WO2022062687A1 - 肿瘤复合抗原、树突状细胞多价疫苗及其应用 - Google Patents

肿瘤复合抗原、树突状细胞多价疫苗及其应用 Download PDF

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WO2022062687A1
WO2022062687A1 PCT/CN2021/110455 CN2021110455W WO2022062687A1 WO 2022062687 A1 WO2022062687 A1 WO 2022062687A1 CN 2021110455 W CN2021110455 W CN 2021110455W WO 2022062687 A1 WO2022062687 A1 WO 2022062687A1
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cells
tumor
ebv
cell
dendritic
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刘慧宁
印泽
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刘慧宁
上海恒赛生物科技有限公司
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    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
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    • A—HUMAN NECESSITIES
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    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
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    • A61K2039/828—Stomach
    • 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

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a tumor composite antigen, a dendritic cell multivalent vaccine and applications thereof.
  • Epstein-Barr virus is a member of the genus Lymphovirus of the family Herpesviridae, with a DNA genome of about 170kb in length. The population is generally susceptible and spreads widely all over the world. The infection rate of adults is as high as 95%. The virus can be carried for life, and there are regional differences in the resulting diseases. EBV infection mostly occurs in childhood and adolescence. After infecting the body, some cases develop into long-term latent infection, but it may also lead to the formation of various malignant tumors in humans, such as Burkitts lymphoma (BL) and nasopharyngeal carcinoma (NPC).
  • BL Burkitts lymphoma
  • NPC nasopharyngeal carcinoma
  • EBV can specifically infect human and some primate B cells in vivo or in vitro, can stimulate the continuous growth of infected cells and cause cells to pass indefinitely to achieve "immortalization", thereby forming lymphoblastoid cell lines (Lymphoblastoid celllines, LCLs), these cell lines are often used to study the occurrence and development of various diseases, making it possible to study the pathogenesis of certain diseases on a large scale and long-term.
  • LCLs lymphoblastoid cell lines
  • EBV-related tumors such as EBV-related gastric cancer and nasopharyngeal cancer with high incidence in southern China.
  • Gastric carcinoma is a malignant tumor originating from gastric mucosa epithelium, and it is one of the common tumors in the world.
  • gastric cancer In addition, the incidence of gastric cancer in the northwestern and eastern coastal regions of China is significantly higher than that in the southern region, and the disease burden is very serious.
  • the pathogenesis of gastric cancer is complex, and the specific etiology has not yet been fully elucidated.
  • a large number of research data show that the occurrence of gastric cancer is the result of a combination of factors, such as geographical environment, dietary and living factors, Helicobacter pylori infection, EB virus infection, and precancerous lesions. , heredity, gene mutation, etc., among which studies have reported that EBV-related gastric cancer accounts for 10% of all gastric cancers.
  • Gastric cancer can occur in any part of the stomach, more than half of which occur in the gastric antrum, and the greater curvature of the stomach, the lesser curvature of the stomach, and the anterior and posterior walls can also be involved.
  • the vast majority of gastric cancers are adenocarcinomas, with no obvious symptoms in the early stage, or non-specific symptoms such as epigastric discomfort and belching, which are often similar to those of chronic gastric diseases such as gastritis and gastric ulcers and are easily overlooked. Therefore, the current early diagnosis of gastric cancer in my country It is imminent to discover gastric cancer-specific markers and improve the early diagnosis rate of gastric cancer.
  • the treatment of gastric cancer has shown a trend of multidisciplinary comprehensive treatment. The comprehensive application of surgery, radiotherapy, targeted therapy, and immunotherapy will help the treatment and prognosis of gastric cancer and reduce the pain of patients.
  • nasopharyngeal carcinoma is a malignant tumor that occurs in the nasopharynx, originates from the epithelium, and belongs to squamous carcinoma. Once onset, it develops rapidly, is highly invasive and metastatic, and is accompanied by headache, tinnitus, and hearing or vision loss.
  • NPC nasopharyngeal carcinoma
  • the incidence of nasopharyngeal carcinoma is very regional, with a high incidence in Southeast Asia, the Middle East, southern Africa and southern China, especially in Guangdong, where the incidence is as high as 30/100,000. It is one of the most common malignant tumors in southern my country.
  • nasopharyngeal carcinoma mainly relies on tissue biopsy with nasal endoscope, which is invasive to a certain extent. Moreover, due to the lack of serological diagnostic markers with high sensitivity and specificity, it is difficult for nasopharyngeal carcinoma to become a hospital. Routine inspection items. At present, due to the special location of nasopharyngeal carcinoma in clinical practice, early detection is difficult, and surgical resection is not suitable. With the development of the disease course, the 5-year survival rate of patients declines rapidly.
  • nasopharyngeal cancer For patients with advanced clinical stage IV, the 5-year survival rate is less than 50%; on the other hand, after radiotherapy and chemotherapy, it is easy to relapse and metastasize, and has serious side effects. Therefore, if not detected and treated early, nasopharyngeal cancer will be life-threatening for the patient. It poses a great threat, and finding a safe and effective treatment for relapsed and refractory advanced nasopharyngeal carcinoma is an urgent need for patients with nasopharyngeal carcinoma.
  • nasopharyngeal carcinoma is still uncertain. Some studies have pointed out that its incidence is mainly related to genetic factors, environmental regionality, dietary habits (preserved food, etc.) and Epstein-Barr virus (EBV) infection. , among which EBV has been confirmed to be highly correlated with the occurrence and development of nasopharyngeal carcinoma.
  • EBV Epstein-Barr virus
  • the treatment of nasopharyngeal carcinoma is mainly based on conventional radiotherapy and chemotherapy. Although certain effects can be achieved (the normal survival rate is 80% after 3 years of treatment), radiotherapy and chemotherapy will bring more benefits to patients during the treatment of nasopharyngeal carcinoma.
  • EBV can latently reside in infected B cells for a long time to form persistent latent infection, which is closely related to the occurrence, development and prognosis of various lymphomas, including Burkitt lymphoma. tumor, diffuse large B-cell lymphoma, Hodgkin lymphoma, NK/T-cell lymphoma, etc. WHO estimates that there were 589,600 new lymphoma cases worldwide in 2018, including 80,000 Hodgkin lymphomas and 509,600 non-Hodgkin lymphomas, with a global standardized incidence rate of 6.6/100,000.
  • EBV-related lymphoma In addition to the selection of sensitive chemotherapy regimens and traditional local radiotherapy according to different lymphoma types, it is also necessary to design a treatment plan based on the characteristics of EBV to effectively eliminate EBV infection and activity, and attenuate EBV in the disease.
  • the driving factors of development, blocking the further development of lymphoma; traditional treatment methods supplemented with specific treatment for EBV, such as antiviral therapy, immunotherapy, etc., are essential to improve the efficacy of EBV-related lymphoma.
  • dendritic cells in patients with EBV-related tumors have a low degree of differentiation, a reduced number, impaired ability to recognize and present antigens, and a low ability to activate initial T cells, which eventually leads to the body's inability to recognize and clear tumor cells, and EB virus infection. It is very likely to be one of the reasons for the impaired dendritic cell function in patients with nasopharyngeal carcinoma and other related cancers.
  • Dendritic cells (DC) were discovered in 1973 by Canadian scientist Ralph M. Steinman, winner of the 2011 Nobel Prize in Medicine and Physiology, and are named for their many dendritic or pseudopodia-like protrusions when they mature .
  • DCs are the most powerful professional antigen-presenting cells (APCs) known to the body. They can efficiently take up, process and present antigens. They are the only naive T cells discovered so far that can activate unsensitized na ⁇ ve T cells. and immature DCs have a strong ability to migrate and take up antigens, and mature DCs can effectively activate na ⁇ ve T cells and are at the center of initiating, regulating, and maintaining immune responses.
  • APCs professional antigen-presenting cells
  • DCs are an important class of innate immune cells and professional antigen-presenting cells, which play an important role in activating the body. It plays a key regulatory role in the process of immune response and maintenance of autoimmune tolerance.
  • DC can effectively present antigen information to T cells, induce T cell activation and lead to a series of immune responses.
  • the MHC molecules on the DC surface can combine with antigens to form peptide-MHC molecule complexes, present antigen signals to T cells, and some dendritic cells highly express costimulatory molecules (CD80/B7-1, CD86/B7-2, CD40 etc.) provide the second signal necessary for T cell activation, and DCs can also directly present antigen peptides to CD8 + T cells, activate CD8 + T cells with the help of CD4 + T cells, and activated DCs can secrete IL-12 in large quantities. , IL-18, Chemotactic Cytokines (CCK), etc.
  • CCK Chemotactic Cytokines
  • NK cells activate T cell proliferation, and initiate MHC-I-restricted CTL response and MHC-II-restricted CD4 + Th1 immune response; in addition, it can also activate perforation
  • the cytotoxicity of NK cells is enhanced by the pathway mediated by cytoplasmic granzyme B and FasL/Fas to enhance the body's anti-tumor immune response and facilitate tumor clearance.
  • DC itself can be used as a natural immune adjuvant to improve the immune capacity of the body by secreting various cytokines, and can also enhance the immune response of various vaccines.
  • dendritic cells with relevant antigen information with vaccine function are called For the dendritic cell vaccine (DC vaccine).
  • Vaccines are prophylactic or therapeutic biological products used for human vaccination, which play an important role in preventing, treating and controlling the occurrence and prevalence of infectious diseases.
  • problems such as too many vaccinations and limited therapeutic effects also arise.
  • Vaccines containing only a single antigenic component are called “monovalent vaccines", and monovalent vaccines can only prevent one infectious disease or one type of pathogen infection; vaccines made by mixing two or more antigenic components in appropriate proportions are called “multivalent vaccines”. vaccines" or “combination vaccines”.
  • HPV 16 18, 31, 33, 35, 39 , 45, 51, 52, 56, 58, 59, 68, 69, 73, 82 are called "high-risk HPV", and others are "low-risk HPV”
  • bivalent HPV vaccine can only prevent human Papillomaviruses 16 and 18, which are classified as “high-risk HPV” and cause 70% of cervical cancers
  • the quadrivalent HPV vaccine protects against HPV types 16, 18, 6, and 11, 6 and Type 11 HPV belongs to "low-risk HPV” and is the main cause of genital warts
  • the nine-valent HPV vaccine can prevent types 16, 18, 31, 33, 45, 52, 58, 6 and 11, and can prevent 7 "high-risk HPV types”.
  • HPV and two "low-risk HPV” are currently the vaccines that can prevent the most types of HPV infection.
  • the development of multivalent vaccines has a history of nearly a hundred years. As early as the 1930s, people started research on combined vaccines. In 1945, the trivalent influenza vaccine was approved for use in the United States for the first time, followed by the 6-valent pneumococcal vaccine, the double- and triple-dip vaccines, and the three-valent oral live attenuated polio vaccine. Results from clinical trials have shown that combined immunization with a multivalent vaccine is often superior to multiple vaccinations with a monovalent vaccine. When the multivalent vaccine is combined for immunization, the immune effect is similar to or better than that of the monovalent vaccine, and the side effects of the vaccine are not increased.
  • the number of dendritic cells in the tumor site is relatively small, and the lack of strong tumor antigen-stimulated dendritic cells-induced anti-inflammatory The immune response of the tumor cannot play a very significant therapeutic effect in the host.
  • the purpose of the present invention is to provide a tumor composite antigen, a dendritic cell multivalent vaccine and its application.
  • a variety of tumor cell lysates (such as SNU-719, YCCEL1, GD1, B95-8, M81, HKNPC1, SNU-719, YCCEL1, GD1, B95-8, M81, HKNPC1, etc., which are highly immunogenic for different EBV-related tumors) are loaded by stimulating the patient's own dendritic cells in vitro.
  • LCLs Human immortalized B lymphoblastoid cell lines
  • EB virus strains such as ⁇ 9 or lysates of EBV-positive tumor cells such as C666-1, HNE1, CCL85, etc.
  • cytokines specific Under the induction of agonists, it becomes mature dendritic cells, forming a complete DC vaccine with corresponding cancer antigens, which is then injected back into the human body to activate the immune system, stimulate innate immunity (such as inducing NK cells) and stimulate lymphocytes to produce an adaptive immune response
  • Generate cytotoxic T cells to kill cancer cells and precisely kill nasopharyngeal cancer cells to achieve personalized treatment; compared with radiotherapy and chemotherapy, it is particularly safe and has almost no side effects; and the production cycle of dendritic cell vaccine is about 1 week, which is short in time and cost. Low.
  • the present invention provides a tumor composite antigen, characterized in that: the composite antigen includes human immortalized B lymphoblastoid tumor cell lysate or/and EBV positive cancer cell lysate derived from different EB virus strains
  • the human immortalized B lymphoblastoid tumor cell lysate derived from different EB virus strains is any one or a combination of GD1, B95-8, M81, HKNPC1 ⁇ 9, SNU-719 or/and YCCEL1
  • the EBV-positive cancer cell lysate is any one or more combinations of C666-1, HNE1 or/and CCL85.
