WO2021027057A1 - B cell vaccine against ebv virus antigen and preparation method therefor - Google Patents

Abstract

Provided are a B cell vaccine against a plurality of human susceptible viruses and a preparation method therefor. The B cell vaccine has anti-tumor and/or anti-virus preventive and/or therapeutic effects. Provided is a B cell composition, comprising B cells and viral antigens, the B cell composition being subjected to a certain dose of ionizing radiation irradiation treatment. Also provided is a B cell vaccine, comprising the B cell composition. Further provided are a preparation method for the B cell vaccine and a system for improving the presentation of B cell antigen.

Description

B cell vaccine against EBV virus antigen and preparation method thereof

This application claims the priority of the Chinese invention patent 201910752830.2 "A B-cell vaccine against EBV virus antigen and its preparation method" filed on August 15, 2019.

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a B cell vaccine against EBV virus and a preparation method thereof.

Background technique

Epstein-Barr virus (EBV) 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%. The incidence of gastric cancer ranks second in China, accounting for 42.6% and 45.0% of global gastric cancer incidence and death, and the five-year survival rate is only 25%-30%. Gastric cancer treatment is currently mainly surgery, chemotherapy, and radiotherapy. Trastuzumab has become the first-line drug for the treatment of gastric cancer for the treatment of HER2-positive gastric cancer patients.

Lymphoma is a malignant tumor that originates from the lymphoid hematopoietic system and is the most common hematological tumor in the world. 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. In recent years, immunotherapy has significantly improved the therapeutic effect of lymphoma. For example, 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%. In 2016, the U.S. Food and Drug Administration (FDA) approved the PD-1 antibody nivolumab for the treatment of patients with relapsed refractory Hodgkin's lymphoma (r/rHL), which is expected to further improve the therapeutic effect of lymphoma. However, whether it is rituximab or PD-1 monoclonal antibody, the price is very expensive, and the cost of treatment for patients is high. Clinically, there is an urgent need for more economical and effective treatment programs or drugs.

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. 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.

Research in recent years has shown that DC vaccines have shown good therapeutic effects on a variety of solid tumors, and allogeneic DC vaccines from healthy people have the same safety as patients’ autologous DC vaccines, and theoretically can stimulate better Anti-tumor immune response has received more and more attention and promotion. However, it is difficult for DC cells to be cultured and expanded in vitro. All current DC vaccine programs require individualized preparation. The operation process is complicated, the individualized preparation process is difficult to be standardized, and the quality control requirements are high.

Summary of the invention

In view of this, one of the objectives of the present invention is to provide a B cell composition.

In order to achieve the above objective, the technical solution of the present invention is:

A B cell composition comprising: B cells and EBV virus antigens; the B cell composition is irradiated with ionizing radiation below the inactivation threshold.

Further, the 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.

Further, the B cells have biological activity but do not proliferate after being irradiated with ionizing rays.

Further, the EBV virus is inactivated after being irradiated with ionizing rays.

Further, the B cell does not carry live virus.

Further, the ionizing rays are one or ^more of X-rays, γ-rays, and Co ⁶⁰ isotopes.

Furthermore, the ionizing radiation dose is about 10-200 Gy.

Further, 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.

Further, the irradiation dose rate of the ionizing rays is about 3-12 Gy/min; the irradiation time is about 200-600s.

Further, 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. min, 7.5Gy/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 is approximately It is 200s, 225s, 250s, 275s, 300s, 325s, 350s, 375s, 400s, 425s, 450s, 475s, 500s, 525s, 550s, 575s or 600s.

Further, the B cells have undergone immortalization treatment.

Further, the immortalization treatment is to infect the B cells with EBV virus.

Further, the B cell is a diploid cell.

Further, before the ionizing radiation is irradiated, the B cell undergoes expansion.

Further, the B cells have undergone EBV infection treatment.

Further, the B cell composition is cultured under serum-free conditions before being irradiated by the ionizing radiation.

Furthermore, the B cell composition is cultured at 37°C and 5% CO ₂ .

