WO2014075631A1 - Procédé de préparation d'un vaccin contre une tumeur autologue et son utilisation - Google Patents

Procédé de préparation d'un vaccin contre une tumeur autologue et son utilisation Download PDF

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WO2014075631A1
WO2014075631A1 PCT/CN2013/087266 CN2013087266W WO2014075631A1 WO 2014075631 A1 WO2014075631 A1 WO 2014075631A1 CN 2013087266 W CN2013087266 W CN 2013087266W WO 2014075631 A1 WO2014075631 A1 WO 2014075631A1
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tumor
vaccine
tumor cells
cell
cell inactivation
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PCT/CN2013/087266
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English (en)
Chinese (zh)
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韩德平
林建华
张鲁榕
张美�
张震寰
张亨山
吕文龙
洪金省
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厦门鹭佳生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells

Definitions

  • the present invention is in the field of biotechnology and immunology, and in particular, the present invention relates to a method for preparing an autologous tumor vaccine and an application thereof. Background technique
  • Tumors are one of the leading causes of death in humans. Although the level of diagnosis and treatment of tumors has been continuously improved and improved in recent years, the programs of chemotherapy and radiotherapy are constantly improving, but in the end, most patients still have difficulty to escape the fate of death.
  • malignant tumors have multiple immune escape mechanisms, including low antigen content in tumor cells, destruction or constant mutation of tumor antigens, poor treatment of tumor antigens, or lack of expression of some immune cofactors, or low immune function. Etc., the clinical treatment effect of existing tumor tumor vaccines is not good. Because of the heterogeneity, diversity, and variability of malignant tumors, the individual tumor antigens of different individuals are very different when they are suffering from the same kind of tumor. Thus, tumor immunotherapy should be individualized to induce active immunity of individual-specific killing autologous tumor cells.
  • mice are usually immunized with inactivated tumor cells to protect the mice from inoculation of the inoculated tumor.
  • the vaccine loses the ability to protect the mouse.
  • the current clinical response to tumor cell vaccines is relatively poor, and it is only suitable for preventing recurrence of tumor patients without special tumor antigens. Very few good results have been obtained in clinical studies for advanced patients.
  • An ideal tumor-specific antigen should be immunogenic, expressed by tumor cells but not expressed in normal cells. Unfortunately, most tumor antigens do not have sufficient immunogenicity to induce an effective immune response.
  • Another object of the present invention is to provide an autologous tumor vaccine and use thereof.
  • a method of preparing an autologous tumor vaccine comprising the steps of:
  • step (1) treating the autologous tumor cells of step (1) by radiotherapy and/or heating to obtain tumor cells having enhanced antigenicity and antigen-promoting properties;
  • the inactivated tumor cells obtained in the step (3) were prepared as an autologous tumor vaccine.
  • the tumor is: a solid tumor or a non-solid tumor.
  • the solid tumor is derived from: brain, lung, head and neck, skin, pancreas, gastrointestinal tract, bladder, genital tract, spinal cord, spleen, kidney, liver, limbs, bones, tongue, Or throat.
  • the non-solid tumor is derived from blood or bone marrow.
  • the tumor is a primary tumor or a secondary metastatic tumor.
  • the tumor is derived from a tissue of a non-human mammalian tissue or a human.
  • the method for obtaining the isolated autologous tumor cells according to the step (1) is selected from the group consisting of: enzymatic digestion, mechanical shearing, or artificial grinding of tumor tissue to treat tumor tissue to be culturable. Isolated autologous tumor cells.
  • the radiation method described in the step (2) is selected from the group consisting of: selected from ⁇ , ⁇ , ⁇ , proton, or heavy ion radiation, optionally selected from the range of 0.1-50 Gy, optionally
  • the autologous tumor cells of step (1) were treated at a dose rate of 0.1-10 Gy/min to obtain antigenic and antigen-presenting tumor cells.
  • the radiation is derived from a natural or artificial source of radiation.
  • heating method described in the step (2) is selected from the group consisting of: heating temperature is selected from the group consisting of
  • the heating time is selected from 1 min to 2 hours (preferably 30 min - l hour).
  • the method further comprises the step of: culturing the tumor cells obtained by the step (2) to obtain antigenicity and antigen-promoting enhancement.
