WO2021027057A1 - Vaccin à lymphocytes b contre l'antigène du virus d'epstein-barr (veb) et son procédé de préparation - Google Patents

Vaccin à lymphocytes b contre l'antigène du virus d'epstein-barr (veb) et son procédé de préparation Download PDF

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WO2021027057A1
WO2021027057A1 PCT/CN2019/111889 CN2019111889W WO2021027057A1 WO 2021027057 A1 WO2021027057 A1 WO 2021027057A1 CN 2019111889 W CN2019111889 W CN 2019111889W WO 2021027057 A1 WO2021027057 A1 WO 2021027057A1
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cell
cells
vaccine
preparation
cell composition
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杨寒朔
魏于全
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成都冕康生物科技有限公司
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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
    • A61K39/46—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
    • 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
    • A61K39/4612—B-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
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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 A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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 A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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 A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a B cell vaccine against EBV virus and a preparation method thereof.
  • Epstein-Barr virus is widely infected in the Chinese population. According to serological surveys, the positive rate of EB virus VCA-lgG antibody in children aged 3 to 5 years in China is over 90%. Most infants have no obvious symptoms after infection, or only cause mild symptoms.
  • EBV is closely related to a variety of malignancies, including nasopharyngeal carcinoma, a variety of lymphomas (Hodgkin's lymphoma (HD), non-Hodgkin's lymphoma (NHD), Burkitt's lymphoma (African childhood lymphoma) , Diffuse large B-cell lymphoma and lymphoma after organ transplantation, etc.), midline malignant reticulum (malignant reticulocytosis), infectious mononucleosis, and a small number of gastric cancers. It is estimated that there are hundreds of thousands of new EBV-positive tumor patients in China.
  • Nasopharyngeal carcinoma is a malignant tumor that occurs on the top and side walls of the nasopharyngeal cavity. It is one of the most common malignant tumors in China. There are about 100,000 patients, and the incidence is the highest among malignant tumors of the ear, nose and throat. The 5-year survival rate after radiotherapy for nasopharyngeal carcinoma stage I to stage II nasopharyngeal carcinoma is more than 60%, and stage III to stage IV is only 20-40%. Among the patients currently treated in the first course of treatment in various cancer centers in China, stage III to IV Accounted for 70 to 80%.
  • Lymphoma is a malignant tumor that originates from the lymphoid hematopoietic system and is the most common hematological tumor in the world.
  • lymphoid hematopoietic system In 2012, there were approximately 385,700 new cases of non-Hodgkin’s lymphoma and approximately 197,700 deaths worldwide.
  • the incidence of lymphoma in China has been on the rise in recent years, and it currently ranks 8th among all types of cancer.
  • immunotherapy has significantly improved the therapeutic effect of lymphoma.
  • CHOP chemotherapy combined with rituximab in the treatment of diffuse large B-cell lymphoma (DLBCL) the median overall survival time of patients reached 4.9 years, and 5 years without The survival rate of disease progression increased from 30% to 54%.
  • DLBCL diffuse large B-cell lymphoma
  • FDA Food and Drug Administration
  • Therapeutic tumor vaccine is a kind of immunotherapy strategy that has attracted much attention. It can achieve the purpose of treating tumors by activating the immune response in patients.
  • Therapeutic tumor vaccines based on dendritic cells (DC) are currently the most studied tumor vaccines.
  • DC dendritic cells
  • Past basic research and development and clinical practice have proved that vaccines prepared using antigen-presenting cells loaded with tumor antigens can produce objective clinical therapeutic effects.
  • Provenge the world's first anti-tumor cell vaccine, was approved by the US FDA in 2010 for the treatment of prostate cancer.
  • one of the objectives of the present invention is to provide a B cell composition.
  • a B cell composition comprising: B cells and EBV virus antigens; the B cell composition is irradiated with ionizing radiation below the inactivation threshold.
  • EBV virus antigen is presented to the surface of the B cell membrane, binds to the HLA molecule, and exists on the surface of the B cell membrane in the form of an antigen peptide-HLA complex.
  • the B cells have biological activity but do not proliferate after being irradiated with ionizing rays.
  • the EBV virus is inactivated after being irradiated with ionizing rays.
  • the B cell does not carry live virus.
  • the ionizing rays are one or more of X-rays, ⁇ -rays, and Co 60 isotopes.
  • the ionizing radiation dose is about 10-200 Gy.