  • the present invention provides a dendritic cell multivalent vaccine, characterized in that: the dendritic cell multivalent vaccine carries the tumor composite antigen of claim 1; Multivalent dendritic cell vaccine loaded with tumor cell lysates or LCLs tumor cell lysates from one or two EBV-related cancers, or tumor cell lysates or LCLs tumor cell lysates from three or more EBV-related cancers simultaneously thing.
  • the human immortalized B lymphoblastoid tumor cell lysates derived from different EB virus strains are human transformed with EBV viruses such as GD1, B95-8, M81, HKNPC1-9, SNU-719, YCCEL1, etc. Any one or more of immortalized B lymphoblastoid cell lines; the cancer cell lysate is C666-1, HNE1 or CCL85.
  • the dendritic cell multivalent vaccine comprises a first adjuvant or other cytokines for adjuvant therapy.
  • the first adjuvant is any one of PloyI:C, LPS or OK432; the cytokine for adjuvant therapy is TNF- ⁇ or IL-12.
  • the first adjuvant is any one of PloyI:C, LPS or OK432; the cytokine for adjuvant therapy is TNF- ⁇ or IL-12.
  • each tumor cell lysate is 2.5*10 7 -2.5*10 9 .
  • the present invention provides an application of the above-mentioned tumor composite antigen in the preparation of a drug for preventing or treating EBV-related tumors.
  • the tumor includes EBV-related gastric cancer, EBV-positive lymphoma, nasopharyngeal carcinoma and other EBV-related epithelial cell cancers.
  • the medicament comprises the dendritic cell multivalent vaccine according to any one of claims 2-7.
  • the dendritic cell multivalent vaccine provided by the present invention can stimulate the immune response of the body to treat EBV-related tumors, especially EBV-related gastric cancer, EBV-positive lymphoma, nasopharyngeal cancer and other EBV-related epithelial cell cancers, and has good curative effect, And the side effects are small, and it can effectively inhibit the growth of EBV-related tumors for a long time and reduce the disease process of cancer, and even achieve a complete remission state.
  • the antigen-sensitized dendritic cell population which has been loaded with the corresponding antigen, is an immunogenic composition.
  • Specific antigens include GD1, B95-8, M81, HKNPC1 ⁇ 9, SNU-719, YCCEL1 and other human immortalized B lymphoblastoid cell lines (LCLs) tumor cell lysates and C666-1, HNE1 derived from different EB virus strains , CCL85 and other cancer cell lysates, the specific dosage of each tumor cell lysate is 2.5*10 7 -2.5*10 9 .
  • the dendritic cell vaccine loaded with tumor complex antigens may be a dendritic cell multivalent vaccine loaded only with tumor cell lysates of two EBV-related cancers or LCLs tumor cell lysates,
  • the dendritic cell multivalent vaccine can also be loaded with three or even multiple EBV-related cancer tumor cell lysates or LCLs tumor cell lysates at the same time.
  • the dendritic cell multivalent vaccine may contain a first adjuvant (Ploy(I:C), LPS, OK432, etc.) or other adjuvant cytokines such as TNF- ⁇ , IL-12, etc.
  • Dendritic cell multivalent vaccines are administered 3-30 times by intravenous, intradermal, intratumoral, intramuscular, intraperitoneal, intranodal, subcutaneous, or topical administration, one or two weeks apart; dendritic cells The amount of cells infused each time for the multivalent vaccine is 5*10 6 -5*10 8 .
  • Dendritic cell multivalent vaccine is a vaccine that fights cancer by activating the patient's own immune system. Compared with the traditional treatment of EBV-related tumors, it has the following advantages:
  • the immune system strengthens the immune system; it can inhibit the evolution of cancer cells, with a low recurrence rate, and the overall side effects are significantly less than traditional chemotherapy and multi-targeted targeted drugs, such as dendritic cell multivalent vaccines.
  • the mechanism of action is to activate the immune system Therefore, the most common side effects are only clinical grade I/II adverse reactions, such as fever, fatigue, dizziness, general muscle pain, drowsiness, etc., which can be treated symptomatically and has a good clinical application prospect.
  • the selection of antigens for dendritic cell multivalent vaccine is more effective and safe for the treatment of EBV-related tumors.
  • the international projects using DC vaccine to treat EBV-related tumors mostly use a specific segment of polypeptide as the antigen.
  • the dendritic cell multivalent vaccine of the present invention selects a variety of tumor cell lysates, including all antigens that activate immune responses against cancer cells, maximizing the load of tumor antigen information in the actual human body, and enhancing the presentation of dendritic cells.
  • the diversity of antigens maximizes the activation of human immune function and induces stronger T cell responses, thereby improving the therapeutic effect of the product.
  • the tumor cell lysate selected for the dendritic cell multivalent vaccine is derived from stable tumor cell lines, which does not require time-consuming and labor-intensive screening of each patient's tumor antigen, and does not need to worry about the patient's human leukocyte antigen (Human Leukocyte Antigen). , HLA) and antigen peptide binding, no HLA restriction, belong to the universal antigen.
  • the antigen is easy to prepare, the process is simplified, and the quality of the tumor lysate can be uniform, eliminating the complicated antigen screening process, and showing outstanding advantages in reducing cost and saving time.
  • the dendritic cell multivalent vaccine has a more durable therapeutic effect and can effectively inhibit the recurrence and metastasis of EBV-related tumors for a long time.
  • the multivalent dendritic cell vaccine can produce a large number of memory T lymphocytes containing a variety of tumor antigen information in the patient after being reinfused into the patient, which exists for several years to several decades. When the corresponding stimulus is encountered again, it can be rapidly activated in the body, kill tumor cells, and effectively prevent tumor recurrence and metastasis.
  • a variety of universal tumor antigens used in dendritic cell multivalent vaccines are suitable for most EBV-related tumor patients with EBV-specific antigen information. Because there is no need to screen each patient's tumor antigens, the whole preparation The cycle is only 1 week, which saves several months compared with the preparation of neoantigen DC vaccine, which is of great significance to cancer patients, especially advanced patients. And the universal antigen makes the preparation of the antigen easy to standardize and standardize, which greatly reduces the cost of production and quality control.
  • Figure 1 The expression level of CD19 on the surface of immortalized human B lymphocyte line LCLs;
  • Figure 2 EBV viral load in plasma and cancer tissue of 4 patients with nasopharyngeal carcinoma
  • Figure 3 Morphology of mature dendritic cells
  • Figure 4 Flow cytogram of dendritic cell surface marker molecule expression
  • Figure 5 CTL-specific lymphocyte killing rate induced by in vitro stimulation
  • T lymphocytes stimulated with dendritic cell multivalent vaccine were compared with T lymphocytes stimulated only with dendritic cell monovalent vaccine loaded with autologous tumor cell lysate or a control group (group aI) by Detecting the killing activity of the collected cells on nasopharyngeal carcinoma cells of tumor patients, it was found that T lymphocytes stimulated by dendritic cell multivalent vaccine showed strong killing of tumor cells in EBV-positive nasopharyngeal carcinoma patients (patients I and II). The more T lymphocytes, the more obvious the killing effect, while the tumor cells of EBV-negative nasopharyngeal carcinoma patients (patients III and IV) cannot be effectively killed.
  • the dendritic cells from different patients The CTL cells stimulated by the vaccine have a significant effect on killing autologous tumor cells.
  • the dendritic cell multivalent vaccine applied to EBV-positive nasopharyngeal carcinoma tumor cells its effect on killing tumor cells is equivalent;
  • the dendritic cells obtained from EBV-positive tumor cell lysate can obtain the same tumor killing effect as the dendritic cell monovalent vaccine loaded with autologous tumor cell lysate, avoiding the tedious process of collecting tumor tissue from each EBV-positive NPC patient. , can widely identify EBV-positive nasopharyngeal carcinoma tumor cells, and stimulate the proliferation of T cells to exert immunological effects.
  • CTL cells in each group were co-cultured with tumor tissue cells of nasopharyngeal carcinoma patients at a 20:1 effector-target ratio, and the amount of interferon ⁇ secreted by lymphocytes was detected. It was found that the dendritic cells loaded with autologous tumor cell lysates were The T lymphocytes of the 4 patients with nasopharyngeal carcinoma stimulated by the vaccine could produce a large amount of interferon ⁇ after co-culture with the tumor cells, which was significantly higher than that of the control group.
  • T cells produced a large amount of interferon ⁇ , while the T cells of two EBV-negative nasopharyngeal carcinoma patients in patients III and IV were stimulated with dendritic cell multivalent vaccine and co-cultured with their own EBV-negative nasopharyngeal carcinoma tumors.
  • Interferon ⁇ could not be produced effectively; it means that the dendritic cell multivalent vaccine loaded with EBV positive tumor cell lysate can strongly promote the differentiation of autologous T lymphocytes in patients with EBV positive nasopharyngeal carcinoma, secrete interferon ⁇ , and promote the body's anti-tumor effect. tumor capacity.
  • FIG. 8 EBV viral load in plasma of 4 gastric cancer patients
  • the EB virus load in the corresponding position of the patients was calculated; EBV numbers A and B were found.
  • the EBV load in the plasma of patients with positive gastric cancer is greater than 6000 copies, and the EBV load in the cancer tissue is greater than 17000 copies, and there is a large amount of EBV virus in the body, while the EBV virus is basically undetectable in the plasma of C and D EBV-negative gastric cancer patients, indicating that There are a lot of EBV in EBV-positive gastric cancer patients.
  • ELISA was used to detect the expression of IL12 in the supernatant of DC vaccine medium in each group. It was found that the expression of IL12 secreted by multivalent and monovalent vaccines from different patients was significantly higher than that of normal mature DCs in the control group. There is little difference in the expression of IL12 among the multivalent vaccines or monovalent vaccines, indicating that both gastric cancer tumor cell lysates and gastric cancer-related tumor antigens can stimulate DCs to secrete IL12, and there are many tumor antigens loaded with EBV-positive gastric cancer information.
  • the monovalent vaccine has the same ability to stimulate DC maturation and secrete IL12 as the monovalent vaccine of the patient's autologous tumor cell lysate.
  • Figure 10 CTL-specific lymphocyte killing rate induced by in vitro stimulation
  • Poly-DC+TA group indicates that the CTL cells stimulated by the DC multivalent vaccine group loaded with various tumor cell lysates are used as effector cells, and the gastric cancer tumor cells of patient A are used as target cells. According to the effect-target ratio of 5:1, 10:1, 20:1 culture effects, and so on; it can be seen from the results that the CTL cells stimulated by the monovalent vaccine group loaded with autologous gastric cancer tumor tissue cell lysate have significant killing effect on autologous gastric cancer tissue tumors.
  • the CTL cells stimulated by the multivalent vaccine group also showed strong tumor killing ability when encountering autologous EBV-positive tumor cells (patient A, patient B); but against EBV-negative gastric cancer cells (patient C, patient B) D), but did not significantly improve the tumor-killing effect of its T cells; it means that when there are EBV antigens similar to the multivalent vaccine itself on the surface of autologous tumor cells, it will induce a strong immune response in the body. On the contrary, if the tumor cells are EBV negative At the same time, the multivalent dendritic cell vaccine loaded with EBV antigen information cannot effectively stimulate and activate the body's anti-tumor immune response. It is proved that the Poly-DC multivalent vaccine can effectively inhibit the growth of EBV-positive gastric cancer tumors, and has a wider application.
  • interferon ⁇ A large amount of interferon ⁇ was secreted, but interferon ⁇ could not be effectively produced when co-cultured with EBV-negative nasopharyngeal carcinoma tumors, and compared with the control group, there was no significant difference; indicating that the general EBV tumor cell lysate was used as the Antigen information can effectively avoid differences in individualized EBV-positive tumor cells, accelerate the industrialization of dendritic cell vaccines, and confirm that multivalent vaccines loaded with EBV-positive tumor cell lysates can stimulate and activate the immune system to recognize and kill EBV-positive gastric cancer tumors. And a wide range of immune function-boosting effects.
  • Example 1 Immunological study of dendritic cell multivalent vaccine in the treatment of nasopharyngeal carcinoma
  • tumor cells from two EBV-positive nasopharyngeal carcinoma patients (marked as I, II) and two EBV-negative nasopharyngeal carcinoma patients (marked as III, IV) were selected (the informed consent of the patients has been obtained). ).
  • peripheral blood mainly contains platelets, mononuclear cells, granulocytes, red blood cells and other cells: the platelet density is 1.030-1.035 kg/m 3 , and the mononuclear cell density is 1.075-1.075 kg/m 3 .
  • 1.090kg/m 3 granulocyte density 1.092kg/m 3
  • red blood cell density 1.093kg/m 3
  • Paque Plus (GE Healthcare) solution density is 1.075-1.089kg/m 3
  • density gradient centrifugation is carried out, different cell components are stratified, and mononuclear cells can be quickly separated from human peripheral blood.
  • the methods for separating and extracting CD14 monocytes include but are not limited to the use of CD14 + magnetic bead sorting in this example, and can also be CD14 negative selection, meltenyi immunomagnetic cell sorting (MACS), cell attachment and other methods .
  • the principle is based on the specific binding characteristics of antigen-antibody.
