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WO2010024719A1 - Procédé de fabrication d’un vaccin antitumoral - Google Patents

Procédé de fabrication d’un vaccin antitumoral Download PDF

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Publication number
WO2010024719A1
WO2010024719A1 PCT/RU2009/000392 RU2009000392W WO2010024719A1 WO 2010024719 A1 WO2010024719 A1 WO 2010024719A1 RU 2009000392 W RU2009000392 W RU 2009000392W WO 2010024719 A1 WO2010024719 A1 WO 2010024719A1
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cells
antigens
thе
antigen
presenting
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PCT/RU2009/000392
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English (en)
Russian (ru)
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Петр Генриевич ЛОХОВ
Елена Евгеньевна БАЛАШОВА
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Lokhov Petr Genrievich
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Publication of WO2010024719A1 publication Critical patent/WO2010024719A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/49—Breast

Definitions

  • the invention relates to biotechnology, pharmaceuticals and medicine and can be used for the manufacture of medicines and the implementation of medical technologies, in particular immunotherapy of cancer.
  • immunotherapy is also used in the treatment of cancer patients.
  • antitumor vaccination which consists in inducing an immune response in a cancer patient against vaccine antigens that are, to one degree or another, identical to tumor antigens.
  • the vaccine-induced immune response cross-damages tumor cells (OK), which determines the therapeutic or prophylactic effects of antitumor vaccination.
  • Sapser Impepe Thearu Surpert Future Strategies (eds. G. G. ap. Waldep R., Johp Wilheu & Sops, Ips, 2002).
  • the main factors determining the effectiveness of vaccines are, firstly, the specificity of the antigens included in it against the tumor cells of the patient and, secondly, the ability to elicit a strong and stable immune response to these antigens.
  • antigens in the preparation of the synthesized peptides are used, in particular gplOO (g2O9) -2M, MART-January 27 _ 35, MAGE-Z.A1, NY-ESO-I, K -Ras / 12, etc. . (see ROSEPBERG S.A. et al.
  • proteins for example, heat shock proteins or a prostate-specific antigen
  • antigens see, for example, Mapjili M.N. et al. "Imperoteruf sapser hepot roteipi”.
  • the main advantages of vaccines obtained by the indicated methods using synthetic peptides and certain proteins include their low allergenicity, safety, and relative ease in standardizing production.
  • the main disadvantage of the known methods for producing vaccines is the inability to obtain vaccines that elicit an immune response with high antitumor efficacy. This circumstance is due to the fact that OK are distinguished by genetic instability and constantly mutate in the patient’s body, which leads to the appearance of new ones having a modified antigenic composition of OK. Thus, a specific antigen to which a vaccine peptide or protein antigen elicits an immune response may or may not be altered or absent in all or part of an OK patient. Moreover, for most types of tumors, individual antigens specific for them that are not suitable for antitumor vaccination are not known.
  • antitumor vaccines use non-tumor antigens as antigens. cells.
  • vascular endothelial cells used as antigens, vaccination of which elicits an immune response aimed at damaging the vessels of solid tumors.
  • xenogenic endothelial cells i.e. cells isolated from the endothelium of the blood vessels of an organism of a different biological species than the vaccinated organism.
  • EC xenogenic endothelial cells
  • the disadvantage of such vaccines is that xenogenic antigens, although more immunogenic, are less specific for the vessels of the tumor of the vaccinated organism, which ultimately leads to a decrease in the effectiveness of the vaccine.
  • allocellular EC as antigens in whole-cell vaccine, i.e. cells of a different organism, but of the same biological species as the vaccinated organism (see, for example, S Vintagearratissi FA, Nolap GP, "Industrial Pharmaceutical Integrated Medical Integrated Pharmaceutical Complex", Pharmaceutical 2003, Pharmaceutical. .23, p. 1165-1672; the authors of the scientific work also compare the effectiveness of vaccination when using EC as antigens from various sources).