  • the culture treatment time is selected from the group consisting of: 6 hours to 2 weeks.
  • a basic cell culture solution is used, and/or a growth factor or a small molecule compound is added.
  • the inactivation treatment described in the step (3) is selected from the group consisting of microwave cell inactivation, electromagnetic wave cell inactivation, laser cell inactivation, high temperature cell inactivation, sonication and cell disruption. Live method, repeated cell freeze-thaw, homogenate, centrifugation, pellet cell inactivation, enzymatic digestion cell inactivation, hypotonic rupture, or a combination thereof.
  • step (4) the inactivated tumor cells obtained in the step (3) are mixed with an adjuvant to obtain an autologous tumor vaccine.
  • the adjuvant is Freund's complete adjuvant or incomplete adjuvant.
  • the adjuvant is selected from the group consisting of liposomes, cytokines (eg, GM-CSF, G-CSF,
  • CSF CSF, IFN, IL-2, etc.
  • various plant proteins DNA, deactivated or inactivated viruses, bacteria, fungi, microorganisms and their extracts, or combinations thereof.
  • an autologous tumor vaccine prepared by the method of the first aspect.
  • the vaccine further comprises an adjuvant.
  • the autologous tumor vaccine of the second aspect which is used for the preparation of a composition for preventing or treating a tumor.
  • the composition is a pharmaceutical composition or a vaccine composition.
  • the composition is in the form of an injection.
  • the injection is administered by a route selected from the group consisting of intradermal single/multiple injection, subcutaneous single/multiple injection, intralymphatic or peripheral injection, intramuscular injection, intrathoracic/peritoneal injection, Intravenous, peritumoral normal tissue injection.
  • it is the distal end of the intradermal limb.
  • the injection site of the injection is selected from the group consisting of: primary tumor, metastases, lymphatic drainage area around the primary tumor, lymphatic drainage area around the metastasis, distal extremities, proximal extremities, chest Back, abdomen, neck, locally accessible lymph nodes or lymphatic drainage areas.
  • the vaccine can be used in combination with a drug selected from the group consisting of cytokines, electromagnetic wave therapy, or a combination thereof.
  • the drug is selected from the group consisting of IFN, TNF, IL-2, or a combination thereof.
  • the inactivation treatment described in the step (3) is selected from the group consisting of microwave cell inactivation, electromagnetic wave cell inactivation, laser cell inactivation, high temperature cell inactivation, sonication and cell disruption. Live method, repeated cell freeze-thaw, homogenate, centrifugation, pellet cell inactivation, enzymatic digestion, cell inactivation, hypotonic rupture, chemical covalent Combined method, chemical denaturing method, or a combination thereof.
  • a method of preventing or treating a tumor comprising administering an effective amount of the tumor vaccine of the second aspect of the invention to a subject is provided.
  • the administration time is selected from the group consisting of: postoperative, pre-chemotherapy, or WBC rebound to >3000 per ul.
  • the number of administrations may be 4 courses (3-4 weeks per course) to life.
  • the method further comprises: monitoring the extent of the immune response using an immunological assay.
  • the immunological assay is selected from the group consisting of a cellular immune response, a humoral immune response, an immunomodulatory response, or a combination thereof.
  • the method for detecting cellular immune response comprises:
  • the anti-tumor delayed allergic reaction can be seen as a cumulus 0.5 cm or more for a good immune response, ⁇ 0.5 cm for poor immune response;
  • Lymphocyte transformation reaction The lymphocytes of the patient one month after immunization are separated, and the vaccine of the second aspect of the invention is added to observe the conversion rate (preferably by BrdU method, MTT method), and the conversion rate is high. Good immune response;
  • the lymphokine is selected from the group consisting of IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IL-18, TNF-a, IFN- ⁇ , Or a combination thereof;
  • the detection method is selected from the group consisting of ELISPOT, ELISA, FCM, or a combination thereof.
  • the subset of immune cells is selected from the group consisting of CD4+, CD8+, CD1 lb+, Grl+, or a combination thereof;
  • the killer activation marker is selected from the group consisting of Fas L, LIGHT, CD25, TNF-a, IFN- ⁇ , perforin or a combination thereof.
  • each subpopulation is detected by flow cytometry.