  • the CD14 + magnetic bead sorting kit can specifically identify and bind to CD14 + cells in PBMC, and is indirectly coupled with magnetic beads through biotin or dextran. The purpose of CD14 + cell separation is achieved under the action of a magnetic field. In this example, EasySep TM CD14 positive selection kit was used.
  • RapidSphere TM solution Add an appropriate amount of RapidSphere TM solution to the flow tube to a final concentration of 100 ⁇ L/mL, fully aspirate and mix, and incubate at room temperature for 3 min.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Interleukin-4 Interleukin-4, IL-4
  • IL-4 Interleukin-4, IL-4
  • IL-4 can inhibit the overgrowth of macrophages, reduce the expression of CD14 molecules on the cell surface, and induce the differentiation of CD14 + monocytes into iDCs.
  • PBMCs were isolated from peripheral blood and resuspended in 2 ml of RPMI1640/10% FBS medium.
  • B95-8 cell supernatant or other EB virus suspensions such as GD1, B95-8, M81, HKNPC1-9, SNU-719, YCCEL1, etc.
  • R20 medium RPMI1640/20% FBS, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin
  • the 96-well plate was placed in an incubator for 6 days, and the cell status was observed every day. Observe whether the cell morphology exhibits lymphoblastoid-like changes: the cell volume increases, the cytoplasm is abundant, spherical, and small colonies are aggregated and distributed, the cell mass at the bottom of the well increases significantly, and the color of the medium turns yellow.
  • EBV viral load (Viral Load, VL) in blood and cancer tissues of patients with nasopharyngeal carcinoma
  • Blood samples were collected from 4 patients with nasopharyngeal carcinoma.
  • DNA extraction and polymerase chain reaction (PCR) were performed using the MagMAX Viral Nucleic Acid Extraction Kit (Thermo A42352) and the EBV Real-TM Quant Kit (Sacace BioTechnologies Srl, Como, Italy), respectively, from 100 ⁇ L of plasma according to the manufacturer’s protocol. DNA was extracted and eluted with 50 ⁇ L of elution buffer.
  • the nasopharyngeal carcinoma tumor tissue cells obtained above were extracted from the total DNA of NPC patient nasopharyngeal carcinoma tissue according to the DNeasy Blood & Tissue Kit (Qiagen, Cat. No. 69506) product instructions, and real-time quantitative PCR (EBV Real-TM Quant Kit) was used to analyze 10 microns of DNA. liter samples for EBV quantification.
  • the coding region of the EBNA1 gene was selected as the amplification target, and the final volume of polymerase chain reaction was 25 ⁇ L, and the reaction was operated according to the instructions.
  • the primer sequences are as follows
  • EBNA1-FP 5'-CCAGACAGCAGCCAATTGTC-3', as shown in SEQ ID NO.1;
  • EBNA1-RP 5'-GGTAGAAGACCCCCTCTTAC-3', as shown in SEQ ID NO.2;
  • the repeated freezing and thawing method is a commonly used mechanical lysis method, which usually consists of freezing and thawing.
  • the principle is that due to the formation of intracellular ice particles and the increase of the salt concentration of the remaining cell fluid, swelling is caused, the cell structure is broken, and the cells are killed, but the immunogenicity of the cells is retained. Freezing is usually performed in liquid nitrogen or on ice at -20°C, and thawing can be performed in a 37°C, 50°C, 65°C, or 100°C water bath by heat shock, which is milder than chemical lysis.
  • the temperature of the water bath is preset to 37°C.
  • C666-1 nasopharyngeal carcinoma cell line Collect C666-1 nasopharyngeal carcinoma cell line, immortalized human B lymphocyte line LCLs constructed from different virus strains, and tumor cells from 4 nasopharyngeal carcinoma patients; .
  • the cells were frozen in liquid nitrogen for 20 s.
  • Dendritic cell monovalent vaccine Co-culture 2.5x10 7 autologous tumor cell lysates and 5x10 6 dendritic cells for at least 2 hours, and then add cytokines such as TNF- ⁇ to stimulate dendritic cell maturation to prepare Dendritic cell monovalent vaccine;
  • Dendritic cell multivalent vaccine Take 2.5x10 7 C666-1 tumor cell lysates or M81-LCLs tumor cell lysates and other different tumor cell lysates, and co-culture with 5x10 6 dendritic cells respectively, the effect is at least For 2 hours, cytokines such as TNF- ⁇ were added to stimulate the maturation of dendritic cells, and multiple dendritic cells loaded with tumor cell antigen information were mixed in dendritic cell culture medium at the same time to prepare tumor-loaded cells.
  • Dendritic cell multivalent vaccine of lysate antigen Take 2.5x10 7 C666-1 tumor cell lysates or M81-LCLs tumor cell lysates and other different tumor cell lysates, and co-culture with 5x10 6 dendritic cells respectively, the effect is at least For 2 hours, cytokines such as TNF- ⁇ were added to stimulate the maturation of dendritic cells, and multiple dendritic cells loaded with tumor cell antigen information were mixed in dendriti
  • Flow cytometry was used to detect the surface molecular markers of immature dendritic cells and mature dendritic cells, such as CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, HLA-ABC, etc.
  • Figure 3 Morphological diagram of mature dendritic cells
  • Figure 4 Flow diagram of the expression of dendritic cell surface marker molecules.
  • DC-I indicates the self-tree of No. I EBV-positive nasopharyngeal carcinoma patients Dendritic cells
  • DC-II represents the EBV-positive nasopharyngeal carcinoma patient's own dendritic cells
  • DC-III represents the III EBV-negative nasopharyngeal carcinoma patient's own dendritic cells
  • DC-IV represents the IV-negative nasopharyngeal carcinoma patient's own dendritic cells dendritic cells
  • Ag-I represents the antigen information of tumor cell lysates of patients with type I nasopharyngeal carcinoma
  • Ag-II represents the antigen information of tumor cell lysates of patients with type II nasopharyngeal carcinoma
  • Ag-III represents the antigen information of patients with type III
  • Tumor cell lysate antigen information indicates the tumor cell lysate antigen information of IV nasopharyngeal carcinoma patients
  • Poly-DC-I indicates dendritic cell multivalent vaccine suitable for I patient
  • Ag-DC-I Indicates the dendritic cell monovalent vaccine for patient I loaded with information on patient I tumor cell lysate, and so on.
  • the magnetic bead separation method can be used to separate T lymphocytes using CD3 + magnetic beads.
  • the cells were first incubated with anti-surface antigen monoclonal antibody for 12 min, and 10 7 cells were incubated with 50 ⁇ L anti-CD3 monoclonal antibody. After washing, the cells were incubated with 100 ⁇ L of biotin-labeled goat anti-mouse secondary antibody for 10 min. After washing, FITC-labeled streptavidin was added. 25 ⁇ L and 25 ⁇ L of and reacted for 8 minutes, and after washing, biotin-labeled magnetic particles (100 ⁇ L magnetic particles added to anti-CD3 monoclonal antibody) were added to react for 8 minutes.
  • T lymphocytes were obtained by immunomagnetic separation using a magnetic cell separator (MACS).
  • MCS magnetic cell separator
  • DC-I denotes DC derived from patient I
  • TI denotes T lymphocytes derived from patient I
  • T-II denotes T lymphocytes derived from patient II
  • T-III denotes T cells derived from patient III Lymphocytes
  • T-IV means T lymphocytes derived from patient IV.
  • IL-2 was 1000U/ml
  • the content of IL-12 was 1500U/ml
  • the content of Poly(I:C) was 10mg/ml
  • the content of TNF- ⁇ was 1000U/ml, etc.
  • IL-2 at a final concentration of 30 U/ml was added, and 2 x 10 5 corresponding T cells or vaccines were added to each group for secondary stimulation , cultured for another week, and the cells were collected on the 21st day.
  • the CTL effector cells of the above groups and the tumor cells of 4 patients with nasopharyngeal carcinoma were mixed in a U-bottom 96-well plate according to the effect-target ratio of 20:1 and cultured for 72 hours. Process to detect the content of IFN- ⁇ in the culture supernatant, as shown in Figure 6.
  • Example 2 Study on the immunological effect of dendritic cell multivalent vaccine against EBV-positive gastric cancer
  • EBV-positive gastric cancer patients A, B
  • EBV-negative gastric cancer patients C, D
  • peripheral blood mainly contains platelets, mononuclear cells, granulocytes, red blood cells and other cells: the platelet density is 1.030-1.035 kg/m 3 , and the mononuclear cell density is 1.075-1.075 kg/m 3 .
  • 1.090kg/m 3 granulocyte density 1.092kg/m 3
  • red blood cell density 1.093kg/m 3
  • Paque Plus (GE Healthcare) solution density is 1.075-1.089kg/m 3
  • density gradient centrifugation is carried out, different cell components are stratified, and mononuclear cells can be quickly separated from human peripheral blood.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Interleukin-4 Interleukin-4, IL-4
  • IL-4 Interleukin-4, IL-4
  • IL-4 can inhibit the overgrowth of macrophages, reduce the ability to express CD14 molecules on the cell surface, and induce CD14+ monocytes to differentiate into iDCs.
  • EBV viral load (Viral Load, VL) in blood and gastric cancer tissue samples from gastric cancer patients: Blood samples from 4 gastric cancer patients were collected. DNA extraction and polymerase chain reaction (PCR) were performed using MagMAX Viral Nucleic Acid Extraction Kit (Thermo A42352) and EBV Real-TM Quant Kit (Sacace BioTechnologies Srl, Como, Italy), respectively. DNA was extracted from 100 ⁇ L of plasma according to the manufacturer’s protocol and eluted with 50 ⁇ L of elution buffer. The gastric cancer tumor tissue cells obtained above were extracted from the total DNA according to the DNeasy Blood & Tissue Kit (Qiagen, Cat. No.
  • the primer sequences are as follows:
  • EBNA1-FP 5'-CCAGACAGCAGCCAATTGTC-3', as shown in SEQ ID NO.1;
  • EBNA1-RP 5'-GGTAGAAGACCCCCTCTTAC-3', as shown in SEQ ID NO.2;
  • LCLs constructed from EBV-related gastric cancer virus strains (such as SNU719-LCLs, YCCEL1-LCLs, etc.) were added to the cells until the fifth day.
  • gastric cancer tumor cell line lysates GT38, PT, etc.
  • Dendritic cell monovalent vaccine Co-culture 2.5x10 7 autologous tumor cell lysates and 5x10 6 dendritic cells for at least 2 hours, and then add cytokines such as TNF- ⁇ to stimulate dendritic cell maturation to prepare Dendritic cell monovalent vaccine;
  • Dendritic cell multivalent vaccine Take 2.5x107 YCCEL1 -LCLs, GCEBV-LCLs tumor cell lysates or GT38, PT and other EBV-positive gastric cancer cell line lysates and other different cell lysates, respectively, and 5x106 dendritic cells The dendritic cells were co-cultured for at least 2 hours, and then cytokines such as TNF- ⁇ were added to stimulate the maturation of dendritic cells, and multiple dendritic cells loaded with tumor cell antigen information were simultaneously mixed in equal amounts in the dendritic cell culture. In the base, a dendritic cell multivalent vaccine loaded with tumor cell lysate antigens was prepared.
  • Ag-Poly indicates that it contains antigen information of YCCEL1-LCLs, GCEBV-LCLs tumor cell lysates or EBV-positive gastric cancer cell lines such as GT38, PT and other tumor cell lysates
  • DC-A indicates patient A's own dendritic cells
  • DC -B stands for patient B's own dendritic cells
  • DC-C stands for C patient's own dendritic cells
  • DC-D stands for D patient's own dendritic cells
  • Ag-A stands for tumor cell lysate antigen information of gastric cancer patient A
  • Ag -B represents the antigen information of tumor cell lysate of gastric cancer patient B
  • Ag-C represents the antigen information of tumor cell lysate of gastric cancer patient C
  • Ag-D represents the antigen information of tumor cell lysate of gastric cancer patient D
  • Poly-DC -A indicates a dendritic cell multivalent vaccine for patient A
  • Ag-DC-A indicates a den
  • T lymphocytes were isolated by magnetic bead separation using CD3 + magnetic beads.
  • the cells were first incubated with anti-surface antigen monoclonal antibody for 12 min, and 10 7 cells were incubated with 50 ⁇ L anti-CD3 monoclonal antibody. After washing, the cells were incubated with 100 ⁇ L of biotin-labeled goat anti-mouse secondary antibody for 10 min. After washing, FITC-labeled streptavidin was added. 25 ⁇ L of cohesin was added to react for 8 min, and after washing, biotin-labeled magnetic beads (100 ⁇ L of magnetic beads added to anti-CD3 monoclonal antibody) were added to react for 8 min.
  • T lymphocytes were obtained by immunomagnetic separation using a magnetic cell separator (MACS).
  • MCS magnetic cell separator
  • DC-A means DC derived from patient A; TA means T lymphocytes derived from patient A, TB means T lymphocytes derived from patient B, TC means T lymphocytes derived from patient C, TD means T lymphocytes derived from D patient's T lymphocytes.
