Patent Application
20230346935
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy is named GBSHSS001_Sequence_Listing.txt, created on Sep. 23, 2022, and is 1,132 bytes in size.
The present disclosure relates to the technical field of biomedicine, and in particular to a tumor complex antigen, a multivalent dendritic cell (DC) vaccine, and a use thereof.
Epstein-Barr virus (EBV) is a member of the lymphotropic virus genus of the herpesvirus family, which has a DNA genome with a length of about 170 kb. People are generally susceptible to EBV, and EBV spreads easily with an infection rate as high as 95% among adults. EBV can be carried for life, and there are regional differences in diseases caused by EBV. EBV infection mostly occurs in the childhood and adolescence. EBV infection in the body may develop into long-term latent infection in some cases and may also lead to various human malignant tumors, such as Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), gastric carcinoma (GC), breast cancer, and tumors in immunodeficiency patients. In addition, EBV can specifically infect human and some primate B cells in vivo or in vitro and can stimulate the continuous growth of infected cells and cause the unlimited passage of cells to achieve immortalization, thereby producing lymphoblastoid cell lines (LCLs). Such cell lines are often used to study the occurrence and development of various diseases, which makes it possible to study the pathogenesis of some diseases on a large scale for a long time. However, the pathogenesis of EBV is still not fully understood, and the research and treatment for EBV-associated tumors (such as EBV-associated GC and NPC with high incidence in Southern China) still need to be improved.
GC is a malignant tumor originating from the gastric mucosa epithelium and is one of the most common tumors worldwide. GC is common in people aged 50 or older, but there is an upward trend in younger populations being diagnosed with GC. According to statistics of the International Agency for Research on Cancer (IARC), in 2012, there were about 951,000 new GC cases and about 723,000 GC deaths worldwide, where the incidence ranked fifth and the mortality ranked third among malignant tumors. More than 70% of the new GC cases occurred in developing countries, and about 50% of the new GC cases occurred in East Asia, mainly in China. In 2012, the new GC cases and the GC deaths in China accounted for 42.6% and 45.0% of the global new GC cases and the global GC deaths, respectively. In 2015, there were approximately 679,000 new GC cases and about 498,000 GC deaths in China. The GC incidences in the northwest and eastern coastal regions of China were significantly higher than the GC incidence in the southern region, causing extremely heavy disease burden. The specific pathogenesis of GC is complex and has not yet been fully understood, but a large number of research data show that the occurrence of GC is a result of the combined action of various factors, such as geographical environment, dietary and living factors, Helicobacter pylori (H. pylori) infection, EBV infection, precancerous lesions, inheritance, and gene mutation. Studies have reported that EBV-associated GCs account for 10% of all GCs. GC can occur in any part of the stomach, but more than half of GCs occur in the gastric antrum although GCs may occur in the greater curvature of stomach, the lesser curvature of stomach, and the anterior and posterior walls as well. The vast majority of GCs are adenocarcinomas and have no obvious symptoms or have non-specific symptoms, such as epigastric discomfort and belching in an early stage, which are often similar to symptoms of chronic gastric diseases, such as gastritis and gastric ulcer, and thus are easily ignored. Therefore, an early diagnosis rate of GC in China is currently low, and it is urgent to discover a GC-specific marker and improve the early diagnosis rate of GC. In addition, the treatment of GC has shown a trend of multidisciplinary treatment. The combined use of surgery, radiotherapy, targeted therapy, and immunotherapy may help the treatment and prognosis of GC and the pain relief of a patient.
Among EBV-associated tumors, NPC is a malignant tumor occurring on an epithelium of the nasopharynx and is almost a squamous cell carcinoma (SCC). NPC progresses rapidly, which is highly invasive and metastatic and is accompanied by headache, tinnitus, and hearing or vision loss. Epidemiologically, the incidence of NPC is very regional and is high in Southeast Asia, the Middle East, Southern Africa, and Southern China. In particular, an incidence of NPC in Guangdong province of China is as high as 30/100,000, and NPC is one of the most common malignant tumors in Southern China. According to the World Health Organization (WHO) in 2012, there are about 33,000 new NPC cases and 20,000 NPC deaths in China every year. At present, a diagnosis method of NPC mainly relies on tissue biopsy by a nasal endoscope, which is invasive to some extent. Moreover, due to the lack of serological diagnostic markers with both high sensitivity and high specificity, it is difficult for NPC to become a routine examination item in the hospital. At present, due to the special location of NPC, the early detection of NPC is difficult in clinical practice, and surgical resection is not suitable. With the progression of NPC, a 5-year survival rate of NPC patients declines rapidly. For example, a 5-year survival rate of NPC patients at an early clinical stage is 90%, while a 5-year survival rate of NPC patients at an advanced clinical stage IV is less than 50%. In addition, after radiotherapy and chemotherapy, NPC easily relapses and metastasizes, and the radiotherapy and chemotherapy have large side effects. Therefore, if not detected and treated early, NPC will pose a great threat to the life of a patient. The development of a safe and effective treatment for relapsed and advanced stage NPC is an urgent need for NPC patients.