  • the ionizing radiation dose is approximately: 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy, 40Gy, 45Gy, 50Gy, 55Gy, 60Gy, 65Gy, 70Gy, 75Gy, 80Gy, 85Gy, 90Gy, 95Gy, 100Gy, 105Gy, 110Gy, 115Gy, 120Gy, 125Gy, 130Gy, 135Gy, 140Gy, 145Gy, 150Gy, 155Gy, 160Gy, 165Gy, 170Gy, 175Gy, 180Gy, 185Gy, 190Gy, 195Gy, 200Gy.
  • the irradiation dose rate of the ionizing rays is about 3-12 Gy/min; the irradiation time is about 200-600s.
  • the ionizing radiation dose rate is approximately 3Gy/min, 3.5Gy/min, 4Gy/min, 4.5Gy/min, 5Gy/min, 5.5Gy/min, 6Gy/min, 6.5Gy/min, 7Gy/min.
  • radiation time is approximately It is 200s, 225s, 250s, 275s, 300s, 325s, 350s, 375s, 400s, 425s, 450s, 475s, 500s, 525s, 550s, 575s or 600s.
  • the B cells have undergone immortalization treatment.
  • the immortalization treatment is to infect the B cells with EBV virus.
  • the B cell is a diploid cell.
  • the B cell undergoes expansion.
  • the B cells have undergone EBV infection treatment.
  • the B cell composition is cultured under serum-free conditions before being irradiated by the ionizing radiation.
  • the B cell composition is cultured at 37°C and 5% CO 2 .
  • the B cell composition also includes T cells.
  • the purpose of the present invention is also to provide a B cell vaccine.
  • a B cell vaccine comprising the above B cell composition.
  • the B cell vaccine also contains a coupling agent and/or an immune adjuvant.
  • the coupling agent in the present invention refers to a general medical sterile coupling agent, which is usually composed of carbomer, glycerin, purified water and other ingredients.
  • the immune adjuvant described in the present invention refers to an auxiliary species that can be injected into the body together or in advance to enhance the body's immune response ability to the antigen or change the type of immune response.
  • Currently commonly used vaccine adjuvants can be divided into aluminum salt adjuvants, protein adjuvants, nucleic acid adjuvants, lipid-containing adjuvants and mixed adjuvants according to their chemical composition.
  • the dosage form of the B cell vaccine is injection.
  • the dosage form of the B cell vaccine is subcutaneous injection, intradermal injection, local injection, intraperitoneal injection or intravenous injection.
  • the B cell vaccine is an allogeneic B cell vaccine.
  • the present invention can also be prepared as an autologous B cell vaccine.
  • the tumor medicine is used to treat tumors carrying EBV virus.
  • the tumor is related to EBV infection.
  • EBV DNA and antigen can be detected in the cancer tissue of patients with nasopharyngeal carcinoma, and EBV antibodies can be detected in their serum.
  • the tumors carrying the EBV virus include nasopharyngeal carcinoma, various lymphomas (Hodgkin's lymphoma (HD), non-Hodgkin's lymphoma (NHD), Burkitt's lymphoma (African childhood lymphoma), diffuse One or more of large B-cell lymphoma and lymphoma after organ transplantation), midline malignant reticulum (malignant reticulocytosis), infectious mononucleosis, and gastric cancer.
  • various lymphomas Hodgkin's lymphoma (HD), non-Hodgkin's lymphoma (NHD), Burkitt's lymphoma (African childhood lymphoma), diffuse One or more of large B-cell lymphoma and lymphoma after organ transplantation
  • midline malignant reticulum malignant reticulocytosis
  • infectious mononucleosis and gastric cancer.
  • the purpose of the present invention is also to provide a method for preparing a B cell vaccine.
  • a method for preparing a B cell vaccine includes the following steps: 1) culturing and expanding the number of B cells that carry EBV antigen; 2) irradiating the B cells obtained in step 1) with a certain dose of ionizing radiation .
  • the culture conditions of the step 1) are: 37°C, 5% CO 2 , and serum-free.
  • the seeding density of B cells in step 1) is 0.5-1 ⁇ 10 5-8 cells/ml.
  • the seeding density of B cells in step 1) is 0.5-1 ⁇ 10 5-6 cells/ml.
  • the number of generations of the number of expanded B cells in step 1) is greater than 5 generations.
  • step 1) the number of generations for the number of expanded B cells is greater than 100 generations.