  • IL-2 was 1000U/ml
  • the content of IL-12 was 1500U/ml
  • the content of Poly(I:C) was 10mg/ml
  • the content of TNF- ⁇ was 1000U/ml, etc. .
  • IL-2 at a final concentration of 30 U/ml was added, and 2 x 10 5 corresponding T cells or vaccines were added to each group for secondary stimulation , continue to culture for one week, and collect the cells on the 21st day to detect the immunological function of the multivalent vaccine.
  • Part of the cells collected above were centrifuged and suspended in RPMI1640 complete medium to adjust the cell concentration and divided into three experimental groups with different effect-target ratios.
  • the three groups were added to a 96-well culture plate with 4 ⁇ 10 5 , 2 ⁇ 10 5 , and 1 ⁇ 10 5 per well respectively.
  • As effector cells tumor cells from different EBV-positive gastric cancer patients were set as target cells, and 2 ⁇ 10 4 tumor cells from different gastric cancer patients were added to each well, with a final volume of 200 ul.
  • the set groups are shown in Table 6.
  • a lymphocyte-free control group and a cell-free blank culture medium control group were set, with 5 replicate wells.
  • the CTL effector cells of the above groups and the tumor cells of 4 different patients were mixed in a U-bottom 96-well plate according to the effect-target ratio of 20:1 and cultured for 72 hours, and then detected using the interferon- ⁇ enzyme-linked immunosorbent assay kit according to the instructions.
  • Figure 11 shows the content of IFN- ⁇ in the culture supernatant.

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Abstract

一种肿瘤复合抗原、树突状细胞多价疫苗及其应用。通过在体外刺激患者自身的树突细胞，负载具有针对不同EBV相关肿瘤的超强免疫原性的多种肿瘤细胞裂解物，并在多种细胞因子、特定激动剂诱导下成为成熟的树突细胞，形成完整的带有相应癌抗原的DC疫苗，回输到人体激活免疫系统，刺激天然免疫(如诱导NK细胞)和刺激淋巴细胞产生获得性免疫反应，产生细胞毒性T细胞杀伤癌细胞，一同精准杀伤癌细胞；相较于放化疗，特别安全，几乎没有副作用；且树突细胞疫苗制作周期1周左右，时间短，成本低。

Description

肿瘤复合抗原、树突状细胞多价疫苗及其应用 技术领域
本发明涉及生物医药技术领域，具体涉及一种肿瘤复合抗原、树突状细胞多价疫苗及其应用。
背景技术
EB病毒(Epstein-Barr virus，EBV)是疱疹病毒科嗜淋巴细胞病毒属的成员，基因组为DNA，长度约为170kb。人群普遍易感，在全世界广泛传播，成人的感染率高达95％，病毒可终身携带，且导致的疾病存在地域性差异。EBV的感染多发生于幼年和青春期，在感染机体后，部分情况发展为长期的潜伏感染，但也有可能导致人类多种恶性肿瘤的形成，如Burkitts淋巴瘤(BL)、鼻咽癌(NPC)、霍奇金淋巴瘤、非Hodgkin性淋巴瘤(NHL)、胃癌、乳腺癌和免疫功能缺陷患者发生的肿瘤等。此外，EBV能专一地在体内或体外感染人及某些灵长类细胞B细胞，能刺激感染细胞持续生长并引起细胞无限传代达到“永生(immortalization)”，从而形成淋巴母细胞样细胞系(lymphoblastoid celllines，LCLs)，这类细胞系常用来研究多种疾病的发生发展，使得大规模长期研究某些疾病的发病机理成为可能。但是目前关于EBV致病机制的理解，还不完全清楚，针对EBV相关肿瘤(如EBV相关胃癌和中国南方高发的鼻咽癌等)的研究与治疗水平还有待提高。
胃癌(gastric carcinoma，GC)是起源于胃黏膜上皮的恶性肿瘤，是全球常见肿瘤之一，好发年龄在50岁以上，且目前呈现年轻化趋势；根据国际癌症研究机构的统计数据，2012年全球胃癌新发病例约95.1万例，死亡病例约72.3万例，分别位于恶性肿瘤发病率第5位、死亡率第3位；其中超过70％的胃癌新发病例发生在发展中国家，约50％的病例发生在亚洲东部，主要集中在中国(2012年中国胃癌发病例数和死亡例数分别占全球胃癌发病和死亡的42.6％和45.0％，2015年中国胃癌新发病例约为67.9万例，胃癌死亡病例约为49.8万例)，且我国的西北与东部沿海地区胃癌发病率比南方地区明显要高，疾病负担十分严重。胃癌的发病机制复杂，具体病因迄今尚未完全阐明，但大量研究资料表明胃癌的发生是多种因素综合作用的结果，如地域环境，饮食生活因素，幽门螺杆菌感染，EB病毒感染、癌前病变，遗传，基因突变等，其中有研究报道EBV相关的胃癌占全部胃癌的10％。胃癌可发生于胃的任何部位，其中半数以上发生于胃窦部，而胃大弯、胃小弯及前后壁也均可受累。绝大多数胃癌属于腺癌，早期无明显症状，或出现上腹不适、嗳气等非特异性症状，常与胃炎、胃溃疡等胃慢性 疾病症状相似，易被忽略，因此，目前我国胃癌的早期诊断率较低，发现胃癌特异性标志物，提高胃癌早期诊断率迫在眉睫。此外，针对胃癌的治疗已呈现多学科综合治疗态势，手术治疗、放射治疗、靶向治疗、免疫治疗等综合应用，将有助于胃癌的治疗与预后，减轻患者病痛。
在EBV相关肿瘤中，鼻咽癌(Nasopharyngeal carcinoma，NPC)是一种发病于鼻咽部的恶性肿瘤，起源于上皮，属于鳞状上皮癌。一旦发病即会快速发展，具有高度的侵袭和转移特性，并伴有头痛、耳鸣、听力或视力下降。流行病学上，鼻咽癌发病极具区域性，高发于东南亚、中东、非洲南部及中国南部地区，特别是广东地区发病率高达30/10万，是我国南方地区最常见的恶性肿瘤之一；2012年WHO发布中国每年新发约3.3万，死亡2万。目前，鼻咽癌的诊断方法主要依赖于借助鼻内窥镜的组织活检，具有一定的侵入性；而且由于缺乏兼顾灵敏度高及特异性好的血清学诊断标志物，使鼻咽癌难以成为医院常规检查项目。目前在临床上由于鼻咽癌发病位置特殊，早期发现有困难，不宜进行手术切除，随着病程的发展，患者的5年生存率急速下降，如临床早期病人的5年生存率达90％，而临床IV期的晚期病人，5年生存率不到50％；另一方面是放化疗后，容易复发转移，且毒副作用大，因此，若不及早发现并治疗，鼻咽癌将对患者生命造成极大威胁，针对复发难治的晚期鼻咽癌寻找安全有效的治疗是当前鼻咽癌患者迫切的需求。
目前，鼻咽癌的病因尚不十分确定，有研究指出，其发病主要与遗传因素、环境地域性、饮食习惯(腌制性食品等)及EB病毒(Epstein-Barr virus，EBV)的感染有关，其中EBV已被证实与鼻咽癌的发生发展高度相关。目前鼻咽癌的治疗以常规的放化疗为主，虽然能取得一定的效果(治疗后3年正常生存率达80％)，但放化疗在治疗鼻咽癌过程中会给患者带来较多的不适，且目前尚不存在特效药，开发预防和治疗性鼻咽癌疫苗一直是研究的热点。另外，由于不同病人癌症之间差异很大，不同病人的癌细胞内生物进程不一样，各种蛋白表达量也不一样；所以，往往目前已知的标志物或特效药的效果并不能在所有病人中得到良好反馈。因此，临床上亟需直接来源于患者本身的、特异性好、用于鼻咽癌诊断治疗的试剂或疫苗。
同时，多年来针对鼻咽癌疫苗的研究主要集中在以EBV gp350位靶抗原的预防性疫苗及以EBV-EBNA1、LMP1和LMP2为靶位点的单一或联合性重组治疗性疫苗。目前，虽然国内的相关疫苗研究取得了一定实质性进展，不少已进行了I、II期临床试验，但鼻咽癌的发生发展无特异的肿瘤标志物，使得后续临床试验较为困难；而目前相关疫苗的研究模型多为小鼠、恒河猴、食蟹猴等，科研依据不充足。
另外，EBV作为一种嗜B细胞的人类疱疹病毒，可长期潜伏于被感染的B细胞内，形 成持续潜伏感染，与多种淋巴瘤的发生、发展、预后有着密切关系，包括伯基特淋巴瘤、弥漫大B细胞淋巴瘤、霍奇金淋巴瘤、NK/T细胞淋巴瘤等。WHO估计，2018年全球新发淋巴瘤病例有58.96万，其中霍奇金淋巴瘤为8万，非霍奇金淋巴瘤为50.96万，全球标化发病率为6.6/10万。其中在我国，霍奇金淋巴瘤患者中EB病毒检出率可达48％-57％。EBV相关的淋巴瘤治疗比较复杂，除了根据不同的淋巴瘤类型选择敏感的化疗方案、传统的局部放疗外，还需要针对EBV特性设计治疗方案，有效清除EBV感染和活动的状况，减弱EBV在疾病发展中的驱动因素，阻断淋巴瘤进一步发展；在传统治疗方法上辅以针对EBV的特异性治疗，如抗病毒治疗、免疫治疗等，对提高EBV相关的淋巴瘤的疗效至关重要。
有研究表明EBV相关肿瘤患者的树突细胞分化程度较低，数量减少，识别及呈递抗原的能力受损，对初始T细胞的激活能力低下，最终导致机体无法识别和清除肿瘤细胞，EB病毒感染极有可能是导致鼻咽癌等相关癌症患者的树突细胞功能受损的原因之一。树突状细胞(Dendritic cells,DC)由2011年诺贝尔医学及生理学奖获得者加拿大科学家Ralph M.Steinman于1973年发现，因其成熟时伸出许多树突样或伪足样突起而得名。DC是目前所知的机体功能最强的专职抗原递呈细胞(Antigen presenting cells,APC)，能高效地摄取、加工处理和呈递抗原，是目前发现的唯一能激活未致敏的初始型T细胞的APC；且未成熟的DC具有较强的迁移和摄取抗原的能力，成熟DC能有效激活初始T细胞，处于启动、调控、并维持免疫应答的中心环节。其数量不足外周血单核细胞的1％，但其表面具有丰富的抗原递呈分子(如MHC-Ⅰ和MHC-Ⅱ)、共刺激因子(CD80/B7-1、CD86/B7-2、CD40等)和粘附因子(ICAM-1、ICAM-2、ICAM-3、LFA-1、LFA-3)等，故DC是一类重要的天然免疫细胞和专职性抗原提呈细胞，在激活机体免疫应答及维持自身免疫耐受过程中发挥着关键性的调控作用。