  • the disadvantage of allogeneic EC antigens is also their low specificity for the vessels of the tumor of the vaccinated organism.
  • the obvious advantages of using whole-cell antitumor vaccines include their polyvalency (i.e., the content of multiple antigens).
  • the multivalency of the vaccine allows you to elicit an immune response not to one but to many antigens on the patient’s OK surface, which significantly increases the likelihood of tumor destruction.
  • the second advantage is the high specificity of antigens, especially if autologous OKs are used as antigens for vaccination, i.e. OK isolated from the tumor of the same patient who is being vaccinated.
  • the benefits of using whole OKs as antigens include the ability to their use in vaccination of patients with tumors for which individual tumor-specific antigens are unknown and, accordingly, monovalent vaccines have not been created.
  • the second significant drawback is that whole cells, in addition to the target antigens that are present on their surface, contain many unsuitable for vaccination antigens and ballast substances derived from intracellular contents.
  • the unsuitability of intracellular contents for vaccination is explained by the fact that intracellular antigens are hidden inside the cells and inaccessible to the body's immune system.
  • the problem of absorption of OK by antigen-presenting cells and the presence of non-targeted antigens and ballast substances in the vaccine significantly worsen the effectiveness of whole-cell vaccines.
  • OK lysates The disadvantage of using OK lysates is that the bulk of the lysate is also made up of non-targeted antigens that elicit an immune response to the intracellular contents of the patient’s OK hidden from the immune system.
  • vaccines contain many ballast substances, in particular, nucleic acids, carbohydrates, lipids, etc.
  • the vaccine obtained in this way is distinguished by the established high content of non-target antigens, in particular intracellular proteins (see, for example, "Proteomis apolysis of the method of the test, the test method of the test.” v.4. Immulogu, 2002, v. 2, p. 569-579).
  • the second drawback is the need to purify antigens before using them from lipids, which are an integral part of exosomes.
  • a common disadvantage of antigens specific for tumor cells or tumor vascular cells is their weak immunogenicity. This is due to the fact that, in contrast to antimicrobial, antiviral or nonspecific antitumor vaccinations, which use antigens that are foreign and, accordingly, immunogenic for the vaccinated organism, in this case they use antigens identical to the antigens of the cells of the vaccinated organism.
  • a normally functioning immune system of the body is tolerant of its own antigens, as a result of which the evoked immune response is weak, short-term, or even absent.
  • the effectiveness of using such tumor-specific antigens depends on the ability to elicit an appropriate immune response to them.
  • antigens There are various ways to increase the immune response to antigens, for example, chemical modification of antigens or mixing them with adjuvants (oils, mineral salts, bacterial extracts, cytokines, etc.).
  • adjuvants oil, mineral salts, bacterial extracts, cytokines, etc.
  • antirepresenting cells i.e. those cells with which antigens introduced into the body usually interact.
  • DCs dendritic cells
  • a dendritic cell that are capable of absorbing antigens, degrading them to small fragments and presenting them on their surface associated with the main histocompatibility complex are used as antipresenting cells.
  • Cells of the immune system for the manifestation of humoral and cellular immunity recognize the main histocompatibility complex and its associated antigens.
  • mature DCs receive ip vitro from CD34 + bone marrow precursors or populations peripheral blood monocytes. After receiving the DC loaded with tumor specific antigens. The antigens themselves are no longer administered to the patient, but DCs loaded with them, i.e. apply the so-called antitumor dendritic cell vaccine.
  • DCs are loaded with synthesized peptides, tumor cells or their lysates, live tumor cells (formation of hybrids of DCs and tumor cells), etc.
  • Examples of the use of DCs loaded with various antigens are described (see, for example, Str. S.E. et al., "Strategies for Strategic Loads for Health Imports Reserves, 2002, 2002, 2002). p. 1884-1898; Martin R. et al., "Autologous Distributors Cells CDF + Prog.
