TUMOR COMPLEX ANTIGEN, MULTIVALENT DENDRITIC CELL (DC) VACCINE, AND USE THEREOF
20230346935 · 2023-11-02
Assignee
Inventors
Cpc classification
A61K2039/5154
HUMAN NECESSITIES
A61K39/464499
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
Abstract
A tumor complex antigen, a multivalent dendritic cell (DC) vaccine, and a use thereof are provided. In the present disclosure, monocytes of a patient are stimulated in vitro, loaded with a variety of tumor cell lysates with strong immunogenicity against different Epstein-Barr virus (EBV)-associated tumors, and induced into mature dendritic cells (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 cytotoxic T cells, thereby accurately killing tumor cells. 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.
Claims
1. A tumor complex antigen, comprising a tumor cell lysate of human immortalized B lymphoblastoid cell lines (B-LCLs) derived from different Epstein-Barr virus (EBV) strains and/or an EBV-positive tumor cell lysate, wherein the tumor cell lysate of the human immortalized B-LCLs derived from different EBV strains is at least one selected from the group consisting of GD1, B95-8, M81, HKNPC1 to HKNPC9, SNU-719, and YCCEL1; and the EBV-positive tumor cell lysate is at least one selected from the group consisting of C666-1, HNE1, and CCL85.
2. A multivalent dendritic cell (DC) vaccine, wherein the multivalent DC vaccine carries the tumor complex antigen according to claim 1; and the multivalent DC vaccine carrying the tumor complex antigen is loaded with at least one EBV-associated tumor cell lysate or at least one lymphoblastoid cell line (LCL) tumor cell lysate.
3. The multivalent DC vaccine according to claim 2, wherein the tumor cell lysate of the human immortalized B-LCLs derived from different EBV strains is a tumor cell lysate of one or more selected from the group consisting of human immortalized B-LCLs resulting from a transformation by EBVs of GD1, B95-8, M81, HKNPC1 to HKNPC9, SNU-719, and YCCEL1; and the EBV-positive tumor cell lysate is C666-1, HNE1, or CCL85.
4. The multivalent DC vaccine according to claim 2, wherein the multivalent DC vaccine comprises a first adjuvant or a cytokine for an adjuvant therapy.
5. (canceled)
6. The multivalent DC vaccine according to claim 4, wherein the first adjuvant is one selected from the group consisting of PloyI:C, LPS, and OK432; and the cytokine for the adjuvant therapy is TNF-α or IL-12.
7. The multivalent DC vaccine according to claim 2, wherein each of the tumor cell lysates is specifically used at an amount of 2.5×10.sup.7 to 2.5×10.sup.9 cells.
8. A method of use of the tumor complex antigen according to claim 1 in a preparation of a drug for preventing or treating an EBV-associated tumor.
9. The method according to claim 8, wherein the EBV-associated tumor comprises EBV-associated gastric carcinoma (GC), EBV-positive lymphoma, and nasopharyngeal carcinoma (NPC).
10. The method according to claim 9, wherein the drug comprises a multivalent DC vaccine, the multivalent DC vaccine carries the tumor complex antigen; and the multivalent DC vaccine carrying the tumor complex antigen is loaded with at least one EBV-associated tumor cell lysate or at least one lymphoblastoid cell line (LCL) tumor cell lysate.
11. The multivalent DC vaccine according to claim 3, wherein the multivalent DC vaccine comprises a first adjuvant or a cytokine for an adjuvant therapy.
12. The multivalent DC vaccine according to claim 3, wherein each of the tumor cell lysates is specifically used at an amount of 2.5×10.sup.7 to 2.5×10.sup.9 cells.
13. The multivalent DC vaccine according to claim 6, wherein each of the tumor cell lysates is specifically used at an amount of 2.5×10.sup.7 to 2.5×10.sup.9 cells.
14. The method according to claim 10, wherein the tumor cell lysate of the human immortalized B-LCLs derived from different EBV strains is a tumor cell lysate of one or more selected from the group consisting of human immortalized B-LCLs resulting from a transformation by EBVs of GD1, B95-8, M81, HKNPC1 to HKNPC9, SNU-719, and YCCEL1; and the EBV-positive tumor cell lysate is C666-1, HNE1, or CCL85.
15. The method according to claim 10, wherein the multivalent DC vaccine comprises a first adjuvant or a cytokine for an adjuvant therapy.