  • the humoral immunoassay method comprises:
  • the prepared tumor vaccine is used as an antigen coating to add plasma before and after autoimmunization, and the antibody titer against autoantitumor antigen is compared before and after immunization. The higher the titer, the stronger the immunoreactivity.
  • the molecular weight of the autoantigen is determined by the WESTERN BLOT method.
  • the immunomodulatory reaction detecting method comprises:
  • Detection of a subset of immunoregulatory cells comprises: regulatory T cells: Treg: CD4 + CD25 + , myeloid-derived suppressor cells MDSC: CD14 - CD11b + , tumor-associated macrophages TAM: Ml/M2, regulatory dendritic cell DCreg: CD11b+/Gr-l+, or a combination thereof.
  • the detection method is flow cytometry.
  • the immunoregulatory factors include: IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IL-18 , TNF-a, IFN- ⁇ , or a combination thereof;
  • the assay comprises: ELISA, LUMINEX bead-array assay, or a combination thereof.
  • the immunological assay is performed within 15-60 days after the first immunization.
  • the method further comprises: determining the therapeutic effect and/or adjusting the treatment regimen based on the degree of immune response measured by the immunological experiment.
  • the adjusted treatment regimen refers to adjusting one or more therapeutic parameters selected from the group consisting of: immunoadjuvant binding, immunization dose, immunization site, immunization time, number of immunizations, low dose radiation, other physics Or biological methods to stimulate immune organs.
  • the immune organ is bone marrow.
  • the method includes:
  • the administration of the vaccine and its effect observation time may vary flexibly due to individual differences. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS
  • Figure 1 shows the results of anti-tumor antibody IgG assay 3 weeks after injection of the auto-tumor vaccine.
  • Figure 2 shows an anti-tumor T cell killing experiment.
  • FIG. 3 shows the experimental grouping and the operational flow.
  • Figure 4 shows the results of real-time dynamic measurement of anti-tumor effect fluorescence (Live Image).
  • Fig. 5 shows the results of the vaccine antigenic cancer (4T1-Luc) effect (test 1) prepared by the method of Example 1 of the present invention.
  • Fig. 6 shows the results of the vaccine antigenic cancer (4T1-Luc) effect (test 2) prepared by the method of Example 1 of the present invention.
  • Figure 7 is a graph showing the results of a comparison between the vaccine prepared by the method of Example 1 of the present invention and the anticancer effect of Taxol (Trial 3, compared with the chemotherapy drug Taxol).
  • Figure 8 shows the killing inhibition effect of the method of one embodiment of the present invention on experimental lung metastasis.
  • FIG. 9 shows the flow of an autologous tumor vaccine prepared in accordance with one embodiment of the present invention.
  • Fig. 10 shows a tumor cell illuminator; wherein, the Y-ray is used to break the DNA and cause DNA error repair, and at the same time, the characteristics of the oncogenic gene which promotes growth in a harsh environment are highly expressed, and the tumor cells are treated with the Y illuminator of the exclusive invention.
  • the oncogene is highly expressed, and the activation of tumor antigen is enhanced to increase the efficacy of the method.
  • Figure 11 shows a bone marrow growth stimulator; wherein, using medium/high frequency electromagnetic waves to stimulate cell growth, the bone marrow growth stimulator is placed in the humerus of tumor-free cells to stimulate hematopoietic stem cell proliferation and differentiation, so that autoantibodies The number of cancer immune cells is increased to increase the efficacy of this method.
  • the present inventors have discovered for the first time that the treatment of radiation and/or heating of autologous tumor cells can greatly stimulate the antigenicity of autologous tumor cells, and heat shock proteins enhance antigen presentation;
  • the cells are inactivated and supplemented with adjuvant to make a vaccine.
  • the vaccine has the special advantages of strong presentation and strong immunogenicity of the tumor, and can strongly stimulate the humoral and cellular immune responses of the tumor patients to the autologous tumor, so as to improve the antigenic and metastatic tumor effect and prolong the survival of the tumor patient.