DC作为最强的抗原呈递细胞，能有效地把抗原信息呈递给T细胞，诱导T细胞活化而导致一系列免疫应答。DC表面的MHC分子能与抗原结合，形成肽-MHC分子复合物，将抗原信号呈递给T细胞，部分树突细胞高表达的共刺激分子(CD80/B7-1、CD86/B7-2、CD40等)提供T细胞活化所必须的第二信号，同时DC也能直接向CD8 +T细胞呈递抗原肽，在CD4 +T细胞辅助下使CD8 +T细胞活化，活化的DC可以大量分泌IL-12、IL-18、趋化因子(Chemotactic Cytokines，CCK)等激活T细胞增殖，以及启动MHC-I类限制性CTL反应和MHC-Ⅱ类限制性的CD4 +Th1免疫应答；此外，还可激活穿孔素P颗粒酶B和FasL/Fas介导的途径增强NK细胞毒作用来增强机体抗肿瘤免疫应答，利于肿瘤清除。DC本身能作为一种天然的免疫佐剂通过分泌各种细胞因子来提高机体免疫能力，也可增强各种疫苗的免疫反应，通常将带有相关抗原信息的具有疫苗 功能的树突细胞，称为树突细胞疫苗(DC疫苗)。
疫苗(Vaccine)是用于人体接种的预防或治疗性生物制品，对于预防、治疗、控制传染病的发生和流行具有重要作用。但随着疫苗生产及使用数量的增加，接种剂次过多、治疗效果有限等问题也随之产生。如我国免疫规划中儿童常规免疫的14种疫苗在3周岁之前共需进行17～19针次(17针：采用乙脑减毒活疫苗、甲肝减毒活疫苗完成免疫接种；19针：采用乙脑灭活疫苗、甲肝灭活疫苗完成免疫接种)的接种，预防接种次数的增加，给家长和医务人员带来了诸多不便，容易因接种时间冲突产生全程接种率降低和预防接种工作成本增高等问题，还会导致接种不良反应的增加，这些问题加大了疫苗监管和临床使用的难度。为了减少接种次数、提高预防治疗效果，降低扩大免疫规划的实施成本，同时发挥疫苗最大的防病治病效果，开发并推广应用多价疫苗或联合疫苗就显得格外重要，对降低实施成本和最大限度治疗疾病具有重要意义。
只含有单一的抗原成分的疫苗被称为“单价疫苗”，单价疫苗只能预防一种传染病或一种类型的病原体感染；两种以上抗原成分按照适当比例混合制成的疫苗称为“多价疫苗”或“联合疫苗”。如人乳头瘤病毒有100多个分型，大部分只引起皮肤疣，但有一些型的人乳头瘤病毒可引起宫颈癌，如人乳头瘤病毒的16、18、31、33、35、39、45、51、52、56、58、59、68、69、73、82型，被人们称为“高危型HPV”，其他则属于“低危型HPV”；二价HPV疫苗只能预防人乳头瘤病毒的16和18，这两型病毒属于“高危型HPV”，70％的宫颈癌由这两型病毒引起；四价HPV疫苗可预防16、18、6和11型HPV感染，6和11型HPV属于“低危型HPV”，是引起生殖器疣的主要病因；九价HPV疫苗可预防16、18、31、33、45、52、58、6和11型，可以预防7个“高危型HPV”和2个“低危型HPV”，是目前能够预防最多种类型HPV感染的疫苗。多价疫苗的发展已经有将近百年的历史，早在上世纪30年代，人们即开始了有关联合疫苗的研究。1945年，3价流感疫苗在美国最早获准使用，随后6价肺炎球菌疫苗、白破二联及百白破三联疫苗和3价口服脊髓灰质炎减毒活疫苗等相继问世。临床试验的结果表明，使用多价疫苗联合免疫往往优于使用单价疫苗多次接种。多价疫苗联合免疫时，免疫效果与单价疫苗相似或者更佳，而且未增加疫苗的副反应。
现有技术的缺陷与不足：鼻咽癌、胃癌等EBV相关肿瘤由于早期发现困难，中晚期治疗还是以传统治疗为主，而传统的手术、放化疗对肿瘤细胞的抑制率在20％左右，而对淋巴细胞的抑制率在90％左右，过度化疗会缩短患者的生存时间；部分肿瘤对药物不敏感，采用化疗没有临床价值，经常出现了有些肿瘤经放化疗后迅速缩小，但由于肿瘤细胞并没有消灭，肿瘤又迅 速长大，同时出现近处及远处广泛转移，在治疗癌症的同时加速了癌细胞的转移，也摧毁了免疫系统，使一些患者不是死于癌症，而是死于过度治疗后的并发症。目前临床上急需寻找新的治疗癌症的药物或方法。与传统的治疗方法相比，免疫治疗由于其疗效显著、副作用小等优势，正逐渐成为新的癌症治疗手段，其中DC疫苗正发挥越来越重要的作用，但单价DC疫苗治疗往往疗效有限。虽然有不同的抗原类型及组合形式用以治疗各种疾病，但是由于肿瘤中的细胞大多来源于人体自身，单一的肿瘤抗原信息容易造成肿瘤细胞的免疫逃逸，引起的免疫反应无法成功对肿瘤造成杀伤，治疗效果有限。另外在体内由于很多肿瘤细胞可以分泌多种抑制树突状细胞成熟的细胞因子，使存在于肿瘤部位的树突状细胞数量相对较少，而缺少强烈肿瘤抗原刺激的树突状细胞诱导的抗肿瘤的免疫反应也无法在宿主体内起到非常显著的治疗效果。
发明内容
针对现有技术的不足，本发明的目的是提供一种肿瘤复合抗原、树突状细胞多价疫苗及其应用。本发明通过在体外刺激患者自身的树突细胞，负载具有针对不同EBV相关肿瘤的超强免疫原性的多种肿瘤细胞裂解物(如SNU-719，YCCEL1，GD1，B95-8，M81，HKNPC1～9等不同EB病毒株来源的人永生化B淋巴样母细胞系(LCLs)肿瘤细胞或C666-1，HNE1、CCL85等EBV阳性的肿瘤细胞的裂解物)，并在多种细胞因子、特定激动剂诱导下成为成熟的树突细胞，形成完整的带有相应癌抗原的DC疫苗，回输到人体激活免疫系统，刺激天然免疫(如诱导NK细胞)和刺激淋巴细胞产生获得性免疫反应，产生细胞毒性T细胞杀伤癌细胞，一同精准杀伤鼻咽癌细胞，实现个性化治疗；相较于放化疗，特别安全，几乎没有副作用；且树突细胞疫苗制作周期1周左右，时间短，成本低。
为实现上述目的，本发明的技术方案如下：
第一方面，本发明提供一种肿瘤复合抗原，其特征在于：所述复合抗原包括不同EB病毒株来源的人永生化B淋巴样母细胞系肿瘤细胞裂解物或/和EBV阳性癌细胞裂解物；所述不同EB病毒株来源的人永生化B淋巴样母细胞系肿瘤细胞裂解物为GD1、B95-8、M81、HKNPC1～9、SNU-719或/和YCCEL1中任一种或几种组合物；所述EBV阳性癌细胞裂解物为C666-1、HNE1或/和CCL85中任一或多种组合物。
第二方面，本发明提供一种树突状细胞多价疫苗，其特征在于：所述树突状细胞多价疫苗载有所述权利要求1中的肿瘤复合抗原；载有所述肿瘤复合抗原的树突状细胞多价疫苗负载一种或两种EBV相关癌症的肿瘤细胞裂解物或LCLs肿瘤细胞裂解物，或者同时负载三种或多种 EBV相关癌症的肿瘤细胞裂解物或LCLs肿瘤细胞裂解物。
作为优选方案，所述不同EB病毒株来源的人永生化B淋巴样母细胞系肿瘤细胞裂解物为GD1，B95-8，M81，HKNPC1～9，SNU-719，YCCEL1等EBV病毒转化得到的人永生化B淋巴样母细胞系中任一种或多种；所述癌细胞裂解物为C666-1、HNE1或CCL85。
进一步地，所述树突状细胞多价疫苗包含第一佐剂或其他辅助治疗的细胞因子。
更进一步地，所述第一佐剂为PloyI:C，LPS或OK432中任一种；所述他辅助治疗的细胞因子为TNF-α或IL-12。
更进一步地，所述第一佐剂为PloyI:C，LPS或OK432中任一种；所述他辅助治疗的细胞因子为TNF-α或IL-12。
更进一步地，所述每种肿瘤细胞裂解物具体用量在2.5*10 7-2.5*10 9个。
第三方面，本发明提供一种如上述的肿瘤复合抗原在制备预防或治疗EBV相关肿瘤药物中的应用。
作为优选方案，所述肿瘤包括EBV相关胃癌，EBV阳性淋巴瘤，鼻咽癌以及EBV相关的其他上皮细胞癌症。
进一步地，所述药物包括如权利要求2-7中任一所述的树突状细胞多价疫苗。
本发明提供的能刺激机体免疫应答来治疗EBV相关肿瘤的树突状细胞多价疫苗，特别是EBV相关胃癌，EBV阳性淋巴瘤，鼻咽癌以及EBV相关的其他上皮细胞癌症，具有良好疗效，且副作用小，能长期高效抑制EBV相关肿瘤的生长和减轻癌症的疾病进程，甚至达到完全缓解状态。
在一些实施方案的抗原致敏的树突细胞群属免疫原性组合物，所述树突状细胞群已被相应抗原负载。具体的抗原包含GD1，B95-8，M81，HKNPC1～9，SNU-719，YCCEL1等不同EB病毒株来源的人永生化B淋巴样母细胞系(LCLs)肿瘤细胞裂解物和C666-1，HNE1,CCL85等癌细胞裂解物，每种肿瘤细胞裂解物具体用量在2.5*10 7-2.5*10 9个。在本发明的另一些方面中,载有肿瘤复合抗原的树突状细胞疫苗，可以是只负载两种EBV相关癌症的肿瘤细胞裂解物或LCLs肿瘤细胞裂解物的树突状细胞多价疫苗，也可同时负载三种，甚至多种EBV相关癌症的肿瘤细胞裂解物或LCLs肿瘤细胞裂解物的树突状细胞多价疫苗。
在某些方面，树突状细胞多价疫苗可以包含第一佐剂(Ploy(I:C)，LPS，OK432等)或其他辅助治疗的细胞因子如TNF-α、IL-12等。树突状细胞多价疫苗以经静脉内、皮内、肿瘤内、肌肉内、腹膜内、结内、皮下或局部施用被给予3-30次，每次间隔一周或两周；树突状细胞多 价疫苗每次回输细胞量在5*10 6-5*10 8个。
本发明的优点及有益效果如下：
树突状细胞多价疫苗是一种通过激活患者自身的免疫系统达到对抗癌症目的的疫苗。与传统EBV相关肿瘤的治疗相比较，有以下优点：
1、手术、化疗还是放疗，在杀死癌细胞的同时都极大伤害病人身体，大大降低患者自身免疫抵抗力；由于每个病人肿瘤异质性的不同，所以绝大多数抗癌药，尤其是新一代的靶向药物，都只对一小部分病人有效；癌细胞进化很快，抗药性很容易出现，导致癌症复发率很高。相对传统化疗或靶向治疗，树突状细胞多价疫苗为肿瘤的治疗提供了新的思路，其在体内直接针对的是免疫细胞，通过激活自身免疫系统杀伤癌症细胞，它不会造成直接损伤，反而增强免疫系统；可以抑制癌细胞进化，复发率低，且副作用整体而言显著小于传统的化疗，也小于多靶点的靶向药物，比如树突状细胞多价疫苗作用机制是激活免疫系统，因此最常见的副作用仅为临床I/II级不良反应，如发热、乏力、头晕、全身肌肉酸痛、嗜睡等，对症处理即可，具有良好的临床应用前景。
2、树突状细胞多价疫苗对抗原的选择对于治疗EBV相关肿瘤更为有效和安全。目前国际上用DC疫苗治疗EBV相关肿瘤的项目，多是选用特定的一段多肽作为抗原。而本发明的树突状细胞多价疫苗选用的是多种肿瘤细胞裂解物，包含激活免疫应答针对癌细胞的全部抗原，最大程度的负载实际人体中的肿瘤抗原信息，加强了树突细胞呈递抗原的多样性，最大限度激活人体免疫功能，可诱导更强的T细胞反应，从而提高产品的治疗效果。
3、树突状细胞多价疫苗所选用的肿瘤细胞裂解物来源于稳定的肿瘤细胞系，无需耗时耗力筛选每个患者的肿瘤抗原，并且无需多虑患者的人类白细胞抗原(Human Leukocyte Antigen，HLA)与抗原肽的结合，没有HLA限制性，属于通用抗原。此抗原易于制备，工艺简化，并可实现肿瘤裂解物的质量均一，免去了复杂的抗原筛选过程，在降低成本、节约时间方面显示了突出的优势。
4、树突状细胞多价疫苗治疗效果更为持久，能长期有效抑制EBV相关肿瘤复发和转移。树突状细胞多价疫苗在回输患者体内后可使患者体内产生数量庞大且含有多种肿瘤抗原信息的记忆T淋巴细胞，其存在时间长达几年至几十年。当再次遇到相应刺激后，可迅速在体内活化，杀伤肿瘤细胞，有效防止肿瘤的复发和转移。
5、树突状细胞多价疫苗采用的多种通用型肿瘤抗原，适用于绝大部分带有EBV特异性抗 原信息的EBV相关肿瘤患者，因无需对每个患者的肿瘤抗原进行筛选，整个制备周期仅需1周，较新抗原DC疫苗的制备节约几个月的时间，对癌症患者尤其是晚期患者具有重大意义。且通用型抗原使得抗原的制备易于标准化、规范化，大大降低生产和质控的成本。
附图说明
图1：永生化人B淋巴细胞系LCLs表面CD19表达水平；
图中：利用流式细胞术检测B95-8细胞(阴性对照，CD19-B95-8(Negative control))，LCLs-Positive control细胞(阳性对照,CD19-LCL(Positive control))，以及构建的EB病毒转化的LCLs细胞(CD19-LCLs)表面FITC-CD19的表达，发现CD19-LCLs细胞表面的CD19表达高于同型对照(Iso-mIgG1-FITC)和CD19-B95-8(Negative control)，同阳性对照CD19-LCL(Positive control)表达量相当，证实已成功构建的新LCLs，可用于下一步实验。
图2：4名鼻咽癌患者血浆和癌组织中EBV病毒载量；
图中：通过提取4名鼻咽癌患者血浆和鼻咽癌组织中的病毒DNA，并利用EB病毒特异性检测试剂盒进行RT-qPCR，计算出患者体内EB病毒载量；发现I,II号EBV阳性鼻咽癌患者血浆和鼻咽癌组织中存在大量EBV病毒，且癌组织中的EBV-DNA拷贝数远高于血浆中存在的EBV数量；而III,IV号EBV阴性鼻咽癌患者体内基本检测不到EB病毒，反映EBV阳性鼻咽癌患者体内EBV的生命活动十分旺盛。
