamine-2 mM, 2-Mercaptoethanol - 5x10 "5 M, Gentamicin - 1000 ⁇ g / ml, and supplemented with 1 ml of medium
  • step e) it is advisable to cultivate in step e) for 20 to 24 hours in a base medium supplemented with 100 ml of base medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, 2-Mercaptoethanol - 5x10 "5 M, Gentamicin - 1000 ⁇ g / ml, with the addition of a tumor cell lysate at the rate of 100 ⁇ g per 1 ml of base medium.
  • step e) it is advisable to cultivate in step e) for 24 hours in the base medium supplemented with 100 ml of the base medium: FCS-10%, HEPES-5mM, Glutamine-2mM, 2-Mercaptoethanol - 5x10 "5 M, Gentamicin - 1000 ⁇ g / ml, with the addition of tumor cell lysate at the rate of 100 ⁇ g per 1 ml of the base medium and recombinant human tumor necrosis factor alpha (rhTNF- ⁇ ) at the rate of 25 ng per 1 ml of the base medium.
  • rhTNF- ⁇ recombinant human tumor necrosis factor alpha
  • step i) it is advisable at step i) to carry out the exposure for 96 hours in the base medium supplemented with 100 ml of the base medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, Gentamicin - 1000 ⁇ g / ml.
  • step k) it is advisable to cultivate in step k) for 48 hours in a basic medium additionally containing 100 ml of the base medium: FCS -10%, HEPES -5 mm, Glutamine-2 mm, Gentamicin-1000 ⁇ g / ml, supplemented based on 1 ml of medium containing 2.5 million cells, recombinant human Interleukin-2 (Roncoleukin, rhIL-2) -100 U / ml and recombinant human Interleukin-12 (rhIL-12) - 10 ng / ml or recombinant human Interleukin-12 (rhIL-12) - Jung / ml and recombinant human Interleukin-18 (rhIL-18) - 100ng / ml.
  • a basic medium additionally containing 100 ml of the base medium: FCS -10%, HEPES -5 mm, Glutamine-2 mm, Gentamicin-1000
  • step l) it is advisable to cultivate in step l) for 48 hours in a basic medium additionally containing 100 ml of medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, Gentamicin - 1000 ⁇ g / ml and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) - 1000 IU / ml per 1 ml of medium containing 2.5 million cells.
  • a basic medium additionally containing 100 ml of medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, Gentamicin - 1000 ⁇ g / ml and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) - 1000 IU / ml per 1 ml of medium containing 2.5 million cells.
  • DMEM DMEM
  • FIG. 1 A is for immature DC (iDC)
  • FIG. 2A, Fig. 2B shows the change in the expression of CD83 + H CD80 + on cells in a monocytic pool at different stages of DC maturation cultured from monocytes of the adherent fraction of blood MNCs of patients with cancer of different localization at stages d), e). and e) a method for producing an autologous cell vaccine according to the invention: FIG. 2A for immature DC (iDC), FIG. 2B for mature DC (mDC);
  • FIG. 3A, FIG. 3B - change in endocytosis ability in terms of average fluorescence intensity (MFI median fluorescence intensity) at different stages of DC maturation cultured from adherent fraction of blood MNCs cancer patients of different localization in incubation conditions at 37 ° C: Fig. ZA - for immature DC (iDC), Fig.ZV - for mature DC (mDC);
  • Figure 4 indicators of phenotypic markers at different stages of DC maturation cultivated by the method of obtaining the vaccine according to the invention from monocytes adhering fraction of blood MNCs of cancer patients;
  • FIG. 5 - indicators of DC endocytosis activity, measured by the average fluorescence intensity (MFI median fluorescence intensity) at different stages of DC maturation, cultivated by the method for producing the vaccine according to the invention from adherent fraction of blood MNCs from cancer patients;
  • Fig.7 the results of tests to determine the content of IFN- ⁇ in the culture of MNCs with the addition of cytokines (rhIL-12 and rhIL-18, rhIL-12 and rhIL-2) and after co-cultivation of MNCs with mature DC antigenactivated during maturation (AA mDC ), and the addition of cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhlL-2), with spontaneous and lysate-activated production;
  • Fig. 8 shows the results of tests to determine the content of IL-4 in the conditioned medium of MNCs cultures when cytokines (rhIL-12 + rhIL-18 or rhIL-12 and rhIL-2) were added to the medium and tests to determine the content of IL-4 in the conditioned medium cultures of MNCs, co-incubated with mature DCs, antigen-activated during maturation (AA mDC) with the addition of cytokines (rhIL-12 + rhIL-18 or rhIL-12 and rhIL-2), with spontaneous and lysate-activated production.