  • the purpose of time On this basis, the invention of a novel autologous tumor vaccine was completed. Its application is also based on monitoring the patient's response to autologous tumor vaccines, changing the amount of vaccine, the location and time of injection, the length and interval of treatment, and truly Active immunotherapy. Preparation
  • the present invention provides a method of preparing an autologous tumor vaccine, and in a preferred embodiment, comprising the steps of: (1) obtaining isolated autologous tumor cells;
  • step (1) treating the autologous tumor cells of step (1) by radiotherapy and/or heating to obtain tumor cells having enhanced antigenicity and antigen-promoting properties;
  • the inactivated tumor cells obtained in the step (3) were prepared as an autologous tumor vaccine.
  • the tumor cells as follows: Obtain the tumor-free, non-necrotic part of the tumor under sterile conditions, store in DMEM or other culture medium, store at 4 ° C, rinse with PBS, and digest by enzyme. Purified tumor cells were obtained by method or / and mechanical separation method, transferred into a culture flask, subcultured at 37 ° C, 5% CO 2 incubator, and reached the required number of cells to enter the tumor whole cell tumor preparation procedure;
  • the tumor cells in culture are treated by radiation and/or heating to excite the antigen, and the increased heat shock protein enhances antigen presentation.
  • the treated tumor cells are inactivated by the following methods: microwave cell inactivation method, electromagnetic wave cell inactivation method, laser cell inactivation method, high temperature cell inactivation method, ultrasonic disruption cell inactivation method, repeated cells At least one of freezing and thawing, homogenization, centrifugation, sedimentation cell inactivation, enzymatic digestion, cell inactivation, hypotonic rupture, and the like;
  • the invention is supplemented by the above tumor whole cell vaccine with an adjuvant
  • the adjuvant may be a complete Freund's adjuvant or an incomplete adjuvant, after a water-in-oil adjuvant, a liposome, a cytokine (such as GM-CSF, various Plant protein DNA), attenuating or inactivating an adjuvant such as a virus, a bacterium, a fungus, a microorganism or an extract thereof. After adjuvanting, it can better enhance the active immune response, reduce the negative regulation of immunity, and enhance the stimulation and memory of anti-tumor immune cells.
  • a cytokine such as GM-CSF, various Plant protein DNA
  • the method of the invention is to treat the tumor cells by antigenic stimulation treatment and enhance the antigen presenting ability, and then inactivate the treatment to lose the tumorigenicity, and retain the immunogenicity after the stimulation, and simultaneously combine the immunosupsis;
  • an individualized tumor vaccine we will establish an individualized treatment method and keep track of individual immunity. State and tumor conditions continue to adjust individualized immunotherapy programs to achieve optimal anti-tumor efficacy. Immune effect monitoring
  • One of ordinary skill in the art can determine the anti-tumor immune response of a vaccine using conventional methods. Including: anti-tumor humoral immune response evaluation, anti-tumor cell immune response evaluation, anti-tumor clinical response evaluation.
  • the anti-tumor humoral immune response was evaluated by using an individualized tumor vaccine as an antigen-coated ELISA method to detect the anti-autoantibody antibody titer in the serum of the individual before and after immunization of the individualized tumor vaccine to objectively judge the anti-tumor humoral immune response.
  • Anti-tumor cell immune response evaluation generally, including the new individualized tumor vaccine prepared by intradermal injection, observe the patient's own pimple size within 1-3 days (late-type individualized tumor vaccine immune response); or self-tumor stimulation specific Sexual lymphocyte transformation experiment: including steps: taking anticoagulation, separating lymphocytes, serum-free culture, adding individualized tumor vaccine or control vector, culturing, judging lymphocyte transformational proliferation level (BrdU incorporation method or MTT method); Individualized tumor vaccine immunomodulatory response.
  • Immunomodulatory responses to individualized tumor vaccines include: detection of leukocyte populations (eg, CD4/CD8/CD1 lb/Gr-l, etc.) and their surface killing or activating markers (eg, FasL/LIGHT/CD25/TNF-a/IFN-) y, etc., according to the results of these in vitro and in vivo experiments can be divided into two types: good response and poor response: high antibody titer, pyel is greater than 0.5 cm, so can maintain the original individualized immunization program; poor response antibody titer Low, pipi is less than 0.5 cm, so you can change the use of individualized tumor vaccine and use different means to enhance the immune response.
  • leukocyte populations eg, CD4/CD8/CD1 lb/Gr-l, etc.