16. The method according to claim 15, wherein the first adjuvant is one selected from the group consisting of PloyI:C, LPS, and OK432; and the cytokine for the adjuvant therapy is TNF-α or IL-12.
17. The method according to claim 10, wherein each of the tumor cell lysates is specifically used at an amount of 2.5×10.sup.7 to 2.5×10.sup.9 cells.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0036]
[0038]
[0040]
[0042]
[0044]
[0046]
[0048]
[0050]
[0052]
[0054]
[0056]
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0058] 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.
Example 1: Immunological Study on the Treatment of NPC with a Multivalent DC Vaccine
[0059] 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).
1. Isolation of PBMCs from Human Venous Blood
[0060] 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/m.sup.3) 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/m.sup.3. The mononuclear cells have a density of 1.075 to 1.090 kg/m.sup.3. The granulocytes have a density of 1.092 kg/m.sup.3. The RBCs have a density of 1.093 kg/m.sup.3. [0061] 1) Peripheral blood was collected from a vein of an NPC patient in a centrifuge tube with a corresponding size, and 4.5 mL of a Ficoll® Paque Plus solution was added by a pipette to each of two new centrifuge tubes. [0062] 2) A blood sample was drawn by a pipette and slowly added to an upper layer of the Ficoll solution along a wall of the centrifuge tube, 10 mL per centrifuge tube. Then the centrifuge tubes were centrifuged at room temperature and 800 g for 20 min. [0063] 3) The centrifuge tubes were taken out. The sample solution in the centrifuge tubes was divided into four layers, including a plasma layer, a mononuclear cell layer, a Ficoll solution layer, an RBC layer, and a granulocyte layer sequentially from top to bottom. [0064] 4) The mononuclear cell layer was carefully transferred to a 15 mL centrifuge tube. A PBS/1% FBS solution was added to 14 mL mark. The resulting mixture was pipetted up and down for thorough mixing and then centrifuged at 800 g for 5 min at room temperature. [0065] 5) A resulting supernatant was discarded. The bottom of the centrifuge tube was flicked to loosen the cells. Then 14 mL of a PBS/1% FBS solution was added to resuspend the cells. The resulting suspension was pipetted up and down for thorough mixing and then centrifuged at 700 g for 5 min at room temperature. [0066] 6) A resulting supernatant was discarded. The bottom of the centrifuge tube was flicked to loosen the cells. Then 14 mL of an RPMI/10% FBS solution was added to resuspend the cells. The resulting suspension was pipetted up and down for thorough mixing and then centrifuged at 400 g for 5 min at room temperature. [0067] 7) A resulting supernatant was discarded. The bottom of the centrifuge tube was flicked to loosen the cells. Then 10 mL of an RPMI/10% FBS solution was added to resuspend the cells. The resulting suspension was pipetted up and down for thorough mixing. [0068] 8) 10 μL of a resulting cell suspension was transferred to a new 1.5 mL centrifuge tube, and 90 μL of an RPMI/10% FBS solution was added to dilute the cell suspension 10-fold. 10 μL of a diluted cell suspension was taken, 10 μL of Trypan Blue was added for staining, and the resulting mixture was added to a hemacytometer and counted under an inverted microscope. [0069] 9) The remaining cell suspension was centrifuged at 700 g for 5 min at room temperature, and a resulting supernatant was discarded. An appropriate amount of PBS/1% FBS was added for subsequent experiments.
2. Isolation of DC and T Lymphocytes
[0070] 2.1 The isolation methods of CD14 mononuclear cells include, but are not limited to, CD14.sup.+ 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.sup.+ magnetic bead separation kit can specifically recognize and bind to CD14.sup.+ cells among PBMCs and is indirectly coupled with magnetic beads through biotin or dextran, such that the CD14.sup.+ cells can be separated under the action of a high-intensity magnetic field. In this example, the EasySep™ CD14 positive selection kit was used. [0071] 1) A PBMC suspension was transferred to a 5 mL FCM tube. [0072] 2) An appropriate amount of a selection cocktail solution was added to the FCM tube to a final concentration of 100 μL/mL. The resulting mixture was pipetted up and down for thorough mixing and then incubated at room temperature for 10 min. [0073] 3) Magnetic beads were vortexed in a RapidSphere™ solution for 30 s, such that the magnetic beads were dispersed evenly. [0074] 4) An appropriate amount of a RapidSphere™ solution was added to the FCM tube to a final concentration of 100 μL/mL. The resulting mixture was pipetted up and down for thorough mixing and then incubated at room temperature for 3 min. [0075] 5) An appropriate amount of a PBS/2% FBS with 1 mM EDTA solution was added to the FCM tube to a total volume of 2.5 mL, and a resulting mixture was pipetted up and down for thorough mixing. [0076] 6) The FCM tube was vertically inserted into the EasySep™ magnet and incubated for 3 min at room temperature. [0077] 7) A magnet was placed invertedly, and a cell solution flowing out from the FCM tube was collected in a 15 mL centrifuge tube, where the magnet was placed invertedly for 3 s. The tube was not shaken. Liquid on the wall of the tube should not be totally removed. [0078] 8) The magnet was placed upright and then the FCM tube was taken out. [0079] 9) Steps 7 to 10 were repeated twice. [0080] 10) 2 mL of RPMI/10% FBS was added to the FCM tube to resuspend cells, and the cells were counted with Trypan Blue.