Further, there are antennae-like protrusions on the edges of the B cells.

Further, 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.

Further, 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.

Further, the dosage form of the B cell vaccine is injection.

Further, the dosage form of the B cell vaccine is subcutaneous injection, intradermal injection, local injection, intraperitoneal injection or intravenous injection.

Further, the B cell vaccine is an allogeneic B cell vaccine. Of course, the present invention can also be prepared as an autologous B cell vaccine.

The application of the above-mentioned B cell vaccine in the preparation of anti-tumor drugs.

Further, the tumor medicine is used to treat tumors carrying EBV virus.

Further, the tumor is related to EBV infection. Taking nasopharyngeal carcinoma as an example, studies have confirmed that 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.

Further, 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.

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 .

Further, the culture conditions of the step 1) are: 37°C, 5% CO ₂ , and serum-free.

Further, the seeding density of B cells in step 1) is 0.5-1×10 ^5-8 cells/ml.

Further, the seeding density of B cells in step 1) is 0.5-1×10 ^5-6 cells/ml.

Further, the number of generations of the number of expanded B cells in step 1) is greater than 5 generations.

Further, in step 1), the number of generations for the number of expanded B cells is greater than 100 generations.

Further, the ionizing radiation in step 2) is one or ^more of X-ray, γ-ray, and Co ⁶⁰ isotope.

Furthermore, the irradiation dose of ionizing radiation in step 2) is about 10-200 Gy.

Further, 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.

Further, the irradiation dose rate of the ionizing rays is about 2-12 Gy/min; and the irradiation time is about 200-600s.

Further, 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. min, 7.5Gy/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 is approximately It is 200s, 225s, 250s, 275s, 300s, 325s, 350s, 375s, 400s, 425s, 450s, 475s, 500s, 525s, 550s, 575s or 600s.

Further, the B cells have been immortalized before the step 1).

Further, the B cell is a diploid cell.

Further, the B cells used for the treatment in step 1) are peripheral blood lymphocytes taken from healthy people.

Further, the B cells have undergone EBV infection treatment before step 1).

The B cell vaccine prepared by the above preparation method.

Further, the B cell vaccine also contains a coupling agent and/or an immune adjuvant.

Further, the dosage form of the B cell vaccine is subcutaneous injection, intradermal injection, local injection, intraperitoneal injection or intravenous injection.

Further, the B cell vaccine can activate immune cells in the human body.

Further, the immune cells include T cells

Further, the B cell vaccine can specifically recognize EBV virus antigen.

The application of the above-mentioned B cell vaccine in the preparation of anti-tumor drugs.

Further, the tumor medicine is used to treat tumors carrying EBV virus.

Further, 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.

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.

Further, the B cells have been immortalized before the step (1).

Further, 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.

Further, the 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.

Further, the viral antigen is derived from a human susceptible virus.

Further, the human susceptible virus includes one or more of herpes virus, HIV virus and hepatitis virus.

Further, for the application of the B cell composition in preventing or treating viral infections, the B cell composition has a specific antiviral effect.

The beneficial effects of the invention

In view of the deficiencies of tumor vaccines in the prior art, 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:

Principle of immunology : In addition to the function of producing antibodies, 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.

Effectiveness: B cells have a clear antigen presentation ability and can efficiently present EBV antigen. In the present invention, B cells need to be infected with EBV virus to achieve immortality. In other words, realizing 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.

Safety : (i) 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; (ii) 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; (iii) In the process of making the vaccine of the present invention, 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.

In summary, 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. In addition, because 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.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative labor.

Figure 1B Microscopic view of normal cell culture;

Figure 2B Microscopic view of normal cell culture;

Figure 3B Cell optimized culture microscope image;

Figure 4B Cell optimization statistics (abscissa: time; ordinate: cell number);

Figure 5B Cell karyotype map;

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 9 Entity photo of the mouse model of vaccine anti-tumor effect;

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.

detailed description

To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

The examples given are to better describe the present invention, but the content of the present invention is not limited to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the embodiments based on the foregoing invention content still belong to the protection scope of the present invention.