  • their surface killing or activating markers eg, FasL/LIGHT/CD25/TNF-a/IFN-
  • the subset of immunoregulatory cells includes: regulatory T cell Treg (CD4+CD25+), myeloid-derived suppressor cell MDSC (CD14-CD1 lb+), tumor-associated macrophage TAM (M1/M2), regulatory dendritic
  • the cells were DCreg (CDl lb+/Gr-l+) and each subpopulation was detected by flow cytometry.
  • Anti-tumor clinical response According to the tumor image size evaluation, according to the experimental results, the patient's immune status, tumor condition and other anti-tumor treatment programs, decide when to strengthen anti-tumor immunity, adjust individualized treatment plan, continue 2, 3, 4 Or more treatments. Efficacy monitoring includes, but is not limited to, imaging criteria, tumor-free survival time, and overall survival time.
  • the immune response effect of individualized tumor vaccine can be monitored by cellular immunity, humoral immunity, and clinical tumor response.
  • the monitoring time can be about 15 days to 60 days after the first immunization, and the data can be evaluated if the reaction
  • individualized immunotherapy regimens can be re-adjusted, such as by adjusting immune adjuvant binding, immunization dose, immune site, immunization time, number of immunizations, and low-dose radiation-stimulated immune organs such as bone marrow.
  • the present invention also provides a composition, which may be a pharmaceutical composition or a vaccine composition.
  • the compositions of the invention may be therapeutic or prophylactic.
  • the compositions of the present invention comprise an effective amount of an autologous tumor vaccine of the invention, and at least one pharmaceutically acceptable carrier, diluent or excipient.
  • compositions of the present invention can be prepared into various conventional dosage forms including, but not limited to, injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.
  • compositions of the present invention comprise an effective amount of an autologous tumor vaccine prepared by the method of the present invention.
  • an effective amount refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. This effect can be detected by, for example, antigen level. Therapeutic effects also include a reduction in physiological symptoms.
  • the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given situation, routine experimentation can be used to determine the effective amount.
  • an effective dose is from about 0.2 micrograms per kilogram to 2 micrograms per kilogram of the subject.
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration to a therapeutic agent, such as a protein, VLP or other therapeutic agent.
  • the term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration.
  • Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid and the like. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • the pharmaceutically acceptable carrier in the composition may include liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the composition can be formulated as an injectable, such as a liquid solution or suspension; it can also be formulated to be suitable for dissolution prior to injection. Solid form of liquid or suspension, liquid excipient. Liposomes are also included in the definition of pharmaceutically acceptable carriers.
  • the vaccine compositions of the invention may be prophylactic or therapeutic.
  • the vaccine composition comprises an immunogenic antigen (autologous tumor vaccine of the invention) and is typically combined with a "pharmaceutically acceptable carrier" which includes antibodies which do not themselves induce the production of antibodies harmful to the individual receiving the composition.
  • a pharmaceutically acceptable carrier which includes antibodies which do not themselves induce the production of antibodies harmful to the individual receiving the composition.
  • Any carrier. Suitable carriers are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid agglutinates (e.g., oil droplets or liposomes), and the like. These vectors are well known to those of ordinary skill in the art.
  • these carriers can function as immunostimulating agents ("adjuvants").
  • the antigen can be coupled to bacterial toxoids (such as toxoids of pathogens such as diphtheria, tetanus, cholera, Helicobacter pylori) or bacterial extracts (such as endotoxin or flagellin).
  • bacterial toxoids such as toxoids of pathogens such as diphtheria, tetanus, cholera, Helicobacter pylori
  • bacterial extracts such as endotoxin or flagellin
  • Preferred adjuvants for enhancing the effect of the immunological composition include, but are not limited to: (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) oil-in-water emulsion formulations, for example, (a) MF59 (see WO 90/14837), (b) SAF, and (c) RibiTM Adjuvant System (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvant; (4 ⁇ 61111 ( 1 Complete adjuvant (?) and ?61111 ( 1 incomplete adjuvant (1?); (5) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc., interferon (such as gamma interferon), macrophage colony-stimulating factor (M-CFS), mononuclear-macrophage colony-stimulating factor (GM-CFS
  • Vaccine compositions including immunogenic compositions (e.g., can include antigens, pharmaceutically acceptable carriers, and adjuvants), typically contain a diluent such as water, saline, glycerol, ethanol, and the like.