Currently, the pathogenesis of NPC is still uncertain. Some studies have pointed out that the incidence of NPC is mainly related to genetic factors, environmental regionality, dietary habits (pickled foods), and EBV infection, among which EBV has been confirmed to be highly correlated with the occurrence and development of NPC. At present, NPC is mainly treated by conventional radiotherapy and chemotherapy, which can achieve some therapeutic effects (normally, a 3-year survival rate is 80% after treatment) but will bring much discomfort to a patient during the treatment; and there is no specific drug for NPC. Therefore, the development of preventive and therapeutic NPC vaccines has always been a research hotspot. In addition, due to great differences among different NPC patients, biological processes in cancer cells of different patients are different, and expression levels of various proteins are also different. Therefore, the effects of currently known markers or specific drugs are often not favorable in all patients. Therefore, clinically, there is an urgent need for a reagent or vaccine with high specificity that is directly derived from a patient and can be used for the diagnosis and treatment of NPC.
The research on NPC vaccines for many years has mainly focused on preventive vaccines with EBV gp350 as a target antigen and single or combined recombinant therapeutic vaccines with EBV-EBNA1, LMP1, and LMP2 as target sites. At present, some substantial progress has been made in the studies of relevant vaccines in China, and the phase I and II clinical trials have been conducted in many of the studies. However, there is no specific tumor marker for the occurrence and development of NPC, making it difficult to conduct subsequent clinical trials. The current research models for the related vaccines are mostly mice, rhesus monkeys, cynomolgus monkeys, and the like, and thus the scientific research basis is insufficient.
In addition, as a B-cell-tropic human herpesvirus, EBV can latently reside in infected B cells for a long time to cause persistent latent infection, which is closely related to the occurrence, development, and prognosis of various lymphomas, including BL, diffuse large B-cell lymphoma (DLBCL), HL, NK/T-cell lymphomas, and the like. The WHO estimated that there were 589,600 new lymphoma cases worldwide in 2018, including 80,000 HL cases and 509,600 NHL cases with a global standardized incidence of 6.6/100,000. In China, a detection rate of EBV in HL patients can reach 48% to 57%. The treatment of EBV-associated lymphomas is complicated. In addition to the selection of sensitive chemotherapy and traditional local radiotherapy according to different lymphoma types, it is necessary to design a treatment plan based on the characteristics of EBV to effectively clear EBV infection and activity from the body, attenuate a driving action of EBV for disease progression, and block the further development of lymphomas. A supplementary EBV-specific treatment based on a traditional treatment method, such as antiviral therapy and immunotherapy, is essential to improve the efficacy for EBV-associated lymphomas.
Some studies have shown that DCs in EBV-associated tumor patients have a low degree of differentiation, a reduced quantity, an impaired ability to recognize and present antigens, and a weak ability to activate naive T cells, which ultimately makes the body fail to recognize and clear tumor cells. EBV infection is very likely one of the reasons for impaired DC functions in patients with NPC and other related cancers. DCs were discovered in 1973 by the Canadian scientist Ralph M. Steinman, winner of the 2011 Nobel Prize in Medicine and Physiology. DCs are named for their dendritic or pseudopod-like protrusions that protrude in a mature stage. DCs are the most powerful professional antigen-presenting cells (APCs) currently known in the body, which can efficiently take up, process, and present antigens. DCs are the only APCs found so far that can activate unsensitized naive T cells. In addition, immature dendritic cells (iDCs) have a strong ability to transfer and take up antigens, and mature dendritic cells (mDCs) can effectively activate naive T cells and are in a central link for initiating, regulating, and maintaining an immune response. A quantity of DCs is less than 1% of a quantity of peripheral blood mononuclear cells (PBMCs). The surface of a DC is rich in antigen-presenting molecules (such as MHC-I and MHC-II), costimulatory factors (such as CD80/B7-1, CD86/B7-2, and CD40), and adhesion factors (such as ICAM-1, ICAM-2, ICAM-3, LFA-1, and LFA-3). Therefore, DCs are an important group of innate immune cells and professional APCs and play a key regulatory role in the activation of an immune response in the body and in the maintenance of autoimmune tolerance.