Unless otherwise specified, the terms "includes" and "includes" and their grammatical variations are used to denote "open-ended" or "includes" language, so that they include the listed technical features but also allow the inclusion of other Technical features listed.

As used in this specification, the term "approximately" (for example, for ionizing radiation dose rate, radiation time), 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.

In this specification, certain embodiments may be disclosed in a format within a certain range. It should be understood that this description of "in a certain range" is only for convenience and brevity, and should not be construed as a rigid limit to the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent digital values within this range (for example, for ionizing radiation dose rate, radiation time). For example, range

The description of should be seen as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and within this range Individual numbers, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the scope, the above rules apply.

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. For example, 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.

Example 1

The characteristics of the vaccine cell of the present invention: 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.

Before irradiation, 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.

Before irradiation, 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.

Table 1 Statistics of the number of B cells

Training time (days)	Number of cells (10 7 )
0	1.4
2	2
4	4.2
6	6.4
8	11.47
10	23.57
12	43.76
14	87.9
16	163.9

Please note that, according to actual needs, the culture conditions of B cells before irradiation can also contain a certain amount of serum. In addition, 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.

Example 2

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. In addition, from the perspective of the principle of vaccine action, 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.

Example 3

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. The results showed that the quantitative detection result of HBV-DNA in B cells was <5.00E+02IU/mL, which was lower than the minimum detection limit of 5.00E+02IU/mL; the quantitative detection result of HCV-RNA was <5.00E+02IU/mL, which was lower than the minimum detection The lower limit is 5.00E+02IU/mL; the qualitative test result of human papillomavirus (HPV16/18-DNA) is negative (-); the qualitative test result of human papillomavirus (HPV6/11-DNA) is negative (-); parvovirus (B19-DNA) The qualitative test result is negative (-); the cytomegalovirus (CMV-DNA) quantitative test result is <1.00E+03IU/mL, which is lower than the minimum detection limit of 1.00E+03IU/mL; Epstein-Barr virus (EB DNA) ) The quantitative detection result is 6.17E+06Copies/mL.

The above test results show that the B cells used to prepare the present invention do not carry viruses other than EBV.

Example 4

The vaccine cells prepared by irradiation and the characteristics of the vaccine cells after irradiation:

After the B cells were resuspended in serum-free medium according to the above method, they were irradiated with the RS-2000 Biological Irradiator. The irradiation conditions are as follows:

Irradiation dose: Irradiate at about 10-200Gy. Specifically, 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.

Other irradiation conditions: no filter, plus reflector,

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.

Table 2 Statistics of number of living cells

Training time (days)	Number of cells (10 6 )
0	3.38
1	3.69
2	3.42
3	3.37
4	3.21

Table 3 Statistics of total cell numbers

Training time (days)	Number of cells (10 6 )
0	4.3
1	4.95
2	4.73
3	5.02
4	4.96

The B cells used to make the vaccine should be irradiated at least once under the above conditions before cryopreservation. When 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.

Example 5

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 ℃, 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.

Example 6

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. At the 5th week, the 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. 150ul 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 results showed that the tumor growth of mice in the immune cell adoptive treatment group was significantly slower than that in the control group, and began to shrink gradually on the 10th day after vaccination, and all tumors in the treatment group subsided on the 20th day. See Figure 8 and Figure 9 for details. Among them, Figure 8 is a statistical diagram of the vaccine's anti-tumor effect, and specific values are shown in Table 4.

Table 4 Statistics of vaccine anti-tumor effects

Please note that the above experiment is different from the use of vaccine cells to treat cancer patients. In order to test whether human immune cells can activate mouse T cells in a mouse model, and then kill human cancer cells in a mouse model, first 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). When treating cancer patients whose immune system is still healthy, 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.

In addition, the above experiments based on mouse models also 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.

And 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.