图3：成熟树突细胞形态图；
图中：取成熟的树突细胞培养皿置于光学显微镜(20X物镜)下观察，可以看到成熟的树突细胞贴壁生长，细胞表面突起增多、变粗且变长，呈长条放射状，有明显树突状形态。
图4：树突细胞表面标志分子表达量流式图；
图中：取部分未成熟DC和成熟DC细胞进行流式细胞术检测
PE-CD11c/FITC-CD14/FITC-CD40/FITC-CD80/FITC-CD83/FITC-CD86/FITC-HLA-DR/FITC-HLA-ABC等细胞表面因子的表达，粉色线条代表相应抗体同型对照的流式图谱，红色代表未成熟DC表面分子的流式图谱，黄色代表成熟DC表面分子的流式图谱。从图中可以看出，成熟树突细胞其表面CD11c,CD40,CD83,CD86,HLA-DR,HLA-ABC等分子表达高于未成熟树突细胞，呈现高表达，证实树突细胞已被诱导成熟。
图5：体外刺激诱导的CTL特异性淋巴细胞杀伤率；
图中：在体外，接受树突状细胞多价疫苗刺激的T淋巴细胞与只接受负载自体肿瘤细胞 裂解物的树突细胞单价疫苗刺激的T淋巴细胞或对照组(a-I组)相比，通过检测收集的细胞对肿瘤患者鼻咽癌细胞的杀伤活性发现，树突状细胞多价疫苗刺激的T淋巴细胞在杀伤EBV阳性鼻咽癌患者的肿瘤细胞(I,II号患者)时表现出强烈的杀伤能力，且T淋巴细胞越多，杀伤效果越明显，而对于EBV阴性鼻咽癌患者的肿瘤细胞(III,IV号患者)则无法有效杀伤，同时，不同患者来源的树突状细胞单价疫苗刺激产生的CTL细胞，在杀伤自体肿瘤细胞的效果都很显著，与树突状细胞多价疫苗应用于EBV阳性鼻咽癌肿瘤细胞相比，其杀伤肿瘤细胞的效果相当；说明负载有多种EBV阳性肿瘤细胞裂解物的树突状细胞，可以获得自体肿瘤细胞裂解物负载的树突状细胞单价疫苗同样的肿瘤杀伤效果，避免了采集每个EBV阳性鼻咽癌患者肿瘤组织的繁琐过程，可广泛识别EBV阳性鼻咽癌肿瘤细胞，并刺激T细胞的增殖，发挥免疫学效应。
图6:干扰素γ的分泌量；
图中：将各组CTL细胞与鼻咽癌患者的肿瘤组织细胞按20:1效靶比共培养，检测淋巴细胞分泌干扰素γ的量，发现负载自体肿瘤细胞裂解物的树突状细胞单价疫苗刺激的4名鼻咽癌患者T淋巴细胞，与肿瘤细胞共培养后，均可产生大量干扰素γ，显著高于对照组，同时树突细胞多价疫苗组也能强烈刺激激活患者I、II的T细胞并产生大量干扰素γ，而患者III、IV两名EBV阴性鼻咽癌患者的T细胞在接受树突细胞多价疫苗的刺激后，与自身EBV阴性鼻咽癌肿瘤共培养时无法有效产生干扰素γ；说明负载有EBV阳性肿瘤细胞裂解物的树突状细胞多价疫苗能强烈促进EBV阳性鼻咽癌患者自体的T淋巴细胞的分化，分泌干扰素γ，促进机体的抗肿瘤能力。
图7：未成熟DC表面共刺激因子的表达情况；
图中：通过流式细胞术检测未成熟树突细胞表面共刺激因子的表达，发现CD11c高于对照呈现弱表达，CD80、CD83未表达，CD40、CD86有不同程度的高表达，单核细胞表面特异性标志CD14为阴性，证实CD14单核细胞已成功分化为未成熟的树突细胞。
图8：4名胃癌患者血浆中EBV病毒载量；
图中：通过提取4名胃癌患者癌组织和血浆中的病毒DNA，并利用EB病毒特异性检测试剂盒进行RT-qPCR，计算出患者相应位置中的EB病毒载量；发现A,B号EBV阳性胃癌患者血浆中EB病毒载量大于6000拷贝，癌组织中的EB病毒载量大于17000拷贝，体内存在大量EBV病毒，而C,D号EBV阴性胃癌患者血浆中基本检测不到EB病毒，说明EBV阳性胃癌患者体内存在大量EB病毒。
图9：各组疫苗培养上清中IL12的表达；
图中：通过ELISA检测各组DC疫苗培养基上清中的IL12表达量，发现不同患者来源的多价疫苗和单价疫苗分泌的IL12表达量明显高于对照组正常成熟DC的IL12分泌，不同患者间的多价疫苗或单价疫苗间，其IL12的表达量差异不大，说明胃癌肿瘤细胞裂解物和胃癌相关肿瘤抗原均可以刺激DC分泌IL12，负载有多种EBV阳性胃癌的肿瘤抗原信息的多价疫苗能与患者自体的肿瘤细胞裂解物的单价疫苗有同样刺激DC成熟分泌IL12的能力。
图10：体外刺激诱导的CTL特异性淋巴细胞杀伤率；
图中：Poly-DC+T-A组表示，负载多种肿瘤细胞裂解物的DC多价疫苗组刺激的CTL细胞作为效应细胞，患者A的胃癌肿瘤细胞作为靶细胞，按照效靶比5：1，10:1，20:1培养作用，依次类推；从结果可以看出，负载有自体胃癌肿瘤组织细胞裂解物的单价疫苗组刺激产生的CTL细胞对于自体的胃癌组织肿瘤均有显著的杀伤效果，同时多价疫苗组刺激产生的CTL细胞在遇到自体来源的EBV阳性肿瘤细胞(患者A，患者B)时，也展现了强大的肿瘤杀伤能力；但针对EBV阴性的胃癌细胞(患者C，患者D)时却没有显著提高其T细胞的肿瘤杀伤效应；说明当自体肿瘤细胞表面存在有与多价疫苗本身相似EBV抗原时，会诱导机体产生强烈的免疫反应，反之，若肿瘤细胞为EBV阴性时，负载有EBV抗原信息的多价树突细胞疫苗无法有效刺激激活机体抗肿瘤免疫反应。证明Poly-DC多价疫苗能有效抑制EBV阳性胃癌肿瘤的生长，具备更为广泛的应用性。
图11：IFN-γ的分泌量；
图中：从图中可以发现单价疫苗组的CTL与自体的胃癌肿瘤细胞共培养时，都产生了大量干扰素γ，多价疫苗组的CTL在与不同患者来源的EBV阳性胃癌肿瘤细胞共培养中，分泌了大量的干扰素γ，而与EBV阴性鼻咽癌肿瘤共培养时无法有效产生干扰素γ，同对照组相比，不存在显著性差异；说明采用通用性EBV肿瘤细胞裂解物作为抗原信息，可有效避免个体化EBV阳性肿瘤细胞差异，加速树突细胞疫苗的产业化，证实负载有EBV阳性肿瘤细胞裂解物多价疫苗能刺激激活免疫系统识别杀伤EBV阳性的胃癌肿瘤，具备强大而广泛的促进免疫功能的效果。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离 本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：树突状细胞多价疫苗治疗鼻咽癌免疫学研究
本实施例中选用的有两名EBV阳性鼻咽癌患者(记为I，II)的肿瘤细胞和两名EBV阴性鼻咽癌患者(记为III，IV)的肿瘤细胞(已获得患者知情同意)。
一、从人体静脉血中分离外周血单核细胞
本实施方式基于外周血中各细胞成分密度的差异性(外周血主要含有血小板、单个核细胞、粒细胞、红细胞等细胞：血小板密度为1.030～1.035kg/m 3，单个核细胞密度为1.075～1.090kg/m 3，粒细胞密度为1.092kg/m 3，红细胞密度为1.093kg/m 3)，在外周血样品中加入
Figure PCTCN2021110455-appb-000001
Paque Plus(GE Healthcare)溶液(密度为1.075～1.089kg/m 3)，进行密度梯度离心，使不同细胞成分分层，能快速从人体外周血中分离得到单核细胞。
1)从鼻咽癌患者的静脉采集外周血，用相应规格的离心管，使用移液管在两个新的离心管中分别加入4.5mL的
Figure PCTCN2021110455-appb-000002
Paque Plus溶液。
2)使用移液管吸取血样并沿着离心管管壁缓慢地注入Ficoll溶液上层，每管10mL。室温，800g离心20min。
3)取出离心管，样品分为四层，从上到下依次为血浆、单核细胞、Ficoll溶液、红细胞与粒细胞。
4)小心吸取单核细胞转移至一个15mL离心管中，加入PBS/1％FBS溶液补齐至14mL，吸打混匀。室温，800g离心5min。
5)去上清，轻弹管底部使细胞松散，加入14mL PBS/1％FBS溶液重悬细胞吸打混，室温，700g离心5min。
6)去上清，轻弹管底部使细胞松散。加入14mL RPMI/10％FBS溶液重悬细胞吸打混，室温，400g离心5min。
7)去除上清，轻弹管底部使细胞松散。加入10mL RPMI/10％FBS溶液重悬细胞，吹打混匀。
8)吸取10μL细胞液至一个新的1.5mL离心管，加入90μL RPMI/10％FBS溶液稀释10倍；吸取10μL稀释细胞液，加入10μL Trypan Blue染色后加至血球计数板，在倒置显微镜下计数。
9)室温，700g离心5min，去除上清，加入适量PBS/1％FBS用于后续试验。
二、分离获取DC及T淋巴细胞
2.1CD14单核细胞的分离提取方法包括但不限于本实施例中采用CD14 +磁珠分选，还可以是CD14阴选，美天旎免疫磁珠细胞分选(MACS)，细胞贴附等方法。其原理是基于抗原抗体的特异性结合特点，CD14 +磁珠分选试剂盒可以特异性识别结合PBMC中的CD14 +细胞，并通过生物素或葡聚糖间接与磁珠偶联，在高强度磁场作用下达到CD14 +细胞分离目的。本实施例选用EasySep TMCD14阳选试剂盒。
1)将PBMC细胞悬液转移至5mL流式管。
2)在流式管中加入适量selection cocktail溶液至终浓度为100μL/mL。充分吸打混匀，室温孵育10min。
3)准备磁珠，将RapidSphere TM溶液涡旋30s使磁珠颗粒均匀分散。
4)在流式管中加入适量RapidSphere TM溶液至终浓度为100μL/mL，充分吸打混匀，室温孵育3min。
5)在流式管中加入适量PBS/2％FBS with 1mM EDTA溶液至总体积为2.5mL，充分吸打混匀。
6)将流式管垂直插入EasySep TM magnet，室温孵育3min。
7)倒置磁铁，收集流式管流出的细胞液至15mL离心管，保持磁铁倒置状态3s，不要摇晃或吸干管壁上的液体。
8)将磁铁正置，取出流式管。
9)重复步骤7-10两次。
10)在流式管中加入2mL RPMI/10％FBS重悬细胞，Trypan Blue细胞计数。
2.2 CD14 +单核细胞诱导产生未成熟树突细胞实验方案
在体外，粒细胞-巨噬细胞集落刺激因子(granμLocyte-macrophage colony stimμLating factor,GM-CSF)能促进iDC的存活，诱导iDC大量增殖。白细胞介素4(Interleukin-4,IL-4)能抑制巨噬细胞的过度生长，降低细胞表面表达CD14分子，诱导CD14 +单核细胞向iDC分化。
1)在超净工作台内使用移液管吸取CD14 +细胞液转移至六孔板中，每孔2x10 6个/mL,往六孔板中继续加入1μL人重组GM-CSF(20-200ng/μL)和1μL人重组IL-4(10-100ng/μL)。
2)将六孔板置于超净工作台台面上，前后左右各轻轻晃动3次使细胞分散均匀。置于细胞培养箱中37℃，5％CO 2培养3天。
3)从培养箱取出六孔板，在超净工作台内向六孔板中继续加入2mL RPMI 1640/10％ FBS、1μL人重组GM-CSF和1μL人重组IL-4。
4)将六孔板置于超净工作台台面上，前后左右各轻轻晃动3次使成分分散均匀。置于细胞培养箱中37℃，5％CO 2培养2天。
2.3负载肿瘤细胞裂解物制备树突细胞多价疫苗
1)构建EB病毒株感染的永生化人B淋巴细胞系LCLs
a)从外周血分离得到PBMC，重悬于2ml RPMI1640/10％FBS培养基中。
b)吸取10μL细胞液，加入90μL RPMI/10％FBS稀释10倍，在显微镜下进行细胞计数。
c)根据计数结果，计算所需B95-8上清液的体积，其中每1×10 6个PBMC细胞对应500μL B95-8上清液。
d)提前两天培养10ml B95-8细胞，初始密度为1x10 6个/ml，置于培养箱培养48h后吸取B95-8细胞上清液转移至离心管中，配平后2000rpm离心15min。剩余B95-8细胞经灭菌处理后丢弃。
e)使用0.45μm滤膜对离心管内的B95-8细胞上清液过滤待用。
f)收集PBMC细胞。1000rpm离心5min，弃去PBMC上清液。
g)根据细胞计数结果，加入适量B95-8细胞上清液或者其他EB病毒悬浮液(如GD1，B95-8，M81，HKNPC1～9，SNU-719，YCCEL1等)重悬PBMC细胞，使细胞液中PBMC细胞浓度为1×10 6/500μL。
h)准备一个无菌96孔板，将B95-8重悬的PBMC细胞液以每孔100μL的标准转移至96孔板中。