  • the disadvantages of using DCs loaded with individual antigens, whole tumor cells or their lysates are identical to the disadvantages described above for monovalent and multivalent vaccines that do not contain DC, namely, low specificity and the presence of non-targeted antigens.
  • the disadvantage of hybrids of DCs and tumor cells in addition is the possibility of the formation of hybrid clones with malignant properties.
  • the closest analogue of the claimed invention is a method for producing an antitumor vaccine based on antigen-presenting cells loaded with antigens, disclosed in international application WO2004012685 ("Shad pharmacy with medical grade", IPC A61K39 / 00, published February 12, 2004).
  • the loading of antigen-presenting cells is carried out by antigens lost by OK during incubation of vitro in serum-free incubation medium (the method for producing the antigens themselves is also described in US patents US 5993829, IPC A61P 35/00, C07K14 / 47, publ. 11/30/1999; US 6338853, IPC AblR 35/00, C07K14 / 47, publ.
  • Antigens lost by cells are a mixture of proteins whose presentation on the surface of antigen-presenting cells, in particular mature DCs, is difficult. Protein antigens must be absorbed by DC, degraded to peptides and only then presented on the surface of DC. Mature DCs are incapable of absorbing antigens.
  • the disadvantages of the closest analogue can also be attributed to the fact that cells spontaneously lose their antigens in the form of membrane vesicles - exosomes containing, in addition to proteins, many lipids. Therefore, the selection of antigens from them requires an additional purification step - delipidization.
  • the technical result achieved by using the patented invention is to create a vaccine capable of increasing the cytotoxic activity (CTA) of the cells of the immune system against target cells by facilitating their perception of a given set of antigens.
  • CTA cytotoxic activity
  • the antigen-presenting cells are loaded with antigens obtained by cleaving a protease from the surface of living cells with providing a representation on the surface of antigen-presenting cells of a collection of antigens identical to the collection of surface antigens of target cells.
  • autologous living cells are used, because provide a combination of both specific intraspecific and individual antigens for the vaccinated organism.
  • allogeneic living cells are used if necessary to obtain a combination of intraspecific antigens common to the biological species.
  • Xenogenic living cells can also be used if it is necessary to obtain a set of interspecific antigens common to different types of organisms.
  • allogeneic and autologous living cells are used in combination.
  • xenogenic and allogeneic living cells in combination can be used.
  • xenogenic and autologous living cells in combination may be used.
  • xenogenic, allogeneic, and autologous living cells in combination may be used.
  • Combinations of various types of living cells are used to increase the totality of specific antigens.
  • freshly isolated living cells are used.
  • Freshly isolated cells are closest in antigenic composition to the cells of the patient's body.
  • the production of living cells without damaging surface antigens, for example, when they are obtained from biological fluids or the non-enzymatic method of obtaining from tissues (with the enzymatic method of obtaining cells from tissues, damage to surface cell antigens occurs) allows the use of freshly isolated cells to produce antigens according to this invention.
  • living cells representing at least one primary culture can be used.
  • Antigens located on the surface of said cells are also suitable for loading antigen-presenting cells.
  • living cells, which are tumor cells are used.
  • living cells which are endothelial cells, are used.
  • live cells which are genetically modified cells
  • Genetic modification that increases the immunogenicity of cell surface antigens contributes to the production of antigens according to this invention with more pronounced immunogenic properties.
  • trypsin is used as the protease.
  • trypsin is used as the protease.
  • the average amino acid mass is 110 Da and the fact that incomplete proteolysis of proteins with a protease gives peptides with both the absence and one or two undersplit sites, the antigens obtained will try to match the required molecular weights with trypsin.
  • the specificity of the amino acid sequences of peptides and the presence of carbohydrate modifications affects the average size of the resulting antigens, and therefore, when selecting specific conditions for the production of antigens, it is necessary to experimentally control the size of the resulting antigens.