2.2 Experimental Scheme for the Induction of CD14.SUP.+ Mononuclear Cells to Produce iDCs
[0081] 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.sup.+ mononuclear cells into iDCs. [0082] 1) In a clean bench, a CD14.sup.+ cell suspension was transferred by a pipette to a six-well plate with 2×10.sup.6 cells/mL in each well. Then 1 μL of human recombinant GM-CSF (20 ng/μL to 200 ng/μL) and 1 μL of human recombinant IL-4 (10 ng/μL to 100 ng/μL) were added to the six-well plate. [0083] 2) The six-well plate was placed on a surface of the clean bench, then gently shaken three times back and forth and three times left and right to make the cells dispersed evenly, and incubated in a cell incubator at 37° C. and 5% CO.sub.2 for 3 d. [0084] 3) The six-well plate was taken out from the incubator, and then 2 mL of RPMI 1640/10% FBS, 1 μL of human recombinant GM-CSF, and 1 μL of human recombinant IL-4 were added to the six-well plate in a clean bench. [0085] 4) The six-well plate was placed on a surface of the clean bench, then gently shaken three times back and forth and three times left and right to make the components dispersed evenly, and incubated in a cell incubator at 37° C. and 5% CO.sub.2 for 2 d.
2.3 Loading a Tumor Cell Lysate to Prepare a Multivalent DC Vaccine
[0086] 1) Construction of an immortalized human B-LCL infected with an EBV strain [0087] a) PBMCs were isolated from peripheral blood and resuspended in 2 mL of an RPMI1640/10% FBS medium. [0088] b) 10 μL of a resulting cell suspension was taken, 90 μL of RPMI/10% FBS was added to dilute 10-fold, and the cells were counted under a microscope. [0089] c) According to a counting result, a required volume of a B95-8 supernatant was calculated, where every 1×10.sup.6 PBMCs corresponded to 500 μL of the B95-8 supernatant. [0090] d) 10 mL of B95-8 cells were cultivated two days in advance with an initial density of 1×10.sup.6 cells/mL. The B95-8 cells were cultivated in an incubator for 48 h. Then a resulting cell supernatant was transferred to a centrifuge tube and centrifuged at 2,000 rpm for 15 min, and the remaining B95-8 cells were sterilized and discarded. [0091] e) The B95-8 cell supernatant in the centrifuge tube was filtered through a 0.45 μm filter membrane for later use. [0092] f) PBMCs were collected and centrifuged at 1,000 rpm for 5 min, and a resulting PBMC supernatant was discarded. [0093] g) According to a cell counting result, the PBMCs were resuspended with an appropriate amount of the B95-8 cell supernatant or another EBV suspension (such as GD1, B95-8, M81, HKNPC1 to HKNPC9, SNU-719, and YCCEL1) to obtain a cell suspension in which a concentration of the PBMCs was 1×10.sup.6/500 μL. [0094] h) A sterile 96-well plate was prepared, and the suspension of PBMCs in B95-8 was added to the 96-well plate at 100 μL/well. [0095] i) The 96-well plate was incubated in a CO.sub.2 incubator for 24 h. [0096] j) The 96-well plate was taken out. Then 100 μL of an R20 medium (RPMI1640/20% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) was added to each well. The resulting mixture was pipetted up and down for thorough mixing. [0097] k) The 96-well plate was further incubated in an incubator for 6 d, during which a cell status was observed every day to determine whether the cell status underwent the following lymphoblastoid changes: increased cell volume, enriched cytoplasm, spherical shape, aggregated distribution of small colonies, significantly increased cell masses at a bottom of the well, and yellowing medium. [0098] 1) After the 6-day cultivation was completed, the medium was changed every 3 d. The upper 100 μL of the medium in each well was carefully removed. Then 100 μL of an R20 medium was added to each well to resuspend the cells in the well. When the medium turned yellow, the medium was changed timely, or the cells were dispensed into new 2 to 4 wells as required. After the number of cells gradually increased, the cells were then combined and transferred to a 24-well plate, a 6-well plate, and a T25 flask. [0099] m) The status of cells after the four-week cultivation was observed under a microscope. An expression level of CD19 on a surface of the cells was analyzed by FCM. The expression level of CD19 on the surface of the immortalized human B-LCL was shown in
[0101] 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% CO.sub.2 incubator. The tumor cells were purified by repeated adherence.