  • the ionizing radiation in step 2) is one or more of X-ray, ⁇ -ray, and Co 60 isotope.
  • the irradiation dose of ionizing radiation in step 2) is about 10-200 Gy.
  • the ionizing radiation dose is approximately: 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy, 40Gy, 45Gy, 50Gy, 55Gy, 60Gy, 65Gy, 70Gy, 75Gy, 80Gy, 85Gy, 90Gy, 95Gy, 100Gy, 105Gy, 110Gy, 115Gy, 120Gy, 125Gy, 130Gy, 135Gy, 140Gy, 145Gy, 150Gy, 155Gy, 160Gy, 165Gy, 170Gy, 175Gy, 180Gy, 185Gy, 190Gy, 195Gy, 200Gy.
  • the irradiation dose rate of the ionizing rays is about 2-12 Gy/min; and the irradiation time is about 200-600s.
  • the ionizing radiation dose rate is approximately 3Gy/min, 3.5Gy/min, 4Gy/min, 4.5Gy/min, 5Gy/min, 5.5Gy/min, 6Gy/min, 6.5Gy/min, 7Gy/min.
  • radiation time is approximately It is 200s, 225s, 250s, 275s, 300s, 325s, 350s, 375s, 400s, 425s, 450s, 475s, 500s, 525s, 550s, 575s or 600s.
  • the B cells have been immortalized before the step 1).
  • the B cell is a diploid cell.
  • the B cells used for the treatment in step 1) are peripheral blood lymphocytes taken from healthy people.
  • the B cells have undergone EBV infection treatment before step 1).
  • the B cell vaccine prepared by the above preparation method.
  • the B cell vaccine also contains a coupling agent and/or an immune adjuvant.
  • the dosage form of the B cell vaccine is subcutaneous injection, intradermal injection, local injection, intraperitoneal injection or intravenous injection.
  • the B cell vaccine can activate immune cells in the human body.
  • the immune cells include T cells
  • the B cell vaccine can specifically recognize EBV virus antigen.
  • the tumor medicine is used to treat tumors carrying EBV virus.
  • the tumors carrying the EBV virus include nasopharyngeal carcinoma, various lymphomas (Hodgkin's lymphoma (HD), non-Hodgkin's lymphoma (NHD), Burkitt's lymphoma (African childhood lymphoma), diffuse One or more of large B-cell lymphoma and lymphoma after organ transplantation), midline malignant reticulum (malignant reticulocytosis), infectious mononucleosis, and gastric cancer.
  • various lymphomas Hodgkin's lymphoma (HD), non-Hodgkin's lymphoma (NHD), Burkitt's lymphoma (African childhood lymphoma), diffuse One or more of large B-cell lymphoma and lymphoma after organ transplantation
  • midline malignant reticulum malignant reticulocytosis
  • infectious mononucleosis and gastric cancer.
  • the purpose of the present invention is also to provide a method for activating T cells in human body with the above-mentioned B cell vaccine.
  • the purpose of the present invention is also to provide a method for treating tumors carrying EBV virus by using the above-mentioned B cell vaccine.
  • the purpose of the present invention is also to provide a method and system for improving B cell antigen presentation.
  • a method for improving the presentation of B cell antigens includes the following steps: (1) culturing and expanding the number of B cells that carry the EBV antigen; (2) irradiating with a certain dose of ionizing radiation after step (1) B cells obtained.
  • the B cells have been immortalized before the step (1).
  • the B cells have biological activity but do not proliferate after being irradiated with ionizing rays.
  • a system for improving the presentation of B cell antigens includes: a cell culture amplification device for amplifying B cells carrying EBV antigen; and an irradiation device for irradiating the B cells with ionizing rays lower than an inactivation threshold.
  • system further includes: a freezing device for freezing and storing the B cells irradiated with ionizing rays.
  • the purpose of the present invention is also to provide a B cell composition, which is characterized by comprising: B cells and viral antigens; the B cell composition is subjected to a certain dose of ionizing radiation irradiation treatment.
  • the viral antigen is derived from a human susceptible virus.
  • the human susceptible virus includes one or more of herpes virus, HIV virus and hepatitis virus.
  • the B cell composition has a specific antiviral effect.
  • the present invention proposes a new B cell composition and a tumor vaccine solution based on the B cell composition, namely, a B cell vaccine.
  • the vaccine has the following characteristics or advantages:
  • B cells are also an important antigen presenting cell.