  • Immunomodulators and cytokines can be added to antigen-presenting cells. Any suitable combination of cytokines, immunomodulators, and nonspecific antigens can be used to differentiate mononuclear leukocytes into immature DCs, as well as to mature immature DCs.
  • a pharmaceutical carrier may be added, which may include one or more substances.
  • the carrier can contribute to the preservation of antigen-presenting cells during storage, transportation and their introduction into the body, nourish cells, provide gas exchange, stabilize cell membranes, provide local or systemic immunological reactions that enhance the effect of antigen-presenting cells.
  • antigen-presenting cells obtained by artificial means can be used, because artificial antigen presenting cells have the appropriate antigen presenting properties and can be loaded with antigens.
  • dendritic cells are used as antigen-presenting cells.
  • Dendritic cells derived from peripheral blood may be used.
  • dendritic cells derived from bone marrow may be used, as bone marrow DCs are complete antigen-presenting cells and can be loaded with ip vitro antigens.
  • autologous dendritic cells are used.
  • allogeneic dendritic cells may be used, as they are capable of performing an antigen-presenting function in relation to allogeneic cells of the immune system.
  • a mixture of autologous and allogeneic dendritic cells may be used.
  • the use of a mixture of cells is preferable if the number of autologous dendritic cells is insufficient for vaccination, and it is possible in view of the ability of allogeneic DCs to perform an antigen-presenting function with respect to allogeneic cells of the immune system.
  • mature antigen-presenting cells are used.
  • Mature antigen-presenting cells can be obtained directly from body tissues, or obtained in vitro from immature antigen-presenting cells.
  • immature antigen-presenting cells may also be used.
  • Immature antigen-presenting cells in particular DCs, are loaded with antigens by incubation in a medium containing antigens. During the incubation process, immature DCs absorb antigens, degrade them and present on their surface.
  • Mature antigen-presenting cells in particular DCs, can be loaded with antigens during incubation with them by direct binding of antigens (without their preliminary absorption of DCs) with the main histocompatibility complex located on the surface of DCs.
  • mature antigen-presenting cells are obtained from immature antigen-presenting cells already loaded with antigens.
  • the type of protease, its concentration, and also the time of exposure to the cells are selected taking into account that the resulting antigens must have a molecular weight of from ⁇ 800 to ⁇ 3500 Yes . This circumstance is connected with the fact that it is precisely such peptides that are capable of binding directly to the main histocompatibility complex located on the surface of antigen-presenting cells.
  • antigens cleaved from the surface of the cells can additionally, separately from the cells, be incubated with a protease to cleave the antigens to the desired molecular weight. Achievements of the desired molecular weight by antigens can be controlled by mass spectrometric analysis.
  • the molecular weight of the antigens should be at least 600 Da.
  • Antigens, which are peptides of lower molecular weight, are weakly or nonspecific, and therefore unsuitable for vaccination. There are no strict restrictions on the maximum molecular weight of antigens, since antigens after absorption by antigen-presenting cells will be degraded by intracellular proteases to the desired size.
  • the incubation time of DC with antigens can also vary significantly. With a load of immature DCs, the incubation time of DCs with antigens can be from 2 to 7 days. With a load of mature DC from 12 hours to 3 days.
  • the dendritic cells are loaded with antigens with a molecular weight of from 800 to 3,500 Da.
  • the load is carried out during the incubation process, the time of which is selected from the interval from 12 hours to 3 days.
  • their load can be carried out by antigens with a molecular weight of at least 600 Da during the incubation process, the time of which is selected from the interval from 2 to 7 days.
  • the final concentration of antigens for loading antigen-presenting cells added to the incubation medium is selected from a range of 10 ⁇ g / ml to 1 mg / ml. If the final concentration of antigens is less than 10 ⁇ g / ml, the totality of antigens will not be presented on surfaces of antigen-presenting cells in the amount necessary for the manifestation of specific cytotoxic activity (CTA) of the cells of the immune system. When antigens with a final concentration exceeding 1 mg / ml are added, the amount of antigens becomes greater than the limit of their amount, which antigen-presenting cells can perceive for presentation on their surface.