  • a diluent such as water, saline, glycerol, ethanol, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present in such carriers.
  • vaccines including immunogenic compositions, comprise an immunologically effective amount of an immunogenic polypeptide, as well as other desired components as described above.
  • immunologically effective amount means that the amount administered to a subject in a single dose or in a continuous dose is effective for treatment or prevention. The amount may be based on the health and physiological condition of the individual being treated, the type of individual being treated (eg, human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical condition by the treating physician, And other relevant factors. This amount is expected to be in a relatively wide range and can be determined by routine experimentation.
  • the vaccine composition or immunogenic composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be formulated as a solid form suitable for solution or suspension, liquid excipient prior to injection.
  • the formulation may also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.
  • the composition can be administered directly to a subject.
  • the subject can be a human or a non-human mammal, preferably a human.
  • When used as a vaccine it can be directly administered to an individual by a known method.
  • These compositions are usually administered by the same route of administration as conventional vaccines.
  • Routes for administering a pharmaceutical composition or vaccine composition of the invention include, but are not limited to: Formulated pharmaceutical compositions can be administered by conventional routes, including but not limited to: such as intradermal single/multiple Point injection, subcutaneous single/multiple injection, intra- or peripheral lymphatic injection, intramuscular injection, intrathoracic/peritoneal injection, intravenous injection, peritumoral/intratumoral injection.
  • routes including but not limited to: such as intradermal single/multiple Point injection, subcutaneous single/multiple injection, intra- or peripheral lymphatic injection, intramuscular injection, intrathoracic/peritoneal injection, intravenous injection, peritumoral/intratumoral injection.
  • the above approaches may be applied separately depending on the specific situation, and may be applied in combination if necessary.
  • the patient's own site may be, but is not limited to, the primary tumor, metastases or surrounding lymphatic drainage areas and distal or proximal extremities, thoracodorsal, abdomen, neck, locally accessible lymph nodes or lymphatic drainage areas
  • dose control should be considered within a safe and effective range.
  • the specific dose should also take into account the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the vaccine of the present invention may also be used in combination with other drugs or physical therapies, including but not limited to: various cytokines such as IFN, TNF, IL-2, GM-CSF, G-CSF, SCF, And electromagnetic waves, etc.
  • various cytokines such as IFN, TNF, IL-2, GM-CSF, G-CSF, SCF, And electromagnetic waves, etc.
  • the main advantages of the present invention are as follows - (1) The present invention actively stimulates tumor antigen expression and antigen processing presentation function by irradiation and/or heating, and then inactivates, supplements with adjuvant, and modulates immunotherapy according to individual immune response. Program, to achieve the purpose of truly individualized tumor immunotherapy;
  • the present invention can promote immunity in the body, especially for cellular immunity and humoral immunity of tumors;
  • the technical method for treating or preventing tumors according to the present invention is suitable for use in the medical field, particularly for preventing or treating tumors, and is also suitable for postoperative tumor residuals and patients with micrometastases, and provides a field in the art.
  • the new choice has broad application prospects.
  • the invention is further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only. The invention is not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions.
  • Example 1 Example 1
  • the tumor vaccine prepared by the method of the present invention has strong antigen presentation ability and ability to elicit an antibody immune response.
  • the specific results are as follows:
  • mice Conventional Balb/c mice were immunized 3 weeks after immunization with the tumor cell antigen prepared in Example 1, and the plasma was diluted and added to the ELISA plate coated with tumor cells to anti-mouse IgG-HRP and TMB substrates. The antibody titer is shown (this experiment is 1:100000).
  • mice were immunized with the tumor cell antigen prepared in Example 1 for 3 weeks, and lymphocytes (LN) and spleen cells (SP) were taken and the constructed Luciferase-containing 4T1 tumor cells (4T1-luc) were 10:1.
  • LN lymphocytes
  • SP spleen cells
  • Fig. 2A is an antitumor T in the adjuvant group.
  • Cell killing experiment, Fig. 2 is the tumor cell antigen anti-tumor cell killing experiment prepared by the method group of Example 1, Rlu refers to the activity of Luciferase; NC refers to the normal control group; LN refers to lymph node cells; SP refers to spleen cells.