  • the anti-tumor vaccine of the present invention uses B cells as antigen presenting cells to present tumor characteristic antigens or tumor-related antigens. Using B cells to prepare anti-tumor vaccines is a new type of anti-tumor vaccine preparation strategy, which has good innovation and broad application prospects.
  • Antigen specificity The immune response activated by the anti-tumor vaccine of the present invention specifically recognizes the EBV virus antigen.
  • the EBV virus antigen only exists on tumor cells and does not exist in normal cells. Therefore, the vaccine has good specificity and corresponding safety. Very high. Similarly, if the vaccine presents other tumor-specific antigens, it will also produce immune responses that target other tumor types.
  • B cells have a clear antigen presentation ability and can efficiently present EBV antigen.
  • B cells need to be infected with EBV virus to achieve immortality.
  • B cells that can be continuously expanded have been infected with EBV virus and can present EBV antigen, so 100% of vaccine cells (immortalized) produced by the present invention can present EBV antigen, which is much higher Provenge (sipuleucel-T), which was approved for marketing by the FDA.
  • the B cells used to prepare the vaccine of the present invention are derived from peripheral blood lymphocytes with normal nucleus (diploid) and no tumorigenicity;
  • the vaccine of the present invention is immunized by subcutaneous or intradermal localization Immunization, the risk of causing a serious systemic non-anti-tumor immune response is low;
  • B cells are irradiated and retain their biological activity, but lose their ability to proliferate. At the same time, the EBV virus also was inactivated.
  • the uniqueness of the production process the B cells used to prepare the vaccine of the present invention can be expanded in a large amount in vitro before being irradiated, which can realize standardized and large-scale preparation, and ensure the quality and standard of the vaccine for clinical trials.
  • the present invention provides a new type of EBV vaccine prepared by using normal karyotype B cells.
  • the vaccine of the present invention is irradiated B cells, most of which are live cells (that is, maintain activity), hardly proliferate, can stimulate an immune response against tumor cells, have no tumorigenicity, and have high safety.
  • the B cells used in the present invention can be expanded in vitro before irradiation, and can effectively activate anti-tumor immune responses in patients after irradiation, the preparation process can be standardized and scaled, making it easier to ensure clinical trials The quality and standards of vaccines used.
  • Figure 1B Microscopic view of normal cell culture
  • Figure 6 Live cell statistics after irradiation (abscissa: time; ordinate: cell number);
  • Figure 7 Total cell statistics after irradiation (abscissa: time; ordinate: cell number);
  • Figure 8 A statistical graph of vaccine anti-tumor effects (abscissa: time; ordinate: tumor size);
  • Figure 10 shows that the APC cross-presentation-related molecules of the vaccine cells undergo significant changes after irradiation
  • Figure 11 shows that the endogenous presentation-related molecules of vaccine cells undergo significant changes after irradiation
  • Figure 12 shows that the exogenous presentation-related molecules of vaccine cells undergo significant changes after irradiation
  • Figure 13 shows that vaccine cells prepared based on allogeneic B cells activate the immune system more efficiently.
  • the term “approximately” is typically expressed as +/-5% of the stated value, more typically of the stated value +/-4%, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even More typical is +/-0.5% of the stated value.
  • the immortalization of cells in the present invention refers to the use of genetic changes or various external stimulating factors in the process of cell culture in vitro to avoid the aging and death process of normal cells, thereby achieving long-term subculture and unlimited division and proliferation.
  • the current methods of cell immortalization include the use of radioactive factors, telomere-telomerase activation, and viral gene transfection.
  • the allogeneic B cells in the present invention refer to B cells derived from different individuals of humans or animals of the same species.
  • the B cells of the present invention are B cells derived from peripheral blood of different healthy people.
  • the experimental results of the present invention show that allogeneic B cells from different people have the same safety and anti-tumor immune response as the B cell vaccines from the patient's own body, but they have more ways to obtain and are more convenient to prepare.
  • the vaccine is the irradiated B cell, most of which are living cells, which hardly proliferate, but can stimulate the anti-tumor cell immune response.
  • vaccine cells are immortalized diploid B cells under serum-free culture conditions (normally 20% serum culture), which can be expanded in large quantities and can present antigens such as EBV.
  • the B cells used to make the vaccine of the invention can carry EBV antigens in a variety of ways: (i) B cells are the natural host of the EBV virus, allowing the B cells to contact the EBV virus and be infected by the EBV virus. At the same time, infection with the EBV virus can also allow B cells realize immortality; (ii) B cells can also express specific EBV antigens through genetic engineering.