  • CTA cytotoxic activity
  • CTA cytotoxic activity
  • the number of living cells is reduced in accordance with the specified proportion.
  • the number of protease treatments can reach 15-20 times. It was experimentally established that the indicated ratios of living cells and dendritic cells in relation to the number of protease treatments are optimal for representing a given set of antigens on the surface of dendritic cells and facilitating their perception by the cells of the immune system.
  • the load can be carried out by antigens obtained by a single cleavage of the protease from the number of living cells twice - five times more than the number of loaded antigen-presenting cells.
  • the load can be carried out by antigens obtained by two to five fold protease cleavage from the number of living cells equal to the number of antigen presenting cells loaded.
  • Fig. 1 shows the results of a study of the cytotoxic activity of lymphocytes against MCF-7 tumor cells
  • the load of dendritic cells is carried out by antigens obtained by trypsin cleavage from the surface of living tumor cells with the representation of a collection of antigens on the surface of dendritic cells that is identical to the collection of surface antigens of tumor target cells.
  • a solution of antigens specific for the target tumor cells is obtained using MCF-7 human breast adenocarcinoma cell culture.
  • the growth medium is removed from the MCF-7 cell culture vial and washed with a monolayer of cells with sterile saline. Rinsing is carried out at least 3 times before removing residues of the growth medium.
  • the next and key stage is the vital protease treatment of MCF-7 cells for cells, where trypsin (activity ⁇ 3000 U / mg) is chosen as the protease.
  • a 0.0001% trypsin solution is added to the cell monolayer using 1 ml of a solution per 25 cm 2 of the surface of the culture vial.
  • Freshly prepared growth medium containing serum (usually 10%) is added to the cells, and their cultivation is continued.
  • the antigen solution is filtered through a filter with a cut-off of 10-15 kDa protein.
  • patented antigens are suitable for loading antigen-presenting cells, in particular mature DCs, which are obtained in vitro from purified fraction of peripheral blood mononuclear leukocytes (monocytes) (Sapser-Research, 2001, v. bl, p. 6445-6450).
  • Donor blood is collected in tubes with an anticoagulant (heparin or sodium citrate), diluted with an equal volume of sterile physiological saline and layered on a ficoll-pack solution having a density of 1.077 g / ml (2 volumes of diluted blood per volume of ficoll solution). Centrifuged for 20 min at 80Og and room temperature.
  • an anticoagulant heparin or sodium citrate
  • the cell fraction is collected with a pipette at the interface between the ficoll solution and blood plasma, the cells are diluted with physiological saline and centrifuged for 10 min at 250 g.
  • the pellet was resuspended in RPMI-1640 medium containing 10% inactivated cattle serum and, at a concentration of 50 million cells / ml, 10 ml each were added to Petri dishes. Petri dishes are incubated under saturated humidity, 5% CO 2 and 37 ° C for 1 hour.
  • Non-adherent cells mainly lymphocytes
  • 10 ml of fresh medium is added to a monolayer of attached cells (mainly monocytes) and incubated under standard conditions.
  • GM-CSF granulocyte macrophage colony stimulating factor, 800 units / ml
  • IL-4 INTERLEYKIN-4, 1000 units / ml
  • culture media RPMI-1640, 10% inactivated cattle serum
  • TNF- ⁇ tumor necrosis factor- ⁇ , 10 ng / ml
  • antigens are added to DCs to mature and load their antigens obtained by cleavage with trypsin from the surface of living tumor cells, as described above (final concentration of antigens 100 ⁇ g / ml), and incubated for 2 days at 5% CO 2 and 37 ° C.
  • mature DCs are collected in a separate container and used as an antitumor dendritic cell vaccine, and their ability to specifically stimulate IP vitro cytotoxic lymphocytes is tested.