  • cytokines rhIL-12 + rhIL-18 or rhIL-12 and rhIL-2
  • a method of obtaining an autologous cell vaccine for the treatment of cancer according to the invention was carried out using known means and known technologies.
  • various well-known basic media can be used, traditionally used for cell cultivation and characterized by a high content of amino acids and vitamins and providing cell viability, including lymphoid cells, for example, DMEM or RPMI-1640 medium.
  • a method for producing an autologous vaccine according to the invention was implemented by the authors using DMEM as a base medium and known components:
  • DMEM medium - a medium with a high content of amino acids and vitamins, includes: transferrin, leucine, glucose, pyruvate and a number of other substances, and has high biological stability, provides normal vital activity of many types and cell lines, including lymphoid ones;
  • HEPES - a buffer that is not toxic to cells, widely used to maintain a pH of 6.8-8.2 in culture studies on the growth of lymphoid cells;
  • 2-Mercaptoethanol an antioxidant that causes the reactivation of many enzymes and is used as an additive in the cultivation of lymphoid cells, increases the output of granulocyte cells - macrophage series;
  • - Glutamine is an essential amino acid needed to nourish growing cells
  • Gentamicin is an antibiotic that suppresses the growth of the bacterial flora, added in a concentration non-toxic to the growing cell culture.
  • rhGM-CSF is a recombinant human analogue of GM-CSF, which is a glycoprotein, a hematopoietic growth factor that regulates the formation and functioning of immunocompetent cells: dendritic cells, macrophages and granulocytes. Since only about 1% DC is contained in human peripheral blood, the production of a sufficient amount of DC in vitro from peripheral blood monocytes can be carried out by culturing in the presence of growth factors, the main of which is GM-CSF.
  • rhIL-4 - recombinant human Interleukin-4
  • rhIL-4 is a differentiation factor for T and B lymphocytes.
  • the most powerful effect of IL-4 has on the regulation of the formation of other cytokines through participation in numerous biological processes, such as the immune response and inflammatory reactions.
  • monocytes under the influence of GM-CSF and IL-4 lose their ability to phagocytosis, acquire the morphology of dendritic cells.
  • - autologous lysate of the patient’s tumor cells a lysate obtained from a fragment of the patient’s autologous tumor by mechanical grinding, filtering, washing in three steps with a serum-free medium containing a double concentration of antibiotics and antimycotics, subsequent processing of the resulting tumor cell suspension with three cycles of freezing-thawing and subsequent filtration through a filter cartridge with a pore diameter of 0.45 ⁇ m (Yu JS. et all. "Vaccination with tumor lysate-pulsed dendritic cells elicits antigen-specific, cytotoxic T-cells in patients with malignant glioma.” Cancer Res. 2004, Jul 15 ; 64 (14): 4973-9; Stift A.et all. “Dendritic cell vaccination in medullary thyroid carcinoma.” Clin. Cancer Res. 2004, May 1; 10 (9): 2944-53);
  • rhTNF-a - recombinant human tumor necrosis factor alpha
  • rhTNF-a a polypeptide.
  • TNF-a is a pro-inflammatory cytokine that causes accelerated maturation of dendritic cells;
  • IL-2 Roncoleukin (rhIL-2) - a preparation of the recombinant human cytokine IL-2.
  • IL-2 causes polyclonal activation of T-lymphocytes and the generation of LAK in vitro.
  • the formation of LAK is the result of the activation of natural killers by IL-2.
  • This cytokine helps to generate and enhance the cytotoxicity of LAKs, which are more specific for tumor cells compared to NK;
  • rhIL-12 recombinant human Interleukin-12
  • rhIL-12 a cytokine that activates the differentiation of T-lymphocytes, increases their cytotoxic activity, enhances the proliferation of DC and T-lymphocytes and the production of other cytokines.
  • IL-12 One of the most important effects of IL-12 is the ability to rotate the differentiation of T-helpers towards T-helpers of 1 way;
  • rhIL-18 recombinant human Interleukin-18 - is involved in the activation of cytotoxic T-lymphocytes, ⁇ cells, macrophages, dendritic cells and contributes to the formation of an effective anti-infection and anti-tumor immune response.