[0102] 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:
TABLE-US-00001 EBNA1-FP: 5′-CCAGACAGCAGCCAATTGTC-3′, as shown in SEQ ID NO: 1; EBNA1-RP: 5′-GGTAGAAGACCCCCTCTTAC-3′, as shown in SEQ ID NO: 2; upstream primer for internal reference β-actin gene: 5′-CTCCATCCTGGCCTCGCTGT-3′, as shown in SEQ ID NO: 3; and downstream primer for internal reference β-actin gene: 5′-GCTGTCACCTTCACCGTTCC-3′, as shown in SEQ ID NO: 4.
[0103] Copy numbers of EBV-DNA in the blood and tumor tissue of the 4 patients were compared, and results were shown in
[0104] 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. [0105] a) A temperature of a water bath was pre-set to 37° C. [0106] b) A culture of each of the C666-1 NPC cell line, the immortalized human B-LCLs constructed by different virus strains, and the tumor cells of 4 NPC patients were collected (at least 3×10.sup.7 cells) and centrifuged at 700 g for 5 min at room temperature. [0107] c) A resulting supernatant was discarded, and resulting cells were resuspended in RPMI/10% FBS to obtain a cell suspension. [0108] d) The cells were counted with trypan blue. [0109] e) The cell suspension was centrifuged at 700 g for 5 min at room temperature, and a resulting supernatant was carefully removed. [0110] f) Resulting cells were resuspended with RPMI/10%FBS in a 1 mL freezing tube with a density of 5×10.sup.6/mL. [0111] g) The cells were frozen in liquid nitrogen for 20 s. [0112] h) The cells were immediately thawed quickly and completely in a 37° C. water bath. [0113] i) Steps g) and h) were repeated 4 times (5 times in total). [0114] j) A tumor cell lysate was stored in liquid nitrogen before use. [0115] 3) Acquisition of a DC vaccine [0116] a) Cytokines GM-CSF and IL-4 were used to make mononuclear cells derived from blood differentiated into iDCs, after cultivated with GM-CSF and IL-4 for 5 days, the mononuclear cells differentiated into iDCs: [0117] monovalent DC vaccine: 5×10.sup.6 iDCs were co-cultivated with a lysate of 2.5×10.sup.7 autologous tumor cells for at least 2 h. Then cytokines such as TNF-α were added to stimulate the maturation of DCs to obtain the monovalent DC vaccine. [0118] multivalent DC vaccine: 5×10.sup.6 DCs were co-cultivated with each of different tumor cell lysates, such as a lysate of 2.5×10.sup.7 C666-1 tumor cells or an M81-LCL tumor cell lysate, for at least 2 h. Then cytokines, such as TNF-α, were added to stimulate the maturation of DCs, and 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.
[0119] 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
TABLE-US-00002 TABLE 1 Composition Ag- DC- DC- DC- DC- Ag- Ag- Ag- Ag- Group Poly I II III IV I II III IV Poly- + + DC-I Poly- + + DC-II Poly- + + DC-III Poly- + + DC-IV Ag-DC-I + + Ag-DC-II + + Ag- + + DC-III Ag- + + DC-IV * Ag-Poly indicates that it carries different tumor cell lysate antigen information, such as a C666-1 tumor cell lysate or tumor cell lysates of B95-8 and M81-LCLs. DC-I represents autologous DCs from the EBV-positive NPC patient I. DC-II represents autologous DCs from the EBV-positive NPC patient II. DC-III represents autologous DCs from the EBV-negative NPC patient III. DC-IV represents autologous DCs from the EBV-negative NPC patient IV. Ag-I represents tumor cell lysate antigen information of the NPC patient I, Ag-II represents tumor cell lysate antigen information of the NPC patient II, Ag-III represents tumor cell lysate antigen information of the NPC patient III, and Ag-IV represents tumor cell lysate antigen information of the NPC patient IV. Poly-DC-I represents a multivalent DC vaccine for patient I, Ag-DC-I represents a monovalent DC vaccine for patient I that is loaded with tumor cell lysate information of the patient I, and so on.