As the strongest APCs, DCs can effectively present antigen information to T cells and induce the activation of T cells, thereby causing a series of immune responses. An MHC molecule on the surface of DCs can bind to an antigen to produce a peptide-MHC molecular complex, thereby presenting an antigen signal to T cells. Some costimulatory factors (such as CD80/B7-1, CD86/B7-2, and CD40) highly expressed on DCs provide a second signal necessary for the activation of T cells. DCs can also directly present an antigenic peptide to CD8+ T cells and activate CD8+ T cells with the help of CD4+ T cells. Activated DCs can secrete IL-12, IL-18, chemotactic cytokines (CCK), and the like in large quantities to activate the proliferation of T cells and initiate an MHC-class I-restricted cytotoxic T lymphocyte (CTL) response and an MHC-class II-restricted CD4+ Th1 immune response. In addition, DCs can also activate perforin-dependent and FasL/Fas-mediated pathways to enhance NK cytotoxicity, thereby enhancing the body's anti-tumor immune response to eliminate tumors. DCs themselves can serve as a natural immune adjuvant to improve the immune competence of the body by secreting various cytokines and can also enhance immune responses of various vaccines. Generally, DCs with relevant antigen information and vaccine functions are called DC vaccine.
Vaccines are preventive or therapeutic biological products for human vaccination, which play an important role in preventing, treating, and controlling the occurrence and prevalence of infectious diseases. However, with the increasing production and use of vaccines, problems such as excessive inoculation doses and limited therapeutic effects also arise. For example, the 14 vaccines in the routine immunization of children in the immunization plan of China require a total of 17 to 19 injections before the age of 3 (17 injections: the Japanese encephalitis virus (JEV) live attenuated vaccine and the hepatitis A live attenuated vaccine are used to complete immunization; and 19 injections: the JEV inactivated vaccine and the hepatitis A inactivated vaccine are used to complete immunization). The large number of immunization injections is inconvenient to parents and medical staff, may decrease all-course vaccination rate, and increase the cost of immunizations due to immunization time inconsistance. There may also be an increase in the likelihood of adverse reactions from immunization, which increases the difficulty in supervision and clinical use of vaccines. In order to reduce the number of immunization injections, improve the prevention and treatment effects, reduce the implementation cost of the expanded programme on immunization (EPI), and maximize the disease prevention and treatment effects of vaccines, multivalent vaccines or combined vaccines must be developed and promoted, which is particularly important in reducing the implementation cost and maximizing the disease treatment effect.
A vaccine with only a single antigenic component is called monovalent vaccine, and a monovalent vaccine can only prevent one infectious disease or one type of pathogen infection. A vaccine prepared by mixing two or more antigenic components in an appropriate ratio is called multivalent vaccine or combined vaccine. For example, there are more than 100 types of human papillomavirus (HPV), most of which only cause skin warts. Some types of HPV can cause cervical cancer, such as HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 69, 73, and 82, which are called high-risk HPV; other types of HPV are low-risk HPV. A bivalent HPV vaccine can only prevent the infection of HPV 16 and 18 (high-risk HPV), and 70% of cervical cancers are caused by these two types of HPV. A quadrivalent HPV vaccine can prevent the infection of HPV 16, 18, 6, and 11, among which the HPV 6 and 11 are low-risk HPV and are the main causes of genital warts. A nine-valent HPV vaccine can prevent the infection of HPV 16, 18, 31, 33, 45, 52, 58, 6, and 11 (namely, 7 high-risk HPV types and 2 low-risk HPV types) and is currently a vaccine that can target the most types of HPV. The development of multivalent vaccines has a history of nearly a hundred years, and the research on combined vaccines began as early as the 1930s. In 1945, the trivalent influenza vaccine was approved for use in the United States for the first time, and then the hexavalent pneumococcal vaccine, the Diphtheria-Tetanus (DT) double vaccine, the Diphtheria-Pertussis-Tetanus (DPT) triple vaccine, and the trivalent oral poliovirus attenuated live vaccine were successively developed. Results of clinical trials have shown that the combined immunization with a multivalent vaccine is often superior to the multi-vaccination with a monovalent vaccine. When a multivalent vaccine is used for combined immunization, an immunization effect is similar to or better than an immunization effect of a monovalent vaccine, and the side effects of the vaccine are not increased.