i)将96孔板置于CO 2培养箱培养24h。
j)取出96孔板，在每孔中继续加入100μL R20培养基(RPMI1640/20％FBS,100U/mL青霉素,100μg/mL链霉素)吸打混匀。
k)将96孔板置于培养箱继续培养6天，每天观察细胞状态。观察细胞形态是否呈现淋巴母细胞样改变：细胞体积增大，胞质丰富，呈球状，呈现小集落聚集分布，孔底细胞团明显增大，培养基颜色变黄。
l)6天培养完成后，每隔3天更换一次培养基。小心地吸取每孔上层100μL培养液弃去，然后每孔加入100μL R20培养基，重悬孔内细胞，注意细胞液变黄时及时更换培养基，或是根据需要分至新的2-4孔中培养，待细胞数慢慢变多，依次合 并转入24孔板，6孔板，T25瓶中。
m)对达到四周培养时长的细胞进行镜检观察细胞状态。使用流式细胞术分析细胞表面CD19表达水平。得图1：永生化人B淋巴细胞系LCLs表面CD19表达量。
2)肿瘤细胞获得
取得患者的肿瘤组织后，立即用含有1000u/ml青霉素、链霉素及两性霉素2μg/ml的培养液漂洗5-10min，再用无血清培养液反复冲洗干净；在超净工作台上取肿瘤组织边缘增殖旺盛的组织，用锋利手术刀片切碎组织，尽可能减少对组织的机械损伤；用0.5％的Ⅳ型胶原酶和0.5％的透明质酸酶消化，37℃消化80min；用200目尼龙网过滤，1000rpm离心5min；用含有10％胎牛血清的RPMI1640完全培养基接种培养板，放置于37℃，5％CO 2培养箱培养；通过反复贴壁法纯化癌细胞。
鼻咽癌患者血液和癌组织中EBV病毒载量(Viral Load,VL)数测定：采集4例鼻咽癌患者血样。分别使用MagMAX病毒核酸提取试剂盒(Thermo A42352)和EBV Real-TM Quant试剂盒(Sacace BioTechnologies Srl，Como，意大利)进行DNA提取和聚合酶链反应(PCR)，按厂家提供的方法从100μL的血浆中提取DNA，用50μL的洗脱缓冲液洗脱。上述获得的鼻咽癌肿瘤组织细胞，根据DNeasy Blood&Tissue Kit(Qiagen,Cat.No.69506)产品说明书提取NPC患者鼻咽癌组织总DNA，用实时定量PCR(EBV Real-TM Quant Kit)对10微升的样品进行EBV定量。本实验选择EBNA1基因编码区作为扩增靶点，聚合酶链反应在最终体积为25μL，反应按照说明书操作。引物序列如下
EBNA1-FP：5’-CCAGACAGCAGCCAATTGTC-3’，如SEQ ID NO.1所示；
EBNA1-RP：5’-GGTAGAAGACCCCCTCTTAC-3’，如SEQ ID NO.2所示；
内参β-actin基因上游引物：5’-CTCCATCCTGGCCTCGCTGT-3’，如SEQ ID NO.3所示；
内参β-actin基因下游引物：5’-GCTGTCACCTTCACCGTTCC-3’，如SEQ ID NO.4所示；
比较4名患者血液和癌组织中EBV-DNA拷贝数，得图2。
反复冻融法，是一种常用的机械裂解方式，通常由冷冻和解冻两部分组成(freezing and thawing)。原理是由于细胞内冰粒形成和剩余细胞液的盐浓度增高引起溶胀，使细胞结构破碎，使细胞死亡，但是又保留有细胞的免疫原性。冷冻通常在液氮或-20℃冰上进行，解冻可以在37℃、50℃、65℃或100℃水浴中进热休克，比化学裂解温和。
a)预先设置水浴温度为37℃。
b)分别收集C666-1鼻咽癌细胞系和不同病毒株构建的永生化人B淋巴细胞系LCLs以及4名鼻咽癌患者的肿瘤细胞；(至少3x10 7个)，700g室温离心5min收获细胞。
c)去除上清液并重悬于RPMI/10％FBS中。
d)用台盼蓝计数细胞。
e)700g室温离心5min，缓慢制动，去除上清液。
f)用RPMI/10％FBS重新悬浮细胞于1ml冻存管中，密度为5x10 6/mL。
g)将细胞置于液氮中冷冻20s。
h)立刻将细胞在37℃水浴中快速完全解冻。
i)重复步骤7和8，4次。(共5次)
j)使用前将肿瘤细胞裂解物储存在液氮中，。
3)树突细胞疫苗的获得
a)应用细胞因子GM-CSF和IL-4使血液中单核细胞分化成不成熟树突状细胞后，培养至第5天时，
·树突细胞单价疫苗：用2.5x10 7个自体肿瘤细胞裂解物与5x10 6个树突状细胞共培养，共同作用至少2小时，再加入TNF-α等细胞因子刺激树突状细胞成熟，制备成树突细胞单价疫苗；
·树突细胞多价疫苗：取2.5x10 7个C666-1肿瘤细胞裂解物或M81-LCLs肿瘤细胞裂解物等不同的肿瘤细胞裂解物，分别与5x10 6个树突状细胞共培养，作用至少2小时，再加入TNF-α等细胞因子刺激树突状细胞成熟，将多个负载有肿瘤细胞抗原信息的树突状细胞同时等量混合在树突状细胞培养基中，制备成负载肿瘤细胞裂解物抗原的树突状细胞多价疫苗；
并利用流式细胞术检测未成熟树突细胞与成熟树突状细胞表面分子标志，如CD11c,CD14,CD40,CD80,CD83,CD86,HLA-DR,HLA-ABC等。得图3：成熟树突细胞形态图，图4：树突细胞表面标志分子表达量流式图。
b)每个DC疫苗中的树突细胞来源于患者自体，制备以下4种疫苗：
表1：
Figure PCTCN2021110455-appb-000003
*Ag-Poly表示包含有C666-1肿瘤细胞裂解物或B95-8、M81-LCLs肿瘤细胞裂解物等不同的肿瘤细胞裂解物抗原信息，DC-I表示I号EBV阳性鼻咽癌患者自身树突细胞，DC-II表示II号EBV阳性鼻咽癌患者自身树突细胞，DC-III表示III号EBV阴性鼻咽癌患者自身树突细胞，DC-IV表示IV号阴性鼻咽癌患者自身树突细胞；Ag-I表示I号鼻咽癌患者的肿瘤细胞裂解物抗原信息，Ag-II表示II号鼻咽癌患者的肿瘤细胞裂解物抗原信息，Ag-III表示III号鼻咽癌患者的肿瘤细胞裂解物抗原信息，Ag-IV表示IV号鼻咽癌患者的肿瘤细胞裂解物抗原信息,Poly-DC-I表示适用于I号患者的树突状细胞多价疫苗，Ag-DC-I表示负载有I号患者肿瘤细胞裂解物信息的适用于I号患者的树突状细胞单价疫苗，依此类推。
2.4.T淋巴细胞制备
1)将分离得到的同一人PBMC，置于37度5％CO 2培养箱2小时后，收集悬浮细胞，制备成1ml细胞悬液；
2)将细胞悬液加入37℃温育的尼龙毛柱，平放柱体，加入200μL预温的含10％FBS的RPMI 1640，用于封口，37℃静置孵育2h；
3)用10％FBS RPMI 1640清洗尼龙毛柱，流速大约为l ml/min，收集最初冼下的10ml细胞悬液，富含T细胞和NK细胞；
4)室温700g离心5min，收集底层细胞。计数并调整细胞浓度至1X l0 7个/ml，置于含80IU/ml的IL-2的RPMI1640完全培养基中备用。
或者可采用磁珠分离法，利用CD3 +磁珠分离T淋巴细胞。细胞先与抗表面抗原的单抗孵育12min，10 7个细胞用50μL抗CD3单抗，细胞经洗涤后与生物素标记的100μL羊抗鼠二抗孵育10min，洗涤后加入FITC标记的链霉亲和素25μL，反应8min，洗涤后加生物素标记的磁颗粒(加抗CD3单抗者加100μL磁颗粒)反应8min。上述每步反应后，均加入1ml的含 1％牛血清白蛋白的PBS洗涤，2000r/min离心10min。使用磁化细胞分离器(MACS)作免疫磁性分离，得到T淋巴细胞。
三、体外刺激诱导CTL细胞的获得
用RPMI完全培养基分别重悬树突状细胞多价疫苗与正常成熟的DC细胞，分别调整密度到2x 10 5个/ml；将分离得到的自体T淋巴细胞悬液，用RPMI完全培养基调整密度到1.6x 10 6个/ml。设置以下实验组，每组各加入1mL相应的树突状细胞疫苗和T淋巴细胞，如表2：
表2：
DC-I +                
Poly-DC-I   +              
Poly-DC-II     +            
Poly-DC-III       +          
Poly-DC-IV         +        
Ag-DC-I           +      
Ag-DC-II             +    
Ag-DC-III               +  
Ag-DC-IV                 +
T-I + +       +      
T-II     +       +    
T-III       +       +  
T-IV         +       +
分组 a b c d E f g h i
*DC-I表示来源于I号患者的DC，T-I表示来源于I号患者的T淋巴细胞，T-II表示来源于II号患者的T淋巴细胞，T-III表示来源于III号患者的T淋巴细胞，T-IV表示来源于IV号患者的T淋巴细胞。
以上实验组均加入同样的辅助细胞因子，IL-2含量为1000U/ml、IL-12含量为1500U/ml、Poly(I:C)含量为10mg/ml和TNF-α含量为1000U/ml等。在37℃、5％CO 2恒温恒湿培养箱中培养2周后，加入终浓度为30U/ml的IL-2，以及各组再加入2x 10 5个相应的T细胞或疫苗进行二次刺激，继续培养一周，于第21天收集细胞。
四、检测收集的细胞对不同患者鼻咽癌细胞的杀伤活性
将上述收集的部分细胞离心后用RPMI1640完全培养基悬浮，调整细胞浓度，分三个不同效靶比实验组，每组分别每孔4x 10 5、2x 10 5、1x 10 5加入96孔培养板作效应细胞；设置不同鼻咽癌患者的肿瘤细胞作为靶细胞，向每孔中加入2x 10 4个鼻咽癌患者的肿瘤细胞作为靶细胞，终体积为200ul，设置组如表3。同时设置有无淋巴细胞对照组和无细胞的空白培养液对照组，均设5复孔。24h后吸去各孔中游离的效应细胞，PBS洗涤2次，每孔各加入含20μl CCK8试剂100μl，继续培养2h，酶标仪检测450nm处吸光度值(OD)，计算特异性淋巴细胞杀伤率(％)，得图5：体外刺激诱导的CTL特异性淋巴细胞杀伤率。
表3：
Figure PCTCN2021110455-appb-000004
五、体外检测干扰素γ的分泌
将上述各组CTL效应细胞和4个鼻咽癌患者的肿瘤细胞，按照效靶比20：1混合于U型底96孔板中培养72h后，使用干扰素γ酶联免疫试剂盒，按照说明书流程检测培养上清中IFN-γ的含量，得图6。
实施例2：树突状细胞多价疫苗抗EBV阳性胃癌免疫学效应研究
本实施例中选用的两名EBV阳性的胃癌患者(A，B)和两名EBV阴性胃癌患者(C，D)，均已获得患者知情同意。
1、从人体静脉血中分离外周血单核细胞
本实施方式基于外周血中各细胞成分密度的差异性(外周血主要含有血小板、单个核细胞、粒细胞、红细胞等细胞：血小板密度为1.030～1.035kg/m 3，单个核细胞密度为1.075～1.090kg/m 3，粒细胞密度为1.092kg/m 3，红细胞密度为1.093kg/m 3)，在外周血样品中加入
Figure PCTCN2021110455-appb-000005
Paque Plus(GE Healthcare)溶液(密度为1.075～1.089kg/m 3)，进行密度梯度离心，使不同细胞成分分层，能快速从人体外周血中分离得到单核细胞。
1)从胃癌患者的静脉采集外周血，用相应规格的离心管，使用移液管在两个新的离心管中分别加入4.5mL的
Figure PCTCN2021110455-appb-000006
Paque Plus溶液。
2)使用移液管吸取血样并沿着离心管管壁缓慢地注入Ficoll溶液上层，每管10mL。室温，800g离心20min。
3)取出离心管，样品分为四层，从上到下依次为血浆、单核细胞、Ficoll溶液、红细胞与粒细胞。
4)小心吸取单核细胞转移至一个15mL离心管中，加入PBS/1％FBS溶液补齐至14mL，吸打混匀。室温，800g离心5min。
5)去上清，轻弹管底部使细胞松散，加入14mL PBS/1％FBS溶液重悬细胞吸打混，室温，700g离心5min。