  • B cells were cultured at 37°C, 5% CO 2 , cell seeding density 0.5-1 ⁇ 10 6 cells/ml, and serum-free. B cells mainly grew in suspension and clusters, and a small part of them were scattered individually. Exist, antennae-like protrusion cells can be seen on the edge of B cells. See Figure 1 and Figure 2 for details.
  • the B cells are cultured according to the above-mentioned optimized culture conditions provided by the present invention. After culture, the B cells grow in a more obvious clonal shape, and can be expanded by more than 100 times in two weeks. See Figure 3 and Figure 4 for details. among them. Figure 4 is a statistical diagram of optimized culture of B cells. After 16 days of culture, the number of cells has increased significantly. See Table 1 for details.
  • the culture conditions of B cells before irradiation can also contain a certain amount of serum.
  • the amplification factor of B cells can also be adjusted. For example, if the amplification factor is more than 40 times, subsequent irradiation treatment can be performed.
  • the B cell karyotype analysis used to make the vaccine of the present invention Take the cultured B cells (expansion>40 generations, preferential expansion>100 generations) to prepare observation slides on the slides, trypsinization treatment for 2-3 minutes, Rinse with 0.9% normal saline to stop the action of pancreatin, and then stain with Giemsa staining solution for 10-15 minutes. After washing and drying the slides, perform karyotype analysis under a microscope to observe whether the morphology is abnormal.
  • the analyzed B cell is a normal diploid karyotype, and the karyotype of the submitted cell is 46,XY. See Figure 5 for details.
  • B cells with a karyotype of 46,XX also have similar immune activation capabilities and can also be used to make vaccine cells of the present invention.
  • Vaccine cell virus detection of the present invention Use cell DNA extraction kit to extract B cell DNA or RNA, use real-time fluorescent quantitative PCR to quantify hepatitis B virus (HBV-DNA), hepatitis C virus (HCV-RNA), human Papillomavirus (HPV16/18-DNA) qualitative, human papillomavirus (HPV6/11-DNA) qualitative, parvovirus (B19-DNA) qualitative, cytomegalovirus (CMV-DNA) quantification, and Epstein-Barr virus (EB DNA) Quantitative testing.
  • HBV-DNA hepatitis B virus
  • HCV-RNA hepatitis C virus
  • HPV16/18-DNA human Papillomavirus
  • HPV6/11-DNA human papillomavirus
  • parvovirus B19-DNA
  • CMV-DNA cytomegalovirus
  • EB DNA Epstein-Barr virus
  • the irradiation conditions are as follows:
  • Irradiation dose Irradiate at about 10-200Gy.
  • the radiation dose can be selected from about 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy, 40Gy, 45Gy, 50Gy, 55Gy, 60Gy, 65Gy, 70Gy, 75Gy, 80Gy, 85Gy, 90Gy, 95Gy, 100Gy, 105Gy, 110Gy , 115Gy, 120Gy, 125Gy, 130Gy, 135Gy, 140Gy, 145Gy, 150Gy, 155Gy, 160Gy, 165Gy, 170Gy, 175Gy, 180Gy, 185Gy, 190Gy, 195Gy or 200Gy.
  • Irradiation dose rate about 3-12Gy/min. Specifically, the irradiation dose rate is approximately 3Gy/min, 3.5Gy/min, 4Gy/min, 4.5Gy/min, 5Gy/min, 5.5Gy/min, 6Gy/min, 6.5Gy/min, 7Gy/min, 7.5 Gy/min, 8Gy/min, 8.5Gy/min, 9Gy/min, 9.5Gy/min, 10Gy/min, 10.5Gy/min, 11Gy/min, 11.5Gy/min or 12Gy/min.
  • Radiation time about 200-600s. Specifically, the radiation time is approximately 200s, 225s, 250s, 275s, 300s, 325s, 350s, 375s, 400s, 425s, 450s, 475s, 500s, 525s, 550s, 575s or 600s.
  • Radiation radiation energy 160KV (kilovoltage); radiation radiation current intensity: 25mA.