  • a method of obtaining an autologous cell vaccine for the treatment of cancer according to the invention was carried out in stages as follows:
  • MNCs mononuclear cells
  • a) mononuclear cells (hereinafter MNCs) were isolated from autologous the peripheral blood of a cancer patient in a known manner on a gradient of ficol-urographin with a density of 1.077 g / cm 3 with a ratio of blood and gradient of 2: 1; b) to obtain populations of monocytes and lymphocytes, cultivation of 2 ⁇ 10 6 in vitro obtained MNCs in vitro was carried out in culture bottles by incubation for 1.0 hour at 37 ° C in an atmosphere of 5% C0 2 in DMEM supplemented with 100 ml of DMEM: FCS -1%, HEPES -5 mm, Glutamine - 2 mm, Gentamicin -1000 ⁇ g / ml;
  • step b) the MNCs obtained in step b) were separated into an adherent fraction of monocytes and an adherent fraction of lymphocytes by collecting the adherent fraction from the walls of the vials after culturing in step b);
  • monocytes were cultured in the adherent MNCs fraction obtained in step c) in vitro for 48 hours in DMEM supplemented with 100 ml DMEM: FCS -10% , HEPES -5 mM, Glutamine - 2 mM, 2-Mercaptoethanol - 5x10 "5 M, Gentamicin - 1000 ⁇ g / ml, and supplemented with 1 ml of medium containing 1 million cells, Neupogen - 50 ng / ml and rhIL- 4 - 100 ng / ml
  • the indicated cultivation time and concentration were chosen experimentally taking into account the maximum output iDC, while the phenotype is determined were added according to the main markers CD83 + and CD 86+, and it was found that adding to the adherent fraction in step d) Neupogen at a concentration of 50 ng / /
  • step g) the fraction obtained in step g) was iodinated for 20-24 hours in DMEM supplemented with 100 ml DMEM: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, 2 -Mercaptoethanol - 5x10 " 5 M, Gentamicin - 1000 ⁇ g / ml, with the addition of 100 ⁇ g per 1 ml of DMEM autologous lysate of tumor cells obtained from a fragment of an autologous tumor of the patient;
  • step e) in order to obtain AA mDC, the fraction obtained in step e) was cultured for 24 hours in vitro in DMEM supplemented with 100 ml DMEM: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, 2-Mercaptoethanol - 5x10 "5 M, Gentamicin - 1000 ⁇ g / ml, with the addition of an autologous tumor cell lysate at the rate of 100 ⁇ g per 1 ml of DMEM and rhTNF-a at the rate of 25 ng per 1 ml of DMEM; and) parallel lymphocytes of non-adherent fraction MNCs obtained in stage c) were kept in DMEM supplemented with 100 ml of DMEM: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, Gentamicin - 1000 ⁇ g / ml for a time sufficient to maintain cell viability, which
  • step j) to obtain T-lymphocytes specifically activated by AA mDC, the adherent fraction obtained in step e) and part of the non-adherent MNCs fraction obtained in step i) were co-cultured in vitro at a ratio of 1: 10 for 48 hours fresh DMEM medium, additionally containing per 100 ml of medium: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, Gentamicin - 1000 ⁇ g / ml, supplemented with 1 ml of medium containing 2.5 million cells, Roncoleukin - 100 IU / ml and rhIL-12 - 10 ng / ml or rhIL-18 - 100 ng / ml and rhIL-12 - 10 ng / ml.
  • step k) in order to obtain LAK, the part of the non-adherent fraction that was not used in step k) was cultured in vitro for 48 hours in DMEM medium additionally containing 100 ml of medium: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, Gentamicin - 1000 ⁇ g / ml, and supplemented with Roncoleukin - 1000 IU / ml per 1 ml of medium containing 2.5 million cells. It was found that increasing the concentration of Roncoleukin to 1000 IU / ml at the stage of LAK production with a reduction in cultivation time to 48 hours allows a higher percentage LAK yield to be obtained in a shorter period;
  • the adherent AA mDC was selected from the cell fraction obtained in step k) and washed twice by centrifugation in physiological saline for 10 minutes at 1500 rpm to obtain one part of the vaccine;
  • non-adherent T-lymphocytes specifically activated by AA mDC, were selected from the cell fractions obtained in step k) and LAK obtained in step k), and they were washed twice by centrifugation in physiological saline for 10 minutes at 1500 rpm. / min to obtain another part of the vaccine.