Defects and deficiencies of the prior art: Due to difficult early detection of EBV-associated tumors such as NPC and GC, these tumors are often found in middle and late stages and thus are mainly treated by traditional methods. The traditional surgery, radiotherapy, and chemotherapy exhibit an inhibition rate of about 20% for tumor cells and an inhibition rate of about 90% for lymphocytes, and excessive chemotherapy will decrease the likelihood of the patient's survival. Some tumors are not susceptible to drugs, and chemotherapy has no clinical value for such tumors. Some tumors often shrink rapidly after radiotherapy and chemotherapy, but because tumor cells are not eliminated, the tumors will grow rapidly and have extensive proximal and distant metastasis, which accelerates the metastasis of tumor cells during treatment and destroys the immune system. Therefore, some patients die not from cancer but from complications after overtreatment. Currently, there is an urgent need to find new drugs or methods for treating cancer in clinical. Compared with traditional treatment methods, immunotherapy has gradually become a new cancer treatment method due to its prominent therapeutic effect and few side effects. DC vaccines are playing an increasingly important role in cancer treatment and prevention, but monovalent DC vaccines often lead to a limited therapeutic effect. Different antigen types and combinations can be used to treat various diseases. However, since most tumor cells are derived from the human body, tumor cells can easily evade treatments based on single tumor antigen, so that the immune response from the treatment does not successfully kill a tumor, resulting in limited therapeutic effects. In addition, because many tumor cells in vivo can secrete a variety of cytokines that inhibit the maturation of DCs, there is relatively a small number of DCs at a tumor site, and an anti-tumor immune response induced by DCs in the absence of strong tumor antigen stimulation cannot lead to a very significant therapeutic effect in a host.
In view of the deficiencies of the prior art, the present disclosure is intended to provide a tumor complex antigen, a multivalent DC vaccine, and a use thereof. In the present disclosure, DCs of a patient are stimulated in vitro, loaded with a variety of tumor cell lysates with strong immunogenicity against different EBV-associated tumors (e.g., a tumor cell lysate of human immortalized B lymphoblastoid cell lines (B-LCLs) derived from different EBV strains, such as SNU-719, YCCEL1, GD1, B95-8, M81, and HKNPC1 to HKNPC9, or an EBV-positive tumor cell lysate, such as C666-1, HNE1, or CCL85), and induced into mDCs by various cytokines and specific agonists to obtain a complete DC vaccine with corresponding tumor antigens. The DC vaccine can be injected back into the patient to activate an immune system, stimulate innate immunity (such as inducing natural killer (NK) cells), and stimulate lymphocytes to produce an acquired immune response and produce cytotoxic T cells, thereby accurately killing EBV-associated tumor cells to achieve personalized treatment. Compared with radiotherapy and chemotherapy, the DC vaccine is particularly safe and has almost no side effects. In addition, the production of the DC vaccine involves a short production cycle of about 1 week and a low cost.
In order to achieve the above objective, the present disclosure adopts the following technical solutions:
In a first aspect, the present disclosure provides a tumor complex antigen, including a tumor cell lysate of human immortalized B-LCLs derived from different EBV strains and/or an EBV-positive tumor cell lysate. The tumor cell lysate of human immortalized B-LCLs derived from different EBV strains is any one or a combination of two or more selected from the group consisting of GD1, B95-8, M81, HKNPC1 to HKNPC9, SNU-719, and YCCEL1. The EBV-positive tumor cell lysate is any one or a combination of two or more selected from the group consisting of C666-1, HNE1, and CCL85.
In a second aspect, the present disclosure provides a multivalent DC vaccine, which carries the tumor complex antigen described above. The multivalent DC vaccine carrying the tumor complex antigen is loaded with one, two, three, or more EBV-associated tumor cell lysates or LCL tumor cell lysates.
As a preferred solution, the tumor cell lysate of human immortalized B-LCLs derived from different EBV strains may be a tumor cell lysate of one or more selected from the group consisting of human immortalized B-LCLs resulting from transformation by EBVs of GD1, B95-8, M81, HKNPC1 to HKNPC9, SNU-719, and YCCEL1. The EBV-positive tumor cell lysate may be C666-1, HNE1, or CCL85.
Further, the multivalent DC vaccine may include a first adjuvant or a cytokine for adjuvant therapy.
Furthermore, the first adjuvant may be any one selected from the group consisting of PloyI:C, LPS, and OK432, and the cytokine for adjuvant therapy may be TNF-α or IL-12.