2.4 Preparation of T Lymphocytes
[0121] 1) PBMCs isolated from the same person were cultivated in a 37° C. and 5% CO.sub.2 incubator for 2 h, and then suspended cells were collected and prepared into 1 mL of a cell suspension. [0122] 2) The cell suspension was added to a nylon wool fiber column incubated at 37° C. The column was laid flat. Then 200 μL of pre-warmed 10% FBS-containing RPMI 1640 was added for sealing, and then the column was incubated at 37° C. for 2 h. [0123] 3) The nylon wool fiber column was subjected to elution with 10% FBS RPMI 1640 at a flow rate of about 1 mL/min, and 10 mL of a cell suspension was collected at the beginning, which was rich in T cells and NK cells. [0124] 4) The cell suspension was centrifuged at 700 g for 5 min at room temperature. A cell pellet was collected, counted, and adjusted to a cell concentration of 1×10.sup.7 cells/mL and placed in an 80 IU/mL IL-2-containing RPMI1640 complete medium for later use.
[0125] Alternatively, the magnetic bead separation method can be used, that is, T lymphocytes can be isolated through CD3.sup.+ 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 10.sup.7 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).
3. Acquisition of CTLs Induced by In Vitro Stimulation
[0126] A multivalent DC vaccine and normal mDCs were each resuspended in an RPMI complete medium with a cell density adjusted to 2×10.sup.5 cells/mL. The isolated autologous T lymphocyte suspension was adjusted with an RPMI complete medium to a cell density of 1.6×10.sup.6/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:
TABLE-US-00003 TABLE 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 + + Group a b c d e f g h i * DC-I represents DCs derived from the patient I. Poly-DC-I represents a multivalent DC vaccine for patient I, Poly-DC-II represents a multivalent DC vaccine for patient II, Poly-DC-III represents a multivalent DC vaccine for patient III, and Poly-DC-IV represents a multivalent DC vaccine for patient IV. Ag-DC-I represents a monovalent DC vaccine for patient I that is loaded with tumor cell lysate antigen of the patient I, Ag-DC-II represents a monovalent DC vaccine for patient II that is loaded with tumor cell lysate antigen of the patient II, Ag-DC-III represents a monovalent DC vaccine for patient III that is loaded with tumor cell lysate antigen of the patient III, and Ag-DC-IV represents a monovalent DC vaccine for patient IV that is loaded with tumor cell lysate antigen of the patient IV. T-I represents T lymphocytes derived from patient I, T-II represents T lymphocytes derived from patient II, T-III represents T lymphocytes derived from patient III, and T-IV represents T lymphocytes derived from patient IV. Group a represents an co-culture of DC-I and T-I, Group b represnets an co-culture of Poly-DC-I and T-I, Group c represnets an co-culture of Poly-DC-II and T-II, Group d represnets an co-culture of Poly-DC-III and T-III, Group e represnets an co-culture of Poly-DC-IV and T-IV, Group f represnets an co-culture of Ag-DC-I and T-I, Group g represnets an co-culture of Ag-DC-II and T-II, Group h represnets an co-culture of Ag-DC-III and T-III, and Group i represnets an co-culture of Ag-DC-IV and T-IV.
[0127] 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% CO.sub.2 constant-temperature and constant-humidity incubator. IL-2 was added with a final concentration of 30 U/mL. Then the corresponding DC vaccines (2×10.sup.5 cells in each group) were added for secondary stimulation, and the cells were further cultivated for one week and harvested on day 21.