  • the load of dendritic cells is carried out by antigens obtained by trypsin cleavage from the surface of living endothelial cells, followed by the presentation on the surface of dendritic cells of a combination of antigens identical to the combination of surface antigens of target endothelial cells.
  • the primary culture of murine microcirculatory dermal ECs (ZAO BioBogemia, Russia) is used as living endothelial cells.
  • the growth medium is removed and the cup is washed 3-5 times with Hanks solution (at the rate of 2 ml per 5 cm 2 of the surface of the Petri dish) to remove traces of serum contained in the growth medium. After each addition of Hanks solution, the cells are kept in it for 2 minutes, after which it is removed.
  • Hanks solution at the rate of 2 ml per 5 cm 2 of the surface of the Petri dish
  • the Petri dish is covered and placed in an incubator for 10 min at 37 ° C, 5% CO 2 . Ensure that the incubator has at least 95% humidity to avoid drying and cell death.
  • a physiological solution is taken with cells detached from the bottom of the vial, centrifuged for 5 min at 20Og.
  • the cells settled by centrifugation are resuspended in freshly prepared growth medium and transferred to a Petri dish for further cultivation and reuse to obtain antigens.
  • the supernatant obtained by centrifugation is selected and filtered through a filter with a cut-off of 10-15 kDa protein to remove traces of trypsin and other proteins.
  • the obtained filtrate which is a solution of surface EC antigens, is used to load mature DCs obtained from the blood of healthy mice of the same breed, similar to that described in Example Na 1.
  • the functional activity of DCs loaded with surface antigens of tumor or EC is tested by their ability to induce ip vitro cytotoxic activity (CTA) of lymphocytes in relation to target cells, namely, MCF-7 tumor cells and murine dermal EC.
  • CTA ip vitro cytotoxic activity
  • Target + lymphocytes radioactivity of samples where target cells were incubated along with stimulated lymphocytes.
  • exosomes of MCF-7 cells were also obtained. In this case, the number of MCF-7 cells equivalent to the number of cells in the experiment was used. Exosomes were obtained by ultracentrifugation of the culture medium according to the method described in the application WO2004012685 ("Shad aptigep vassi desperate with despritzell adjuvapt", IPC A61K39 / 00, published on February 12, 2004).
  • lymphocytes were stimulated with DCs loaded with surface antigens obtained by trypsin cleavage from the surface of living tumor cells of MCF-7 (s).
  • DC A
  • DC loaded with exosomes of MCF-7 cells
  • ⁇ surface EC antigens
  • cytotoxic lymphocytes obtained after co-cultivation with dendritic cells loaded with antigens obtained by trypsin cleavage from the surface of living MCF-7 tumor cells had higher cytotoxic activity against tumor cells compared to control lymphocytes stimulated by intact dendritic cells or dendritic cells, loaded with other antigens, including antigens obtained according to the closest prototype of the invention.
  • lymphocytes were stimulated with DCs loaded with antigens obtained by trypsin cleavage from the surface of living endothelial cells (m).
  • Intact DCs (A-) or DCs loaded with EC lysate (•) or surface antigens of MCF-7 (F) cells were used in the control. Shown are mean values +/- standard deviation.
  • EC lysate was obtained by abrupt triple freezing of cells in liquid nitrogen and subsequent thawing at 37 ° C in a water bath.
  • cytotoxic lymphocytes after co-cultivation with DC loaded with surface EC antigens had higher cytotoxic activity against EC (target cells) compared to control lymphocytes stimulated by intact dendritic cells or dendritic cells loaded with other surface antigens or EC lysate.
  • the antigen-presenting cells When antigen-presenting cells are loaded with antigens obtained by protease cleavage from the surface of living cells, the antigen-presenting cells are represented on the surface of the antigens associated with the main histocompatibility complex as identical to the surface antigens of the target cells. T-lymphocytes recognize the main histocompatibility complex and its associated antigens. As a result, the cytotoxic activity (CTA) of lymphocytes is manifested not only in relation to these antigens, but also in relation to target cells.