  • MNCs isolated from the peripheral blood of an oncological patient first produce immature dendritic cells (iDC) with a pronounced ability to only absorb antigen (step g), and then, after adding an autologous tumor lysate (step e) to obtain antigen-activated immature DCs (AA iDC) ) and the subsequent addition of components that stimulate the maturation of these iDCs (step e), helps to produce AA mDC (step e), which effectively present antigens and activate T-helpers (step k), which leads to the production of T-lymphocytes specifically activated AA mDC.
  • iDC immature dendritic cells
  • step k) of the fraction of MNCs obtained in step e) and a portion of the non-adherent fraction of MNCs obtained in step i), with the production of T-lymphocytes specifically activated by AA mDC, was carried out in the presence of Roncoleukin or rhIL-18 , while the process of differentiation of T-helpers is directed along the T-helper 1 way (YOU are the cellular version of the immune response), which, when both parts of the vaccine are administered to the patient, leads to the deployment of a specific antitumor immune response, activated cytotoxic Kie T-killer (CD8 +) T lymphocytes and delayed-type hypersensitivity (DTH T - CD4 +), which are the main effectors of the cellular immune response path.
  • YOU are the cellular version of the immune response
  • LAK from a portion of the non-adherent fraction of MNCs was obtained in step l) parallel to the co-cultivation step in step k), but separately, which eliminates competition between the specific and non-specific activation of lymphocytes and leads to an increase in the output of LAK.
  • step b) inactivated fetal calf serum (FCS) eliminates the possibility of suppressive effects on tumor cells of tumor growth products contained in the serum of a patient with cancer.
  • FCS inactivated fetal calf serum
  • DC maturation was assessed by the expression of surface differentiation markers (CD 14+, CD80 +, CD83 +, CD205 +) and the change in DC endocytosis activity at different stages of DC maturity by flow cytofluorimetry using appropriate fluorochrome-labeled antibodies and FITC-labeled dextran (fluorescein isocyanothiocyanate )
  • the degree of capture of FITC-labeled dextran by the studied cells was determined as the relative value of the corresponding average fluorescence intensity (hereinafter MFI) of the labeled cells.
  • MFI average fluorescence intensity
  • FIGLA, 1B, FIGS. 2A, 2B and FIGS. 3A, 3B illustrate the induction processes of maturation of DC cultured according to steps d), e) and e) of a method for producing a vaccine according to the invention from monocytes of adherent fraction of blood MNCs of patients with cancer of different localization:
  • FigLA for iDC and FigLV for mDC shows the change in the expression of CD 14+ on cells in a monocytic pool at different stages of DC maturation, while: “CD14-PE” is the luminescence intensity of the phycoerythrin labeled CD 14 marker (phycoerythrin, PE); “CD3-FITC” - the intensity of the glow of the marker CD 3, labeled fluorescein isothiocyanate (fluorescein isothiocyanate, FITC); “Cd 14 + mon” is the population of MNCs carrying the CD 14 monocyte marker, “cd 3 + mon” is the population of MNCs carrying the CD 3 marker. From FigLA and 1B it follows that for iDC the maximum number of CD14 + was 11.2% (FigLA ), and for mDC the maximum amount was 3.3% (FigLV);
  • CD83 + and CD80 + on cells at different stages of DC maturation shows the change in the expression of CD83 + and CD80 + on cells at different stages of DC maturation, while; "CD80-PE” is the luminosity of the marker CD 80 labeled with phycoerythrin (phycoerythrin, PE); “CD83-FITC” is the luminosity of the CD 83 marker labeled with fluorescein isothiocyanate (fluorescein isothiocyanate, FITC); "Cd80 + mon” - mononuclear cells carrying the marker CD 80; "Cd83 + mon” - mononuclear cells carrying the marker CD 83; “Cd83 + cd80 + mon” are mononuclear cells that simultaneously carry markers for CD83 and CD80. From Figures 2A and 2B, it follows that for iDC the maximum number of CD83 + / CD80 + was 34% ( Figure 2A), and for mDC the maximum number of CD83 + /
  • SV shows the change in endocytosis activity in terms of the average fluorescence intensity (MFI median fluorescence intensity) at different stages of DC maturation cultivated from the adherent fraction of blood MNCs of patients with cancer of different localization
  • “Dextran-FITC” is the luminescence intensity absorbed bacterial polysaccharide labeled with fluorescein isothiocyanate (fluorescein isothiocyanate, FITC): in FIG. 3A for iDC under incubation conditions at 37 ° C, the endocytosis activity (according to MFI) was 1754, in FIG.