  • B cells After irradiation, B cells have a normal morphology, and live cells account for about 80%. The number of live cells and total cell numbers remain unchanged for 4-5 days, indicating that B cells have no obvious proliferation ability after irradiation. See Figure 6 and Figure 7 for details. Among them, Figure 6 is a statistical diagram of live cells after irradiation, and the specific values are shown in Table 2. Figure 7 is a statistical diagram of total cells after irradiation, and the specific values are shown in Table 3.
  • the B cells used to make the vaccine should be irradiated at least once under the above conditions before cryopreservation.
  • cryopreserving use the freezing solution (BioLife Sulution, USA) to freeze the vaccine cells (i.e. cells that have been irradiated) at 1 ⁇ 10 7 cells/ml.
  • Vaccine cell tumorigenicity test 6-8 weeks old, BALB/c nude mice, female, 10 were used in the experiment, kept in a standard SPF animal room, the room temperature was maintained at 25 °C, free eating and drinking during the experiment. The irradiated vaccine cells were washed twice with PBS, and the cells were resuspended in serum-free medium to a final concentration of 108/ml. BALB/c nude mice were inoculated subcutaneously with 100ul of 1x107 vaccine cell suspension. After subcutaneous inoculation of vaccine cells, observe the survival of nude mice and whether there is tumor formation at the inoculation site every week for 9 weeks. No nude mice had subcutaneous tumor formation, indicating that the vaccine cells were not tumorigenic.
  • Anti-tumor effect Take well-grown human B cells carrying EBV antigen, irradiate them according to the above experimental parameters to make vaccine cells, and then resuspend the vaccine cells in serum-free medium to a final concentration of 5 ⁇ 10 7 /ml, respectively 4 weeks to multi-point injection vaccine 200ul cell suspension (1x10 7 cells) into BALB / c mice (6-8 weeks old, female) subcutaneous groin.
  • BALB/c mice that had been immunized three times were sacrificed, the mouse spleen was taken out after aseptic treatment, splenic lymphocytes were separated, and the lymphocytes were resuspended in RPMI-1640 medium at a concentration of 10 8 /ml.
  • the spleen lymphocyte suspension (containing 1.5x10 7 lymphocytes) C666-1 nasopharyngeal carcinoma cells inoculated by tail vein injection into (EBV +) tumor-bearing mice were treated three times. Measure the tumor every two days, calculate the tumor volume, and draw the tumor growth curve.
  • the vaccine cells of the present invention are injected into BALB/c mice with a sound immune system to activate T cells in BALB/c mice, and then the activated T cells are injected into tumor-bearing mice that have been inoculated with human tumors (immune system Flawed).
  • vaccine cells can be injected directly into the patient's body to activate T cells in the patient's body, thereby eliminating tumor/cancer cells carrying EBV virus antigens-in addition to nasopharyngeal carcinoma, it also includes: A variety of lymphomas (Hodgkin's lymphoma (HD), non-Hodgkin's lymphoma (NHD), Burkitt's lymphoma (African childhood lymphoma), diffuse large B-cell lymphoma, and lymphoma after organ transplantation, etc.), Midline retinal cell disease (malignant reticulocytosis), infectious mononucleosis, and gastric cancer.
  • HD Hodgkin's lymphoma
  • NHL non-Hodgkin's lymphoma
  • Burkitt's lymphoma Africann childhood lymphoma
  • diffuse large B-cell lymphoma diffuse large B-cell lymphoma
  • lymphoma after organ transplantation etc.
  • Midline retinal cell disease malignant
  • the above experiments based on mouse models show that although the vaccine cells of the present invention are derived from humans, they can still activate the immune system of mice-in other words, the vaccine cells of the present invention can play a role in heterogeneous organisms. In the allogeneic (all humans, but different individuals) should also be able to activate the immune system and produce effects.
  • Figure 13 further shows that vaccine cells prepared based on allogeneic B cells can not only activate T cells in allogenes, but also have better effects – in the experiment in Figure 13, the B cells used to prepare vaccine cells were taken from individual 01, and 01 After vaccine cells were co-cultured with 02, 03, and 04 peripheral blood mononuclear cells (PBMC) or lymphocytes (PBL), the number of activated lymphocytes was significantly higher than the number of activated lymphocytes in individual 01 .
  • Figure 13 shows the number of positive spots of vaccinated individuals in Table 5.
  • the above experimental results show that the ability of B cell antigen presentation is improved after irradiation. Therefore, not only can the present invention be used to efficiently present EBV virus antigens, but also human herpes virus, HIV virus, hepatitis virus and other viral antigens can be efficiently presented. Prepare the B cell-virus antigen composition to activate the immune response against the corresponding virus in the body and produce a specific antiviral effect.