Furthermore, each of the tumor cell lysates may be specifically used at an amount of 2.5×107 to 2.5×109 cells.
In a third aspect, the present disclosure provides a use of the tumor complex antigen described above in the preparation of a drug for preventing or treating an EBV-associated tumor.
As a preferred solution, the EBV-associated tumor may include EBV-associated GC, EBV-positive lymphoma, NPC, and other EBV-associated epithelial tumors.
Further, the drug may include the multivalent DC vaccine described above.
The multivalent DC vaccine provided by the present disclosure can stimulate an immune response of the body to treat an EBV-associated tumor, especially EBV-associated GC, EBV-positive lymphoma, NPC, and other EBV-associated epithelial tumors with prominent efficacy and small side effects. The multivalent DC vaccine can effectively inhibit the growth of an EBV-associated tumor and slow down the progression of the tumor for a long time and can even achieve a complete remission state.
In some embodiments, an antigen-sensitized DC population belongs to an immunogenic composition and is loaded with a corresponding antigen. Specific antigens may include a tumor cell lysate of human immortalized B-LCLs derived from different EBV strains, such as GD1, B95-8, M81, HKNPC1 to HKNPC9, SNU-719, and YCCEL1 and a tumor cell lysate of C666-1, HNE1, or CCL85. Each of the tumor cell lysates may be specifically used at an amount of 2.5×107 to 2.5×109 cells. In other aspects of the present disclosure, the DC vaccine carrying the tumor complex antigen may be a multivalent DC vaccine loaded with only two EBV-associated tumor cell lysates or LCL tumor cell lysates or a multivalent DC vaccine loaded with three or more EBV-associated tumor cell lysates or LCL tumor cell lysates.
In some aspects, the multivalent DC vaccine may include a first adjuvant (such as Ploy(I:C), LPS, and OK432) or a cytokine for adjuvant therapy (such as TNF-α or IL-12). The multivalent DC vaccine may be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, intranodally, subcutaneously, or topically 3 to 30 times, at an interval of one or two weeks. The multivalent DC vaccine may be injected each time at an amount of 5×106 to 5×108 cells.
The present disclosure has the following advantages and beneficial effects:
The multivalent DC vaccine is a vaccine that fights against a tumor by activating an immune system of a patient. Compared with the traditional treatment for EBV-associated tumors, the multivalent DC vaccine has the following advantages:
The preferred implementations of the present disclosure will be described in detail below in conjunction with examples. It should be understood that the following examples are provided merely for the purpose of illustration and are not intended to limit the scope of the present disclosure. Those skilled in the art can make various modifications and substitutions to the present disclosure without departing from the purpose and spirit of the present disclosure. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, and the like used in the following examples are all commercially available unless otherwise specified.
In this example, tumor cells were collected from each of two EBV-positive NPC patients (denoted as I and II) and two EBV-negative NPC patients (denoted as III and IV) (the patients had signed an informed consent form).
In this example, based on density differences among cell components in peripheral blood, a Ficoll® Paque Plus (GE Healthcare) solution (with a density of 1.075 to 1.089 kg/m3) was added to a peripheral blood sample. Density gradient centrifugation was performed, such that different cell components were separated into different layers so the mononuclear cells could be quickly isolated from human peripheral blood. The peripheral blood mainly includes platelets, mononuclear cells, granulocytes, and red blood cells (RBCs). The platelets have a density of 1.030 to 1.035 kg/m3. The mononuclear cells have a density of 1.075 to 1.090 kg/m3. The granulocytes have a density of 1.092 kg/m3. The RBCs have a density of 1.093 kg/m3.
2.1 The isolation methods of CD14 mononuclear cells include, but are not limited to, CD14+ magnetic bead separation in this example, CD14 negative selection, Miltenyi immunomagnetic cell sorting (MACS), and cell attachment. The isolation principle is based on the specific binding of an antigen and an antibody. A CD14+ magnetic bead separation kit can specifically recognize and bind to CD14+ cells among PBMCs and is indirectly coupled with magnetic beads through biotin or dextran, such that the CD14+ cells can be separated under the action of a high-intensity magnetic field. In this example, the EasySep™ CD14 positive selection kit was used.
In vitro, granulocyte-macrophage colony-stimulating factor (GM-CSF) can promote the survival of iDCs and induce the massive proliferation of iDCs. IL-4 can inhibit the overgrowth of macrophages, reduce the expression of CD14 molecule on a cell surface, and induce the differentiation of CD14+ mononuclear cells into iDCs.