  • FIG. 4. presents the indicators of phenotypic markers at different stages of DC maturation cultivated by the method of producing the vaccine according to the invention from monocytes of adherent fraction of blood MNCs of cancer patients in coordinates along the b axis percent (M ⁇ m), moreover: for iDC obtained from MNCs monocytes in step d) of the method in the presence of Neupogen and rhIL-4, the expression of CD 14+ was 10.4 + 0.9 (left column in region a), the expression of CD83 + was 25.2 + 2.0 (the left column in region b is shown), the expression of CD83 + / CD80 + was 32.7 + 8.5 (the left column in region c was shown), the expression of CD205 + was 78.2 + 6.2 (the left column is shown in region d), and for AA mDC obtained in step e) of the method from AA iDC by subsequent cultivation in the presence of rhTNF- ⁇ , the expression of CD 14+ was 4 + 0.7 (the right column in region
  • Figure 5 presents the indicators of DC endocytosis activity, measured by the average fluorescence intensity (MFI median fluorescence intensity) at different stages of DC maturation, cultivated by the method of obtaining the vaccine according to the invention from adherent fraction of blood MNCs of cancer patients, in axial coordinates units of MFI, (M + w), moreover: for iDC obtained from MNCs in step d) in the presence of Neupogen and rhIL-4, the MFI was 1878.0 + 227.3 (left column is shown); for AA mDC obtained in step e) of the method from AA iDC by culturing in the presence of rhTNF- ⁇ , the MFI was 116.8 + 1 1.0 (right column shown). Arrows indicate significant differences between groups (p ⁇ 0.05);
  • Table 1 shows the phenotypic markers and endocytotic activity of DC (according to the MFI indicator) at different stages of DC maturation cultivated by the method of obtaining the vaccine according to the invention from the adherent fraction of blood MNCs of cancer patients, (M ⁇ t), wherein: iDC were obtained from MNCs on step g) of the method in the presence of Neupogen and rhIL-4, and AA mDC were obtained in step e) of the method from AA iDC followed by cultivation in the presence of rhTNF-a.
  • p is an indicator of the significance of differences between groups i DC and mDC.
  • the cytotoxicity test of the non-adherent fraction of MNCs to the autologous tumor cells of the patient was carried out using a Promega kit according to the manufacturer's instructions.
  • step e) of the method for preparing the vaccine according to the invention we used the AA mDC obtained in step e) of the method for preparing the vaccine according to the invention, which were then subjected to co-cultivation in step k) of the non-adherent fraction of MNCs obtained in step i) in a ratio of 1: 10, respectively, in an atmosphere of 5% ⁇ 02 and 37 ° ⁇ in fresh DMEM medium, additionally containing: FCS, HEPES, Glutamine, Gentamicin, supplemented with Roncoleukin and rhIL-12 or supplemented with rhIL-18 and rhIL-12.
  • Figure 6 and table 2 presents the cytotoxicity indices of the MNCs culture against tumor cells in the presence of AA mDC and cytokines (rhIL-12 and rhlL-18 or rhIL-12 and rhIL-2), (M ⁇ m), with confidence compared with cytotoxicity indices in the MNCs groups with the addition of cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2) in the absence of AA mDC, MNCs in the presence of AA mDC, without the addition of cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2).
  • P2 is a confidence indicator compared with group 2
  • RB is a confidence indicator in comparison with group 6.
  • T-helper Type 1 For analysis of antigen-specific stimulation of the immune response by T-helper Type 1 was determined by the content of IFN- ⁇ in the conditioned medium of the culture of MNCs co-cultivated with AA mDC before and after additional stimulation with tumor antigens (lysate of autologous tumor cells at a concentration of 100 ⁇ g / ml) and cytokines.
  • tumor antigens lysate of autologous tumor cells at a concentration of 100 ⁇ g / ml
  • cytokines cytokines.