6)去上清，轻弹管底部使细胞松散。加入14mL RPMI/10％FBS溶液重悬细胞吸打混，室温，400g离心5min。
7)去除上清，轻弹管底部使细胞松散。加入10mL RPMI/10％FBS溶液重悬细胞，吹打混匀。
8)吸取10μL细胞液至一个新的1.5mL离心管，加入90μL RPMI/10％FBS溶液稀释10倍；吸取10μL稀释细胞液，加入10μL Trypan Blue染色后加至血球计数板，在倒置显微镜下计数。
9)室温，700g离心5min，去除上清，加入适量PBS/1％FBS用于后续试验。
2、CD14 +单核细胞诱导产生未成熟树突细胞实验方案
在体外，粒细胞-巨噬细胞集落刺激因子(granμLocyte-macrophage colony stimμLating factor,GM-CSF)能促进iDC的存活，诱导iDC大量增殖。白细胞介素4(Interleukin-4,IL-4)能抑制巨噬细胞的过度生长，降低细胞表面表达CD14分子的能力，诱导CD14+单核细胞向iDC分化。
1)在超净工作台内使用移液管吸取CD14 +细胞液转移至六孔板中，每孔2x10 6个/mL,往六孔板中继续加入1μL人重组GM-CSF(20-200ng/μL)和1μL人重组IL-4(10-100ng/μL)。
2)将六孔板置于超净工作台台面上，前后左右各轻轻晃动3次使细胞分散均匀。置于细胞培养箱中37℃，5％CO 2培养3天。
3)从培养箱取出六孔板，在超净工作台内向六孔板中继续加入2mL RPMI 1640/10％FBS、1μL人重组GM-CSF和1μL人重组IL-4。
4)将六孔板置于超净工作台台面上，前后左右各轻轻晃动3次使成分分散均匀。置于细胞培养箱中37℃，5％CO 2培养2天。收集部分未成熟DC，通过流式细胞术检测其表面共刺激因子的表达情况，得图7。
3、EBV阳性胃癌患者肿瘤组织处理与肿瘤细胞裂解物制备方法同上。
取得患者的肿瘤组织后，立即用含有1000u/ml青霉素、链霉素及两性霉素2μg/ml的培养液漂洗5-10min，再用无血清培养液反复冲洗干净；在超净工作台上取肿瘤组织边缘增殖旺盛的组织，用锋利手术刀片切碎组织，尽可能减少对组织的机械损伤；用0.5％的Ⅳ型胶原酶和0.5％的透明质酸酶消化，37℃消化80min；用200目尼龙网过滤，1000rpm离心5min；用含有10％胎牛血清的RPMI1640完全培养基接种培养板，放置于37℃，5％CO 2培养箱培养；通过反复贴壁法结合机械划除纯化癌细胞。
胃癌患者血液和胃癌组织样本中EBV病毒载量(Viral Load,VL)数测定：采集4例胃癌患者血样。分别使用MagMAX病毒核酸提取试剂盒(Thermo A42352)和EBV Real-TM Quant试剂盒(Sacace BioTechnologies Srl，Como，意大利)进行DNA提取和聚合酶链反应(PCR)。按厂家提供的方法从100μL的血浆中提取DNA，用50μL的洗脱缓冲液洗脱。上述获得的胃癌肿瘤组织细胞，根据DNeasy Blood&Tissue Kit(Qiagen,Cat.No.69506)产品说明书提取其总DNA，用实时定量PCR(EBV Real-TM Quant Kit)对10微升的样品进行EBV定量。本实验选择EBNA1基因编码区作为扩增靶点，聚合酶链反应在最终体积为25μL，反应按照说明书操作。
引物序列如下：
EBNA1-FP：5’-CCAGACAGCAGCCAATTGTC-3’，如SEQ ID NO.1所示；
EBNA1-RP：5’-GGTAGAAGACCCCCTCTTAC–3’，如SEQ ID NO.2所示；
内参β-actin基因上游引物：5-CTCCATCCTGGCCTCGCTGT-3’，如SEQ ID NO.3所示；
内参β-actin基因下游引物：5’-GCTGTCACCTTCACCGTTCC-3’，如SEQ ID NO.4所示；
比较4名患者血浆和肿瘤组织中的EBV-DNA拷贝数，得图8。
4、EBV阳性胃癌的树突状细胞疫苗制备
应用细胞因子GM-CSF和IL-4使血液中单核细胞分化成不成熟树突状细胞后，孵育至第5天加入EBV相关胃癌病毒株构建的LCLs(如SNU719-LCLs，YCCEL1-LCLs等)或胃癌肿瘤细胞系裂解物(GT38、PT等)与树突细胞共培养2小时后，再加入TNF-α等细胞因子刺激树突状细胞成熟。
·树突细胞单价疫苗：用2.5x10 7个自体肿瘤细胞裂解物与5x10 6个树突状细胞共培养，共同作用至少2小时，再加入TNF-α等细胞因子刺激树突状细胞成熟，制备成树突细 胞单价疫苗；
·树突细胞多价疫苗：取2.5x10 7个YCCEL1-LCLs、GCEBV-LCLs肿瘤细胞裂解物或GT38、PT等EBV阳性胃癌细胞系裂解物等不同的细胞裂解物，分别与5x10 6个树突状细胞共培养，共同作用至少2小时，再加入TNF-α等细胞因子刺激树突状细胞成熟，将多个负载有肿瘤细胞抗原信息的树突状细胞同时等量混合在树突状细胞培养基中，制备成负载肿瘤细胞裂解物抗原的树突细胞多价疫苗。
按照表4制备以下4种疫苗，并收集正常培养的树突状细胞和各组疫苗培养基上清检测IL12的表达，得图9：
表4：
Figure PCTCN2021110455-appb-000007
*Ag-Poly表示包含有YCCEL1-LCLs、GCEBV-LCLs肿瘤细胞裂解物或GT38、PT等EBV阳性胃癌细胞系等多种肿瘤细胞裂解物抗原信息，DC-A表示A患者自身树突细胞，DC-B表示B患者自身树突细胞，DC-C表示C患者自身树突细胞，DC-D表示D患者自身树突细胞,，Ag-A表示A号胃癌患者的肿瘤细胞裂解物抗原信息，Ag-B表示B号胃癌患者的肿瘤细胞裂解物抗原信息，Ag-C表示C号胃癌患者的肿瘤细胞裂解物抗原信息，Ag-D表示D号胃癌患者的肿瘤细胞裂解物抗原信息,Poly-DC-A表示适用于A号患者的树突状细胞多价疫苗，Ag-DC-A表示负载有A号患者肿瘤细胞裂解物信息的适用于A号患者的树突状细胞单价疫苗，依此类推。
5、T淋巴细胞制备
采用磁珠分离法，利用CD3 +磁珠分离T淋巴细胞。细胞先与抗表面抗原的单抗孵育12min，10 7个细胞用50μL抗CD3单抗，细胞经洗涤后与生物素标记的100μL羊抗鼠二抗孵育10min，洗涤后加入FITC标记的链霉亲和素25μL，反应8min，洗涤后加生物素标记的磁珠颗粒(加抗CD3单抗者加100μL磁珠颗粒)反应8min。上述每步反应后，均加入1ml的含1％牛血清白蛋白的PBS洗涤，2000r/min离心10min。使用磁化细胞分离器(MACS)作免疫磁性分离， 得到T淋巴细胞。
6、体外刺激诱导CTL细胞的获得：
用RPMI完全培养基分别重悬树突状细胞多价疫苗、树突状细胞单价疫苗与正常成熟的DC细胞，分别调整密度到2x 10 5个/ml；将分离得到的自体T淋巴细胞悬液，用RPMI完全培养基调整密度到1.6x 10 6个/ml。设置以下实验组，每组各加入1mL相应的树突状细胞疫苗和T淋巴细胞，如表5：
表5：
DC-A +                
Poly-DC-A   +              
Poly-DC-B     +            
Poly-DC-C       +          
Poly-DC-D         +        
Ag-DC-A           +      
Ag-DC-B             +    
Ag-DC-C               +  
Ag-DC-D                 +
T-A + +       +      
T-B     +       +    
T-C       +       +  
T-D         +       +
分组 a b c d e f g h i
*DC-A表示来源于A患者的DC；T-A表示来源于A患者的T淋巴细胞，T-B表示来源于B患者的T淋巴细胞，T-C表示来源于C患者的T淋巴细胞，T-D表示来源于D患者的T淋巴细胞。
以上实验组均加入同样的辅助细胞因子，IL-2含量为1000U/ml、IL-12含量为1500U/ml、Poly(I:C)含量为10mg/ml和TNF-α含量为1000U/ml等。在37℃、5％CO 2恒温恒湿培养箱中培养2周后，加入终浓度为30U/ml的IL-2，以及各组再加入2x 10 5个相应的T细胞或疫苗进行二次刺激，继续培养一周，于第21天收集细胞，用于检测多价疫苗的免疫学功能。
7、检测收集的CTL细胞对不同患者胃癌细胞的杀伤活性：
将上述收集的部分细胞离心后用RPMI1640完全培养基悬浮，调整细胞浓度，分三个不同效靶比实验组，三组分别每孔4x 10 5、2x 10 5、1x 10 5加入96孔培养板作效应细胞；设置不同EBV阳性胃癌患者的肿瘤细胞作为靶细胞，向每孔中加入2x 10 4个不同胃癌患者的肿瘤细 胞，终体积为200ul，设置组如表6。同时设置无淋巴细胞对照组和无细胞的空白培养液对照组，均设5复孔。24h后吸去各孔中游离的效应细胞，PBS洗涤2次，每孔各加入含20μl CCK8试剂100μl，继续培养2h，酶标仪检测450nm处吸光度值(OD)，计算特异性淋巴细胞杀伤率(％)，得图10：体外刺激诱导的CTL特异性淋巴细胞杀伤率。
表6：
Figure PCTCN2021110455-appb-000008
8、体外检测干扰素γ的分泌
将上述各组CTL效应细胞和4个不同患者的肿瘤细胞，按照效靶比20：1混合于U型底96孔板中培养72h后，使用干扰素γ酶联免疫试剂盒，按照说明书流程检测培养上清中IFN-γ的含量，得图11。

Claims (10)

  1. 一种肿瘤复合抗原，其特征在于：所述复合抗原包括不同EB病毒株来源的人永生化B淋巴样母细胞系肿瘤细胞裂解物或/和EBV阳性癌细胞裂解物；所述不同EB病毒株来源的人永生化B淋巴样母细胞系肿瘤细胞裂解物为GD1、B95-8、M81、HKNPC1～9、SNU-719或/和YCCEL1中任一种或几种组合物；所述EBV阳性癌细胞裂解物为C666-1、HNE1或/和CCL85中任一或多种组合物。
  2. 一种树突状细胞多价疫苗，其特征在于：所述树突状细胞多价疫苗载有所述权利要求1中的肿瘤复合抗原；载有所述肿瘤复合抗原的树突状细胞多价疫苗负载一种EBV相关癌症的肿瘤细胞裂解物或LCLs肿瘤细胞裂解物，或者同时负载两种或多种EBV相关癌症的肿瘤细胞裂解物或LCLs肿瘤细胞裂解物。
  3. 根据权利要求2所述的的树突状细胞多价疫苗，其特征在于：所述不同EB病毒株来源的人永生化B淋巴样母细胞系肿瘤细胞裂解物为GD1，B95-8，M81，HKNPC1～9，SNU-719，YCCEL1等EBV病毒转化得到的人永生化B淋巴样母细胞系中任一种或多种；所述癌细胞裂解物为C666-1、HNE1或CCL85。
  4. 根据权利要求2或3所述的树突状细胞多价疫苗，其特征在于：所述树突状细胞多价疫苗包含第一佐剂或其他辅助治疗的细胞因子。
  5. 根据权利要求2或3所述的树突状细胞多价疫苗，其特征在于：所述第一佐剂为PloyI:C，LPS或OK432中任一种；所述他辅助治疗的细胞因子为TNF-α或IL-12。
  6. 根据权利要求4所述的树突状细胞多价疫苗，其特征在于：所述第一佐剂为PloyI:C，LPS或OK432中任一种；所述他辅助治疗的细胞因子为TNF-α或IL-12。
  7. 根据权利要求2或3或6所述的树突状细胞多价疫苗，其特征在于：所述每种肿瘤细胞裂解物具体用量在2.5*10 7-2.5*10 9个。
  8. 一种如权利要求1所述的肿瘤复合抗原在制备预防或治疗EBV相关肿瘤药物中的应用。
  9. 根据权利要求8所述的肿瘤复合抗原在制备预防或治疗EBV相关肿瘤药物中的应用，其特征在于：所述肿瘤包括EBV相关胃癌，EBV阳性淋巴瘤，鼻咽癌以及EBV相关的其他上皮细胞癌症。
  10. 如权利要求9所述的肿瘤复合抗原在制备预防或治疗EBV相关肿瘤药物中的应用，其特征在于：所述药物包括如权利要求2-7中任一所述的树突状细胞多价疫苗。
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