  • CTA cytotoxic activity
  • 0.1 ml of a solution loaded with antigens DK (approximately 100 thousand DK) was injected into the tail vein twice with an interval of three days.
  • the animals of the control group were injected with a solution of DCs not loaded with antigens.
  • the experimental mice were inoculated with a subcutaneous injection of 1 million hepatoma H22 tumor cells into the armpit. The size of the tumor and the day of death of the mice were recorded. The average life expectancy of animals immunized with the vaccine obtained by the patented method increased by 52%.
  • Tumor size measurements are shown in FIG. 3, which implies a slowdown in the growth of tumors in animals from the experimental group, which indicates a pronounced antitumor effect of the vaccine obtained by the patented method.
  • the patented method can be used to obtain an antitumor vaccine containing antigen-presenting cells, namely, DC, which can stimulate the cytotoxic activity of lymphocytes in order to selectively damage target cells of both ip vitro and ip vivo.
  • antigens that are peptides with a molecular weight that determines their ability to be presented on the surface of antigen-presenting cells in a form suitable for full perception by the cells of the immune system, as evidenced by an increase in the CTA of lymphocytes.
  • the loading of antigen-presenting cells with antigens obtained by protease cleavage from the surface of living cells allows one to obtain, by single or multiple cleavage, a given set of antigens specific for target cells in an amount optimal for presenting the given set to cells of the immune system.
  • the applied soft (vital for cells) treatment of cells with a protease allows one to obtain antigens from living cells, i.e. with cells with retained barrier function of the cytoplasmic membrane ("wall" of the cell). This circumstance allows the protease to act only on the surface of the cells and, accordingly, only surface antigens are cleaved.
  • the preservation of the cytoplasmic membrane also prevents the intracellular contents from being able to contaminate the resulting antigens with non-target intracellular antigens and other ballast substances (proteins, lipids, nucleic acids, etc.), which makes it possible to present only antigens that are specific for cells to cells of the immune system - targets, excluding the presence on the surface of antigen-presenting cells of other antigens that are not specific.
  • antigen-presenting cells when antigen-presenting cells are loaded with antigens obtained by protease cleavage from the surface of living cells (with the barrier function of the cytoplasmic membrane preserved), the antigens presenting the antigens associated with the main histocompatibility complex identical to the surface cell antigens of the target cells are facilitated. full presentation to T-lymphocytes exclusively of antigens that are specific for cells target current, which increases the cytotoxic activity (CTA) lymphocytes against target cells.
  • CTA cytotoxic activity

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Abstract

L’invention concerne la biotechnologie, la pharmaceutique et la médecine et peut s’utiliser dans la fabrication de médicaments et notamment pour la mise en oeuvre de technologies médicales, notamment de la thérapie immunitaire de maladies cancéreuses. Le procédé de fabrication d’un vaccin antitumoral sur la base de cellules présentatrices d’antigènes, qui sont chargées d’antigènes, consiste à charger les cellules présentatrices d’antigènes, le chargement étant effectué à l’aide d’antigènes obtenus par séparation au moyen d’une protéase depuis la surface de cellules vivantes, la présentation étant assurée à la surface de l’ensemble de cellules présentatrices d’antigènes identique à l’ensemble des antigènes de surface des cellules cibles. L’invention permet de créer un vaccin capable d’augmenter l’activité cytotoxique (CTA) des lymphocytes vis-à-vis des cellules cibles grâce à la réception facilitées par celles-ci d’une séquence prédéterminée d’antigènes.
PCT/RU2009/000392 2008-08-26 2009-08-05 Procédé de fabrication d’un vaccin antitumoral WO2010024719A1 (fr)

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EA200801751A EA200801751A1 (ru) 2008-08-26 2008-08-26 Способ получения противоопухолевой вакцины
EA200801751 2008-08-26

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