  • the IFNy content was determined using commercial ELISA kits (Vector-Best) according to the manufacturer's instructions.
  • cytokines rhIL-12 and rhlL-18 or rbb-12 and rbb-2:
  • the content of IFN- ⁇ in the culture of mononuclear cells co-cultivated with cytokines (rhIL-18 and rhIL-12 or rhIL-2 and rhIL-12)
  • P2 is a confidence indicator compared to group 8
  • rb is a confidence indicator in comparison with group 12.
  • rhIL-18 and rhIL-12 or rhIL-2 and rhIL-12 and rhlL-12 cytokines significantly increased IFN- ⁇ production in response to additional antigenic stimulation compared to the group of MNCs and AA mDC without adding cytokines. This indicates a stimulating effect of cytokines on the modulation of the immune response according to the T-helper type 1.
  • IL-4 The content of IL-4 in the conditioned culture medium of MNCs co-cultured with AA mDC before and after additional stimulation with tumor antigens (lysate of augologous tumor cells at a concentration of 100 ⁇ g / ml) and cytokines was determined.
  • the content of IL-4 was determined using commercial ELISA kits (Vector-Best) according to the manufacturer's instructions.
  • Fig and table 4 presents the results of tests to determine the content of IL-4 in the conditioned medium of the culture of MNCs (13th group of tests), (M ⁇ t), and the content of IL-4 in the conditioned medium of the culture of MNCs with the addition of cytokine medium (rhIL-12 + rhIL-18 or rhIL-12 and rhIL-2):
  • IL-4 The content of IL-4 in the culture of mononuclear cells co-cultivated with cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2), (M ⁇ m) and / or AA mDC in spontaneous conditions
  • P1 is a confidence indicator in comparison with group 13, p2 - confidence indicator compared with group 14,
  • rb is a confidence indicator in comparison with group 18,
  • a method for generating antitumor cytotoxic cells using antigen-activated autologous dendritic cells and rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2 in vitro to produce antigen-activated immature and mature dendritic cells antigenactivated during maturation, from mononuclear cells of patients with cancer of various localization, which are characterized by the corresponding functional activity and phenotype.
  • an increase in the expression of co-stimulatory molecules on the surface of antigen-activated dendritic cells was achieved, which indicates an increase in the effective process of antigen presentation.
  • the method for producing an autologous vaccine according to the invention allows, at the method steps, to increase the yield of immature dendritic cells, increase the efficiency of capture and presentation of antigen, increase the yield of specifically activated killers, eliminates competition between specific and non-specific activation of cells, allows a higher percentage to be obtained in a shorter time. yield of non-specifically activated killers (LAC) and specifically activated killers (T-lymphocytes).
  • LAC non-specifically activated killers
  • T-lymphocytes specifically activated killers
  • the autologous cell vaccine according to the invention for the treatment of cancer obtained by the method according to the invention in comparison with known vaccines, is more effective, does not require additional administration of cytokines directly into the patient’s body, and can be used to treat cancer patients with tumors of different localization .
  • the method of obtaining an autologous cell vaccine according to the invention for the treatment of cancer by simultaneously affecting a specific and non-specific link of the antitumor immune response can be implemented using known techniques and materials, and the vaccine obtained in this way is highly effective and can be used in the field of oncology for the treatment of patients with various oncopathology.

Abstract

L'invention concerne un vaccin autologue pour traiter des affections cancéreuses en agissant simultanément sur des maillons spécifiques et non spécifiques de la réponse immunitaire anticancéreuse, lequel comprend, pour une part, des cellules dendritiques mûres ayant une activité antigène lors du processus de maturation (AA mDC) et, pour l'autre part, des lymphocytes T spécifiquement activés par ces cellules dendritiques mûres ayant une activité antigène lors du processus de maturation (AA mDC), ainsi que des cellules tueuses activées par lymphokine (LAK); l'invention concerne également un procédé de production de ce vaccin à partir de mononucléaires autologues du sang de malades présentant des pathologies oncologiques.
PCT/RU2012/000537 2012-07-04 2012-07-04 Vaccin cellulaire autologue pour traiter des affections cancéreuses et procédé de production WO2014007669A1 (fr)

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CN113151166A (zh) * 2021-01-26 2021-07-23 广州润生细胞医药科技有限责任公司 一种个体化肿瘤新生抗原特异性cd8细胞的获取方法及应用
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