Table 5 Number of positive spots

Example 7

B cell antigen presentation test after irradiation

Divide the cultured vaccine cells into 2 parts, one part of 5×10 ⁶ vaccine cells by centrifugation at 600g for 5 minutes, discard the supernatant, add 1ml of ice TRIzol to resuspend, repeatedly pipette until the liquid is clear, and freeze at -80°C. The other part contains 1.5×10 ⁷ vaccine cells. After γ-radiation irradiation, it is divided into 3 parts. Each 5×10 ⁶ vaccine cells are cultured in an incubator. After 6 hours, 12 hours and 24 hours, 600g After centrifugation for 5 minutes, the supernatant was discarded, and 1ml of ice TRIzol was added to resuspend. After repeated pipetting until the liquid was clear, it was frozen at -80℃. Shipping on dry ice, the second-generation high-throughput sequencing RNA-Seq, the results are as follows:

1) Analyze the changes of B cell transcriptome before and after irradiation by RNA-seq transcriptome high-throughput sequencing, and related molecules involved in antigen presentation by B cells after irradiation (for example, CD36, SEC61A2, TAP1, SEC61A1, SEC61B, SEC61G) Up-regulation of expression: Except for CD36, the other molecules showed up-regulation at the 6-hour and 12-hour measurement points, and showed a downward-regulation trend at the 24-hour measurement point; CD36 was present at the 6-hour, 12-hour, and 24-hour measurement points The amount of expression gradually increased (Figure 10).

2) Flow cytometric detection of the changes of MHC-I, MHC-II, CD80, CD86, CD40, CD54 and other antigen presentation-related molecules on the surface of B cells before and after irradiation (Figure 11, Figure 12). In Figure 11, the expression of ASB2–UBE2C molecules showed a downward-regulated trend over time; the expression of HLA-F–FBXO44 molecules showed an upward-regulated trend over time. In Figure 12, the expression of KIF11-KIF23 molecules showed a downward-regulated trend (but fluctuated) over time, and the expression of HLA-DMB-HLA-DOA molecules showed an upward-regulated trend over time (but the expression at the 24-hour measurement point The amount is lower than the expressed amount at the 12-hour measurement point).

3) Flow cytometry detection of the expression of Granzyme B, IFN-γ, IL2 and other intracellular cytokines of T cells trained by B cells after irradiation indirectly proves the function of the trained CTL (Cytotoxic T Cell).

4) As shown in Figure 13, Elispot's measurement of T cell IFN-γ secretion indirectly proves the function of the trained CTL.

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. Although existing studies have shown that 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.

For example, 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. In some clinical trials, the use of LCL-CTL has produced good therapeutic effects, achieving a 30-50% complete remission (Completely Recover, CR) for relapsed and refractory lymphoma. However, the shortcomings of this treatment plan are also very obvious: (i) Because it is a reinfusion of activated T cells outside the body, the entire process takes a long time, and many patients cannot wait for such a long time or lose the treatment window; (ii) because it needs to pass from outside the body Infused LCL-CTL to kill EBV-infected cancer cells can only be individualized cell preparation and treatment to prevent rejection problems, and multiple rounds of stimulation are required to generate sufficient CTL cells, which is technically cumbersome; part (iii) The patient cannot cultivate enough CTL cells to complete the treatment.

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.

In addition, although 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. -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. In practice, in the present invention, 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.

It should be noted that in this article, the terms "include", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, It also includes other elements not explicitly listed, or elements inherent to the process, method, article, or device. If there are no more restrictions, the element defined by the sentence "including a..." does not exclude the existence of other identical elements in the process, method, article or device that includes the element.

The embodiments of the present invention are described above with reference to the accompanying drawings, but the present invention is not limited to the above-mentioned specific embodiments. The above-mentioned specific embodiments are only illustrative and not restrictive. Those of ordinary skill in the art are Under the enlightenment of the present invention, many forms can be made without departing from the purpose of the present invention and the protection scope of the claims, and these all fall within the protection of the present invention.