A tumor tissue was collected from a patient, immediately rinsed with a medium including 1,000 U/mL penicillin and streptomycin and 2 μg/mL amphotericin for 5 min to 10 min, and then repeatedly rinsed with a serum-free medium. On a clean bench, a vigorously-proliferating tissue at an edge of the tumor tissue was collected, chopped with sharp surgical blades with the mechanical damage minimized, and digested with 0.5% collagenase type IV and 0.5% hyaluronidase at 37° C. for 80 min. The resulting mixture was filtered through a 200-mesh nylon mesh, and the resulting filtrate was centrifuged at 1,000 rpm for 5 min. The resulting cells were resuspended in an RPMI1640 complete medium with 10% FBS, inoculated in a culture plate, and cultivated in a 37° C. and 5% CO2 incubator. The tumor cells were purified by repeated adherence.
Determination of EBV loads in the blood and tumor tissue of NPC patients: Blood samples were collected from the 4 NPC patients. DNA extraction and PCR were conducted respectively with the MagMAX viral nucleic acid extraction kit (Thermo A42352) and the EBV Real-™ Quant kit (Sacace BioTechnologies Srl, Como, Italy). DNA was extracted from 100 μL of plasma according to a method provided by the manufacturer and eluted with 50 μL of an elution buffer. The NPC tissue cells obtained above were subjected to total DNA extraction according to instructions of the DNeasy Blood & Tissue Kit (Qiagen, Cat. No. 69506) product, and 10 μL of a sample was subjected to EBV quantification by RT-qPCR (EBV Real-™ Quant Kit). In this experiment, a coding region of the EBNA1 gene was selected as an amplification target, and the PCR was conducted according to instructions with a final volume of 25 μL. Primer sequences were as follows:
Copy numbers of EBV-DNA in the blood and tumor tissue of the 4 patients were compared, and results were shown in
The repeated freezing-thawing method is a common mechanical lysis method, which usually consists of freezing and thawing. A principle of the method is as follows: The generation of intracellular ice particles and the increase of salt concentration of the remaining cell solution cause swelling, such that the cell structure is broken and cells die, but the immunogenicity of the cells is retained. The freezing is usually conducted in liquid nitrogen or on ice at −20° C., and the thawing can be conducted through heat shock in a water bath at 37° C., 50° C., 65° C., or 100° C., which is milder than the chemical lysis.
Surface molecular markers on iDCs and mDCs were detected by FCM, such as CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC. A morphological image of mDCs is shown in
Alternatively, the magnetic bead separation method can be used, that is, T lymphocytes can be isolated through CD3+ magnetic beads. Cells were first incubated with an anti-surface antigen monoclonal antibody (mAb) for 12 min (50 μL of an anti-CD3 mAb was used for every 107 cells), then washed and incubated with 100 μL of a biotin-labeled goat anti-mouse secondary antibody for 10 min, then washed and incubated with 25 μL of FITC-labeled streptavidin for 8 min, and then washed and incubated with biotin-labeled magnetic beads (100 μL of magnetic beads was added when the anti-CD3 mAb was added) for 8 min. After the above reactions were completed, 1 mL of 1% BSA-containing PBS was added for washing, and a resulting mixture was centrifuged at 2,000 r/min for 10 min. T lymphocytes were isolated through immunomagnetic separation of a magnetic cell separator (MACS).
A multivalent DC vaccine and normal mDCs were each resuspended in an RPMI complete medium with a cell density adjusted to 2×105 cells/mL. The isolated autologous T lymphocyte suspension was adjusted with an RPMI complete medium to a cell density of 1.6×106/mL. The following experimental groups were set, and 1 mL of a corresponding DC vaccine and T lymphocytes were added in each group, as shown in Table 2:
The same helper cytokines were added in each of the above experimental groups with an IL-2 content of 1,000 U/mL, an IL-12 content of 1,500 U/mL, a Poly(I:C) content of 10 mg/mL, and a TNF-α content of 1,000 U/mL. Cells were cultivated for 2 weeks in a 37° C. and 5% CO2 constant-temperature and constant-humidity incubator. IL-2 was added with a final concentration of 30 U/mL. Then the corresponding DC vaccines (2×105 cells in each group) were added for secondary stimulation, and the cells were further cultivated for one week and harvested on day 21.