4. Assay of Killing Activities of Collected Cells on NPC Cells in Different Patients
[0128] 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 4×10.sup.5/well, 2×10.sup.5/well, and 1×10.sup.5/well to set three experimental groups with different effector-target ratios. Tumor cells from different NPC patients were adopted as target cells. 2×10.sup.4 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
TABLE-US-00004 TABLE 3 Effector cell Group Group Group Group Group Group Group Group Group Target cell a b c d e f g h i Tumor cells of patient I + + + Tumor cells of patient II + + Tumor cells of patient III + + Tumor cells of patient IV + + Group a-I b-I c-II d-III e-IV f-I g-II h-III i-IV * Activated T cells from Groups a-i of Table 2 were used to conduct killing experiments on autologous tumor cells derived from patients I-IV, respectively. Group a-I represents an co-culture of tumor cells of patient I and CTL cells of group a in Table 2, Group b-I represents an co-culture of tumor cells of patient I and CTL cells of group b in Table 2, Group c-II represents an co-culture of tumor cells of patient II and CTL cells of group c in Table 2, Group d-III represents an co-culture of tumor cells of patient III and CTL cells of group d in Table 2, Group e-IV represents an co-culture of tumor cells of patient IV and CTL cells of group e in Table 2, Group f-I represents an co-culture of tumor cells of patient I and CTL cells of group f in Table 2, Group g-II represents an co-culture of tumor cells of patient II and CTL cells of group g in Table 2, Group h-III represents an co-culture of tumor cells of patient III and CTL cells of group h in Table 2, and Group i-IV represents an co-culture of tumor cells of patient IV and CTL cells of group i in Table 2. Among them, Group a-I indicate a killing effect of DC vaccine without antigen loading, Groups b-I, c-II, d-III, and e-IV indicate killing effects of multivalent DC vaccine, and Groups f-I, g-II, h-III, and i-IV indicate killing effects of monovalent DC vaccine.
5. In Vitro Detection of Secretion of IFN-γ
[0129] 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
Example 2: Immunological Study on the Resistance of a Multivalent DC Vaccine to EBV-Positive GC
[0130] 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.
1. Isolation of PBMCs from Human Venous Blood
[0131] 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/m.sup.3) 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/m.sup.3, the mononuclear cells have a density of 1.075 to 1.090 kg/m.sup.3, the granulocytes have a density of 1.092 kg/m.sup.3, and the RBCs have a density of 1.093 kg/m.sup.3. [0132] 1) Peripheral blood was collected from a vein of a GC patient in a centrifuge tube with a corresponding size, and 4.5 mL of a Ficoll® Paque Plus solution was added by a pipette to each of two new centrifuge tubes. [0133] 2) A blood sample was drawn by a pipette and slowly added to an upper layer of the Ficoll solution along a wall of the centrifuge tube, 10 mL per centrifuge tube; then the centrifuge tubes were centrifuged at 800 g for 20 min at room temperature. [0134] 3) The centrifuge tubes were taken out. The sample solution in the centrifuge tubes was divided into four layers, including a plasma layer, a mononuclear cell layer, a Ficoll solution layer, an RBC layer, and a granulocyte layer sequentially from top to bottom. [0135] 4) The mononuclear cell layer was carefully transferred to a 15 mL centrifuge tube, a PBS/1% FBS solution was added to 14 mL, and a resulting mixture was pipetted up and down for thorough mixing and then centrifuged at 800 g for 5 min at room temperature. [0136] 5) The resulting supernatant was discarded. The bottom of the centrifuge tube was flicked to loosen the cells. Then 14 mL of a PBS/1% FBS solution was added to resuspend the cells. The resulting suspension was pipetted up and down for thorough mixing and then centrifuged at room temperature at 700 g for 5 min. [0137] 6) A resulting supernatant was discarded. The bottom of the centrifuge tube was flicked to loosen the cells. Then 14 mL of an RPMI/10% FBS solution was added to resuspend the cells. The resulting suspension was pipetted up and down for thorough mixing and then centrifuged at room temperature and 400 g for 5 min. [0138] 7) The resulting supernatant was discarded. The bottom of the centrifuge tube was flicked to loosen the cells. Then 10 mL of an RPMI/10% FBS solution was added to resuspend the cells, and a resulting suspension was pipetted up and down for thorough mixing. [0139] 8) 10 μL of a resulting cell suspension was transferred to a new 1.5 mL centrifuge tube, and 90 μL of an RPMI/10% FBS solution was added to dilute the cell suspension 10-fold. 10 μL of a diluted cell suspension was taken, 10 μL of Trypan Blue was added for staining, and a resulting mixture was added to a hemacytometer and counted under an inverted microscope. [0140] 9) The remaining cell suspension was centrifuged at room temperature at 700 g for 5 min. The resulting supernatant was discarded, and an appropriate amount of PBS/1% FBS was added for subsequent experiments.