Claims (61)

1. A B cell composition, which is characterized by comprising: B cells and EBV virus antigens; the B cell composition is subjected to a certain dose of ionizing radiation irradiation treatment.
2. The B cell composition of claim 1, wherein the EBV virus antigen is presented on 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.
3. The B cell composition according to claim 1, wherein the B cell has biological activity but does not proliferate after being irradiated with ionizing rays.
4. The B cell composition of claim 1, wherein the EBV virus is inactivated after being irradiated with ionizing rays.
5. The B cell composition of claim 1, wherein the B cell carries or does not carry a live virus.
6. The B cell composition of claim 1, wherein the ionizing radiation is one or ^more of X-rays, γ-rays, and Co ⁶⁰ isotope.
7. The B cell composition of claim 1, wherein the ionizing radiation dose is about 10-200 Gy.
8. The B cell composition of claim 7, wherein 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.
9. The B cell composition of claim 1, wherein the irradiation dose rate of the ionizing rays is about 3-12 Gy/min; and the irradiation time is about 200-600s.
10. The B cell composition of claim 9, wherein the ionizing radiation dose rate is about 3Gy/min, 3.5Gy/min, 4Gy/min, 4.5Gy/min, 5Gy/min, 5.5Gy /min, 6Gy/min, 6.5Gy/min, 7Gy/min, 7.5Gy/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 is about 200s, 225s, 250s, 275s, 300s, 325s, 350s, 375s, 400s, 425s, 450s, 475s, 500s, 525s, 550s, 575s or 600s.
11. The B cell composition of claim 1, wherein the B cell has been immortalized.
12. The B cell composition of claim 11, wherein the immortalization treatment is to infect the B cells with EBV virus.
13. The B cell composition of claim 1, wherein the B cell is a diploid cell.
14. The B cell composition of claim 1, wherein the B cell has undergone expansion before being irradiated with the ionizing radiation.
15. The B cell composition of claim 1, wherein the B cell has undergone EBV infection treatment.
16. The B cell composition of claim 1, wherein the B cell composition is cultured under serum-free conditions before being irradiated with the ionizing radiation.
17. The B cell composition according to claim 16, wherein the B cell composition is cultured at 37°C and 5% CO ₂ .
18. The B cell composition of claim 17, wherein the B cell edge has antennae-like protrusions.
19. The B cell composition of claim 1, wherein the B cell composition further comprises T cells.
20. A B cell vaccine, characterized by comprising the B cell composition according to claim 1.
21. The B cell vaccine according to claim 20, which further contains a coupling agent and/or an immune adjuvant.
22. The B-cell vaccine of claim 20, wherein the dosage form of the B-cell vaccine is an injection.
23. The B-cell vaccine of claim 22, wherein the dosage form of the B-cell vaccine is subcutaneous and/or intradermal injection.
24. 22. The B cell vaccine of claim 20, wherein the B cell vaccine is an allogeneic B cell vaccine.
25. The B cell vaccine according to claim 20, wherein the B cell vaccine is an autologous B cell vaccine.
26. The use of the B cell vaccine according to claim 20 in the preparation of anti-tumor drugs.
27. The use of the B cell vaccine in the preparation of anti-tumor drugs according to claim 26, characterized in that the tumor drugs are used to treat tumors carrying EBV virus.
28. The application of the B-cell vaccine in the preparation of anti-tumor drugs according to claim 27, wherein the tumors carrying EBV virus include nasopharyngeal carcinoma, various 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 reticulocyte disease), infectious mononucleus One or more of cytosis and gastric cancer.
29. A method for preparing a B cell vaccine is characterized in that it comprises the following steps:

    (1) Culture and expand the number of B cells, which carry EBV antigen;