Some of the cells collected above were centrifuged and resuspended in an RPMI1640 complete medium. A cell concentration was adjusted, and the cells were added as effector cells to a 96-well culture plate at 4×105/well, 2×105/well, and 1×105/well to set three experimental groups with different effector-target ratios. Tumor cells from different NPC patients were adopted as target cells. 2×104 tumor cells of an NPC patient were added as target cells to each well with a final volume of 200 μL. The set of experimental groups are shown in Table 3. A control group without lymphocytes and a blank medium control group without cells were set, and 5 replicate wells were also set for each of the control groups. 24 h later, free effector cells in each well were removed, the plate was washed twice with PBS, 100 μL of a reagent including 20 μL of CCK8 was added to each well, and the cells were further cultivated for 2 h. The absorbance (OD) at 450 nm was determined by a microplate reader, and a killing rate (%) of specific lymphocytes was calculated. The killing rates of CTLs induced by in vitro stimulation were shown in
The CTL effector cells of each of the above groups and the tumor cells of each of the 4 NPC patients were mixed in a U-bottom 96-well plate according to an effector-target ratio of 20:1 and cultivated for 72 h. A content of IFN-γ in a culture supernatant was detected with an IFN-γ ELISA kit according to instructions, and the results are shown in
In this example, two EBV-positive GC patients (A and B) and two EBV-negative GC patients (C and D) who signed an informed consent form were selected.
In this example, based on density differences among cell components in peripheral blood, a Ficoll® Paque Plus (GE Healthcare) solution (with a density of 1.075 to 1.089 kg/m3) was added to a peripheral blood sample, and then the density gradient centrifugation was conducted, such that different cell components were separated into different layers and thus the mononuclear cells could be quickly isolated from human peripheral blood. The peripheral blood mainly includes platelets, mononuclear cells, granulocytes, and RBCs, where the platelets have a density of 1.030 to 1.035 kg/m3, the mononuclear cells have a density of 1.075 to 1.090 kg/m3, the granulocytes have a density of 1.092 kg/m3, and the RBCs have a density of 1.093 kg/m3.
In vitro, GM-CSF can promote the survival of iDCs and induce the massive proliferation of iDCs. IL-4 can inhibit the overgrowth of macrophages, reduce the expression of CD14 molecule on a cell surface, and induce the differentiation of CD14+ mononuclear cells into iDCs.
A tumor tissue was collected from a patient, immediately rinsed with a medium including 1,000 U/mL penicillin and streptomycin and 2 μg/mL amphotericin for 5 min to 10 min, and then repeatedly rinsed with a serum-free medium. On a clean bench, a vigorously-proliferating tissue at an edge of the tumor tissue was collected, chopped with sharp surgical blades with the mechanical damage minimized, and digested with 0.5% collagenase type IV and 0.5% hyaluronidase at 37° C. for 80 min. The resulting mixture was filtered through a 200-mesh nylon mesh, and the resulting filtrate was centrifuged at 1,000 rpm for 5 min. Resulting cells were resuspended in an RPMI1640 complete medium with 10% FBS, inoculated into a culture plate, and cultivated in a 37° C. and 5% CO2 incubator. The tumor cells were purified by repeated adherence combined with mechanical scraping.
Determination of EBV loads in the blood and GC tissue samples of GC patients: Blood samples were collected from the 4 GC patients. DNA extraction and PCR were conducted respectively with the MagMAX viral nucleic acid extraction kit (Thermo A42352) and the EBV Real-™ Quant kit (Sacace BioTechnologies Srl, Como, Italy). DNA was extracted from 100 μL of plasma according to a method provided by the manufacturer and eluted with 50 μL of an elution buffer. The GC tissue cells obtained above were subjected to total DNA extraction according to instructions of the DNeasy Blood & Tissue Kit (Qiagen, Cat. No. 69506) product, and 10 μL of a sample was subjected to EBV quantification by RT-qPCR (EBV Real-™ Quant Kit). In this experiment, a coding region of the EBNA1 gene was selected as an amplification target, and the PCR was conducted according to instructions with a final volume of 25 μL.
Sequences of the primers were as follows:
Copy numbers of EBV-DNA in the plasma and tumor tissue of the 4 patients were compared, and results were shown in
The cytokines GM-CSF and IL-4 were used to make mononuclear cells in blood differentiated into iDCs. The mononuclear cells were cultivated with GM-CSF and IL-4 for 5 d to differentiate into iDCs. A lysate of LCLs constructed from EBV-associated GC virus strains (such as SNU719-LCLs and YCCEL1-LCLs) or a lysate of a GC tumor cell line (such as GT38 and PT) was added to allow an cultivation for 2 h, and then cytokines, such as TNF-α, were added to stimulate the maturation of DCs.