2. Experimental Scheme for the Induction of CD14.SUP.+ Mononuclear Cells to Produce iDCs
[0141] 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.sup.+ mononuclear cells into iDCs. [0142] 1) In a clean bench, a CD14.sup.+ cell suspension was transferred by a pipette to a six-well plate with 2×10.sup.6 cells/mL in each well. Then 1 μL of human recombinant GM-CSF (20 ng/μL to 200 ng/μL) and 1 μL of human recombinant IL-4 (10 ng/μL to 100 ng/μL) were added to the six-well plate. [0143] 2) The six-well plate was placed on a surface of the clean bench, then gently shaken three times back and forth and three times left and right to make the cells dispersed evenly, and incubated in a cell incubator at 37° C. and 5% CO.sub.2 for 3 d. [0144] 3) The six-well plate was taken out from the incubator. Then 2 mL of RPMI 1640/10% FBS, 1 μL of human recombinant GM-CSF, and 1 μL of human recombinant IL-4 were added to the six-well plate in a clean bench. [0145] 4) The six-well plate was placed on a surface of the clean bench, then gently shaken three times back and forth and three times left and right to make the components dispersed evenly, and incubated in a cell incubator at 37° C. and 5% CO.sub.2 for 2 d. Some iDCs were collected, and the expression of costimulatory factors on the cell surface thereof was detected by FCM. Detection results are shown in
3. A Method for Treating a Tumor Tissue of an EBV-Positive GC Patient and a Method for Preparing a Tumor Cell Lysate were the Same as Above
[0146] 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% CO.sub.2 incubator. The tumor cells were purified by repeated adherence combined with mechanical scraping.
[0147] 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.
[0148] Sequences of the primers were as follows:
TABLE-US-00005 EBNA1-FP: 5′-CCAGACAGCAGCCAATTGTC-3′, as shown in SEQ ID NO: 1; EBNA1-RP: 5′-GGTAGAAGACCCCCTCTTAC-3′, as shown in SEQ ID NO: 2; upstream primer for internal reference β-actin gene: 5′-CTCCATCCTGGCCTCGCTGT-3′, as shown in SEQ ID NO: 3; and downstream primer for internal reference β-actin gene: 5′-GCTGTCACCTTCACCGTTCC-3′, as shown in SEQ ID NO: 4.
[0149] Copy numbers of EBV-DNA in the plasma and tumor tissue of the 4 patients were compared, and results were shown in
4. Preparation of a DC Vaccine for EBV-Positive GC
[0150] 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.
[0151] Monovalent DC vaccine: 5×10.sup.6 DCs were co-cultivated with a lysate of 2.5×10.sup.7 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.
[0152] Multivalent DC vaccine: 5×10.sup.6 DCs were co-cultivated with each of different cell lysates, such as a lysate of 2.5×10.sup.7 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.
[0153] 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
TABLE-US-00006 TABLE 4 Composition Ag- Group Poly DC-A DC-B DC-C DC-D Ag-A Ag-B Ag-C Ag-D Poly-DC-A + + Poly-DC-B + + Poly-DC-C + + Poly-DC-D + + Ag-DC-A + + Ag-DC-B + + Ag-DC-C + + Ag-DC-D + + * Ag-Poly indicates that it carries antigen information of various tumor cell lysates, such as tumor cell lysates of YCCEL1-LCLs and GCEBV-LCLs or EBV-positive GC cell lines, such as GT38 and PT. DC-A represents DCs of patient A, DC-B represents DCs of patient B, DC-C represents DCs of patient C, and DC-D represents DCs of patient D. Ag-A represents tumor cell lysate antigen information of GC patient A, Ag-B represents tumor cell lysate antigen information of GC patient B, Ag-C represents tumor cell lysate antigen information of GC patient C, and Ag-D represents tumor cell lysate antigen information of GC patient D. Poly-DC-A represents a multivalent DC vaccine for patient A. Ag-DC-A represents a monovalent DC vaccine for the patient A that is loaded with tumor cell lysate information of the patient A, and so on.
5. Preparation of T Lymphocytes
[0154] The magnetic bead separation method can be used, that is, T lymphocytes can be isolated through CD3.sup.+ 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 10.sup.7 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).