    (2) Irradiate the B cells obtained in step (1) with ionizing rays lower than the inactivation threshold.
30. The preparation method according to claim 29, wherein the culture conditions of the step (1) are: 37°C, 5% CO ₂ , and serum-free.
31. The preparation method of claim 29, wherein the seeding density of B cells in step (1) is 0.5-1×10 ^5-8 cells/ml.
32. The preparation method of claim 31, wherein the seeding density of B cells in step (1) is 0.5-1×10 ^5-6 cells/ml.
33. The preparation method according to claim 29, wherein the number of generations of the expanded B cells in step (1) is greater than 5 generations.
34. The preparation method according to claim 33, wherein the number of generations of the expanded B cells in step (1) is greater than 100 generations.
35. The preparation method according to claim 29, wherein the ionizing radiation in step (2) is one or ^more of X-ray, γ-ray, and Co ⁶⁰ isotope.
36. The preparation method according to claim 29, wherein the ionizing radiation dose in step (2) is about 10-200 Gy.
37. The preparation method of claim 36, wherein 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.
38. The preparation method of claim 29, wherein the ionizing radiation dose rate is about 2-12 Gy/min; and the radiation time is about 200-600s.
39. The preparation method of claim 38, wherein the ionizing radiation dose rate is about 3Gy/min, 3.5Gy/min, 4Gy/min, 4.5Gy/min, 5Gy/min, 5.5Gy/min , 6Gy/min, 6.5Gy/min, 7Gy/min, 7.5Gy/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 is about 200s, 225s, 250s, 275s, 300s, 325s, 350s, 375s, 400s, 425s, 450s, 475s, 500s, 525s, 550s, 575s or 600s.
40. The preparation method of claim 29, wherein the B cells have been immortalized before step (1).
41. The preparation method of claim 29, wherein the B cells are diploid cells.
42. The preparation method according to claim 29, wherein the B cells used for the treatment in the step (1) are peripheral blood lymphocytes taken from a healthy person.
43. The preparation method according to claim 42, wherein the B cells have undergone EBV infection treatment before step (1).
44. A B cell vaccine prepared by the preparation method of any one of claims 29-43.
45. The B cell vaccine of claim 44, wherein the B cell vaccine further contains a coupling agent and/or an immune adjuvant.
46. The B-cell vaccine of claim 44, wherein the dosage form of the B-cell vaccine is subcutaneous injection, intradermal injection, local injection, intraperitoneal injection or intravenous injection.
47. The B-cell vaccine of claim 44, wherein the B-cell vaccine can activate immune cells in the human body.
48. The B cell vaccine of claim 44, wherein the immune cells comprise T cells.
49. The B cell vaccine of claim 44, wherein the B cell vaccine can specifically recognize EBV virus antigens.
50. The use of the B cell vaccine according to claim 44 in the preparation of anti-tumor drugs.
51. The application of the B cell vaccine in the preparation of anti-tumor drugs according to claim 44, wherein the tumor drugs are used to treat tumors carrying EBV virus.
52. The application of the B cell vaccine in the preparation of anti-tumor drugs according to claim 51, characterized in that the tumors carrying EBV virus include nasopharyngeal carcinoma, various 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 reticulocyte disease), infectious mononucleus One or more of cytosis and gastric cancer.
53. A method for improving the presentation of B cell antigens, which is characterized by comprising the following steps: (1) culturing and expanding the number of B cells, which carry EBV antigen; (2) irradiating with a certain dose of ionizing radiation after the steps (1) The obtained B cells.
54. The method of claim 53, wherein the B cells have been immortalized before step (1).
55. The method of claim 53, wherein the B cells have biological activity but do not proliferate after being irradiated with ionizing rays.
56. A system for improving the presentation of B cell antigens, which is characterized by comprising: a cell culture expansion device for amplifying B cells carrying EBV antigen; an irradiation device for irradiating the plant with ionizing rays below the inactivation threshold The B cells.
57. The system for improving B cell antigen presentation according to claim 56, which is characterized in that it further comprises: a freezing device for freezing and storing the B cells irradiated with ionizing rays.
58. 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.
59. The B cell composition of claim 58, wherein the viral antigen is derived from a human susceptible virus.
60. The B cell composition according to claim 59, wherein the human susceptible virus comprises one or more of herpes virus, HIV virus and hepatitis virus.
61. The use of the B cell composition in the prevention or treatment of viral infections according to claim 58, said B cell composition having specific antiviral effects.

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