  • B cells have the ability to present antigens. They can present EBV antigens to the cell surface, bind to HLA (Human Leukocyte Antigen) molecules, and exist on the cell membrane in the form of antigen peptide-HLA complexes.
  • HLA Human Leukocyte Antigen
  • B cells transformed by EBV virus can activate T cells in vitro, and train T cells to become CTL cells that can specifically recognize EBV antigens. After a large number of expansion and transfusion to tumor patients, they can produce significant anti-tumor effects. However, this process is cumbersome to operate, long in cycle, high in cost, and difficult in clinical application.
  • the CTL adoptive infusion therapy that targets EBV antigens in the prior art is in clinical trials: prepare T lymphocytes (LCL-CTL) that can recognize EBV antigens in vitro, and then inject LCL-CTL cells back to the patient Get treatment.
  • LCL-CTL T lymphocytes
  • the use of LCL-CTL has produced good therapeutic effects, achieving a 30-50% complete remission (Completely Recover, CR) for relapsed and refractory lymphoma.
  • the innovation of the present invention is to use the irradiated B cells as a vaccine to immunize patients and exert the antigen presentation ability of B cells in the human body, thereby activating the immune response in the body and producing anti-tumor effects-this avoids in vitro culture , Training and expansion of the long cycle of T cells.
  • the present invention can use individualized treatment (that is, use the patient's own B cells to make a vaccine), in the present invention, it is also technically feasible to use allogeneic B cells to make a vaccine to train T cells in the patient's body.
  • allogeneic B cells to make a vaccine to train T cells in the patient's body.
  • -Because T cells are trained in the body, the human body will naturally not reject the activated T cells in the body; and allogeneic B cells may be more conducive to activating the human immune system.
  • the effect of allogeneic B cells and T cell training is better, which will produce better anti-tumor effects. Therefore, the present invention can also be prepared into standardized cell lines, the therapeutic effect may be better, and large-scale production can be realized.

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Abstract

L'invention concerne un vaccin à lymphocytes B contre une pluralité de virus pouvant infecter l'être humain et son procédé de préparation. Le vaccin à lymphocytes B présente des effets anti-tumoraux et/ou anti-viraux et/ou thérapeutiques. L'invention concerne une composition de lymphocytes B, comprenant des lymphocytes B et des antigènes viraux, la composition de lymphocytes B étant soumise à une certaine dose de traitement d'irradiation par rayonnement ionisant. L'invention concerne également un vaccin à lymphocytes B, comprenant la composition de lymphocytes B. L'invention concerne en outre un procédé de préparation du vaccin à lymphocytes B et un système pour améliorer la présentation de l'antigène des lymphocytes B.
PCT/CN2019/111889 2019-08-15 2019-10-18 Vaccin à lymphocytes b contre l'antigène du virus d'epstein-barr (veb) et son procédé de préparation WO2021027057A1 (fr)

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WO2007063415A2 (fr) * 2005-08-23 2007-06-07 Iq Corporation Procedes de production de lymphocytes b stables
CN102165057A (zh) * 2008-05-16 2011-08-24 法国血液机构 用于主动或过继性细胞疗法的类浆细胞树突细胞系

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JPS61177984A (ja) * 1985-02-04 1986-08-09 Mitsubishi Chem Ind Ltd Ｂ細胞の培養法
US4804627A (en) * 1985-05-09 1989-02-14 Sloan-Kettering Institute For Cancer Research Method for cloning lymphoblastoid cells
CN1537161A (zh) * 2001-07-30 2004-10-13 ϣ��������ҩ��ҵ��˾ 抗原呈递细胞、其制备方法及其用于癌症疫苗的用途
WO2007063415A2 (fr) * 2005-08-23 2007-06-07 Iq Corporation Procedes de production de lymphocytes b stables
CN102165057A (zh) * 2008-05-16 2011-08-24 法国血液机构 用于主动或过继性细胞疗法的类浆细胞树突细胞系

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LIU, LUYAO ET AL.: "Research Progress on the Immune Mechanism Controlling EB Virus Infection", CHINESE JOURNAL OF EVIDENCE-BASED PEDIATRICS, vol. 12, no. 3, 30 June 2017 (2017-06-30), pages 219 - 232, XP009525986, DOI: 103969 / jissn1673-5501201703012 *
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