6. Acquisition of CTLs Induced by In Vitro Stimulation
[0155] 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 2×10.sup.5 cells/mL. The isolated autologous T lymphocyte suspension was adjusted with an RPMI complete medium to a cell density of 1.6×10.sup.6/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:
TABLE-US-00007 TABLE 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 + + Group a b c d e f g h i *DC-A represents DC derived from patient A. Poly-DC-A represents multivalent DC vaccine suitable for patient A, Poly-DC-B represents multivalent DC vaccine suitable for patient B, Poly-DC-C represents multivalent DC vaccine suitable for patient C, and Poly-DC-D represents multivalent DC vaccine suitable for patient D. Ag-DC-A represents monovalent DC vaccine of patient A that is loaded with tumor cell lysate antigen of the patient A, Ag-DC-B represents monovalent DC vaccine of patient B that is loaded with tumor cell lysate antigen of the patient B, Ag-DC-C represents monovalent DC vaccine of patient C that is loaded with tumor cell lysate antigen of the patient C, and Ag-DC-D represents monovalent DC vaccine of patient D that is loaded with tumor cell lysate antigen of the patient D. T-A represents T lymphocytes derived from patient A, T-B represents T lymphocytes derived from patient B, T-C represents T lymphocytes derived from patient C, and T-D represents T lymphocytes derived from patient D. Group a represents an co-culture of DC-A and T-A, Group b represents an co-culture of of Poly-DC-A and T-A, Group c represents an co-culture of Poly-DC-B and T-B, Group d represents an co-culture of Poly-DC-C and T-C, Group e represents an co-culture of Poly-DC-D and T-D, Group f represents an co-culture of Ag-DC-A and T-A, Group g represents an co-culture of Ag-DC-B and T-B, Group h represents an co-culture of Ag-DC-C and T-C, and Group i represents an co-culture of Ag-DC-D and T-D.
[0156] 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% CO.sub.2 constant-temperature and constant-humidity incubator. IL-2 was added with a final concentration of 30 U/mL. Then the corresponding DC vaccines (2×10.sup.5 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.
7. Assay of Killing Activities of Collected CTLs on GC Cells in Different Patients
[0157] 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 4×10.sup.5/well, 2×10.sup.5/well, and 1×10.sup.5/well to set three experimental groups with different effector-target ratios. Tumor cells from different EBV-positive GC patients were adopted as target cells. 2×10.sup.4 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
TABLE-US-00008 TABLE 6 Effector cell Target cell a b c d e f g h i Tumor cells of + + + patient A Tumor cells of + + patient B Tumor cells of + + patient C Tumor cells of + + patient D Group DC + T- Poly- Poly- Poly- Poly- Ag- Ag- Ag- Ag- A DC + T- DC + T- DC + T- DC + T- DC + T- DC + T- DC + T- DC + T- A B C D A B C D * Activated T cells from Groups a-i of Table 5 were used to conduct killing experiments on autologous tumor cells derived from patients A-D, respectively. Group DC + T-A represents an co-culture of tumor cells of patient A and CTL cells of group a in Table 5, Group Poly-DC + T-A represents an co-culture of tumor cells of patient A and CTL cells of group b in Table 5, Group Poly-DC + T-B represents an co-culture of tumor cells of patient B and CTL cells of group c in Table 5, Group Poly-DC + T-C represents an co-culture of tumor cells of patient C and CTL cells of group d in Table 5, Group Poly-DC + T-D represents an co-culture of tumor cells of patient D and CTL cells of group e in Table 5, Group Ag-DC + T-A represents an co-culture of tumor cells of patient A and CTL cells of group f in Table 5, Group Ag-DC + T-B represents an co-culture of tumor cells of patient B and CTL cells of group g in Table 5, Group Ag-DC + T-C represents an co-culture of tumor cells of patient C and CTL cells of group h in Table 5, and Group Ag-DC + T-D represents an co-culture of tumor cells of patient D and CTL cells of group i in Table 5. Among them, Group DC + T-A indicate a killing effect of DC vaccine without antigen loading, Groups Poly-DC + T-A, Poly-DC + T-B, Poly-DC + T-C, and Poly-DC + T-D indicate killing effects of multivalent DC vaccine, and Groups Ag-DC + T-A, Ag-DC + T-B, Ag-DC + T-C, and Ag-DC + T-D indicate killing effects of monovalent DC vaccine.
8. In Vitro Detection of Secretion of IFN-γ
[0158] 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