Monovalent DC vaccine: 5×106 DCs were co-cultivated with a lysate of 2.5×107 autologous tumor cells for at least 2 h, and then cytokines, such as TNF-α, were added to stimulate the maturation of DCs to obtain the monovalent DC vaccine.
Multivalent DC vaccine: 5×106 DCs were co-cultivated with each of different cell lysates, such as a lysate of 2.5×107 YCCEL1-LCL or GCEBV-LCL tumor cells or a lysate of a GT38 or PT EBV-positive GC cell line, for at least 2 h. Then cytokines, such as TNF-α, were added to stimulate the maturation of DCs. Multiple types of DCs loaded with tumor cell antigen information were mixed in equal amounts in a DC medium to obtain the multivalent DC vaccine loaded with tumor cell lysate antigens.
The following 4 vaccines were prepared according to Table 4, and culture supernatants of the normally-cultivated DCs and the vaccine groups were each collected to detect the expression of IL12. Results were shown in
The magnetic bead separation method can be used, that is, T lymphocytes can be isolated through CD3+ magnetic beads. Cells were first incubated with an anti-surface antigen mAb for 12 min (50 μL of an anti-CD3 mAb was used for every 107 cells), then washed and incubated with 100 μL of a biotin-labeled goat anti-mouse secondary antibody for 10 min, then washed and incubated with 25 μL of FITC-labeled streptavidin for 8 min, and then washed and incubated with biotin-labeled magnetic beads (100 μL of magnetic beads was added when the anti-CD3 mAb was added) for 8 min. After the above reactions were completed, 1 mL of 1% BSA-containing PBS was added for washing, and a resulting mixture was centrifuged at 2,000 r/min for 10 min. T lymphocytes were isolated through immunomagnetic separation of a magnetic cell separator (MACS).
A multivalent DC vaccine, a monovalent DC vaccine, and normal mDCs were each resuspended in an RPMI complete medium with a cell density adjusted to 2×105 cells/mL. The isolated autologous T lymphocyte suspension was adjusted with an RPMI complete medium to a cell density of 1.6×106/mL. The following experimental groups were set, and 1 mL of a corresponding DC vaccine and T lymphocytes were added in each group, as shown in Table 5:
The same helper cytokines were added in each of the above experimental groups with an IL-2 content of 1,000 U/mL, an IL-12 content of 1,500 U/mL, a Poly(I:C) content of 10 mg/mL, and a TNF-α content of 1,000 U/mL. Cells were cultivated for 2 weeks in a 37° C. and 5% CO2 constant-temperature and constant-humidity incubator. IL-2 was added with a final concentration of 30 U/mL. Then the corresponding DC vaccines (2×105 cells in each group) were added for secondary stimulation, and the cells were further cultivated for one week and harvested on day 21. Then the immunological function of the multivalent vaccine was detected.
Some of the cells collected above were centrifuged and resuspended in an RPMI1640 complete medium. A cell concentration was adjusted, and the cells were added as effector cells to a 96-well culture plate at 4×105/well, 2×105/well, and 1×105/well to set three experimental groups with different effector-target ratios. Tumor cells from different EBV-positive GC patients were adopted as target cells. 2×104 tumor cells of a GC patient were added as target cells to each well with a final volume of 200 μL. The set of experimental groups are shown in Table 6. A control group without lymphocytes and a blank medium control group without cells were set, and 5 replicate wells were also set for each of the control groups. 24 h later, free effector cells in each well were removed, the plate was washed twice with PBS, 100 μL of a reagent including 20 μL of CCK8 was added to each well, and the cells were further cultivated for 2 h. The absorbance (OD) at 450 nm was determined by a microplate reader, and a killing rate (%) of specific lymphocytes was calculated. The killing rates of CTLs induced by in vitro stimulation were shown in
The CTL effector cells of each of the above groups and the tumor cells of each of the 4 different patients were mixed in a U-bottom 96-well plate according to an effector-target ratio of 20:1 and cultivated for 72 h. A content of IFN-γ in a culture supernatant was detected with an IFN-γ ELISA kit according to instructions, and the results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011017224.5 | Sep 2020 | CN | national |
This application is the national phase entry of International Application No. PCT/CN2021/110455, filed on Aug. 4, 2021, which is based upon and claims priority to Chinese Patent Application No. 202011017224.5, filed on Sep. 24, 2020, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/110455 | 8/4/2021 | WO |