  • Example 3
  • mice anti-tumor immunity test is divided into two steps:
  • Tumor immunosensitization The treated tumor antigen novel tumor vaccine was first inoculated subcutaneously in the test mice, and 14 days later, the immunization was boosted once again, and the homologous mouse tumor cells were inoculated 7 days later (Fig. 3).
  • a. Inhibition of carcinoma in situ BALB/c mice and 4T1 breast cancer cell lines were used; b. Inhibition of metastatic tumors: C57BL/6 mice and Lewis lung cancer cell lines were used. Lewis and 4T1 cancer cells are highly malignant, grow fast, are difficult to control, and can form lung metastases.
  • a, living tumor fluorescence quantitative method the above 4T1 cells with Luciferase gene, so 4T1-Luc tumor size can be perfused into the anesthesia tumor-bearing mice peritoneal injection of Luciferin substrate, 5 minutes after the determination of fluorescence intensity; b, vernier caliper to determine the tumor diameter; Both methods can objectively reflect tumor size.
  • Anti-pulmonary metastasis effect Due to the rapid growth of experimental Lewis lung cancer, the cancer nodules are rapidly fused into a piece, and the number of nodules cannot be calculated. Therefore, the results are calculated by whole lung weighing.
  • mice immunized with the tumor antigen prepared in Example 1 were subjected to intravenous or subcutaneous injection of live tumor cells to form metastatic cancer and carcinoma in situ for the antitumor effect.
  • the tumor-injected Balb/c mouse tumor fluorescence imaging showed that compared with the control group (adjuvant immunization) and the original method of preparation of tumor vaccine (adjuvant + inactivated tumor cells) group,
  • the vaccine group prepared in Example 1 had a small number of tumors and a small volume.
  • the tumor growth curve (Fig. 5) showed that the growth rate of the in situ carcinoma of the new tumor vaccine group was significantly slower than that of the original tumor vaccine preparation group, the adjuvant group alone, and the saline group (p ⁇ 0.01). Similar results were obtained (Fig. 6, Fig. 7). It is worth noting that the fastest growing tumor of 4T1-Luc cells could not be inhibited by Taxol 5 mg/kg (daily injection) but was inhibited by the vaccine prepared by the method of the present invention.
  • Metastasis is the main cause of tumor death.
  • experimental lung metastasis of Lewis lung cancer cells on homologous C57BL/6 mice was used as a detection model.
  • A saline control group, subcutaneous injection of simulated saline immunization process
  • Taxol group (Paclitaxel group): Conventional chemotherapy drugs, as a positive control group.
  • mice Four groups of mice were injected with lx lO 5 live Lewis lung cancer cells 21 days after receiving different immunotherapy. After 23 days, the lung tumor weight of the mice was weighed to obtain the mean and standard deviation.

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Abstract

L'invention concerne un procédé de préparation d'un vaccin contre une tumeur autologue et ses utilisations, comprenant en particulier : l'irradiation et/ou le chauffage de cellules tumorales autologues pour obtenir les cellules tumorales ayant une antigénicité et une présentation d'antigène améliorées, la culture des cellules tumorales obtenues, la désactivation des cellules tumorales cultivées et l'ajout d'adjuvant pour préparer le vaccin antitumoral.
PCT/CN2013/087266 2012-11-15 2013-11-15 Procédé de préparation d'un vaccin contre une tumeur autologue et son utilisation WO2014075631A1 (fr)

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WO2021016261A3 (fr) * 2019-07-22 2021-04-08 Todd Alamin Traitements de cellules et méthodes thérapeutiques de reperfusion
WO2024069531A1 (fr) * 2022-09-30 2024-04-04 Novocure Gmbh Compositions, systèmes et procédés de traitement du cancer à l'aide de champs électriques alternatifs et de vaccination par cellules cancéreuses apoptotiques

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CN108175853A (zh) * 2018-01-24 2018-06-19 上海赛群生物科技有限公司 一种肿瘤细胞疫苗及其制备方法
CN109999187A (zh) * 2019-05-09 2019-07-12 英诺激光科技股份有限公司 一种利用激光制备癌细胞疫苗的方法
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