There are a large number of immune cells in immune tolerance state in the tumor microenvironment. The theoretical basis of this clinical study is to use ultraviolet inactivated oncolytic herpes simplex virus type 2 (UV-oHSV2) to activate PBMC or immune cells in immune tolerance state in malignant pleural and peritoneal fluid in vitro, and to transfusion the activated immune cells back to the patient's peripheral blood or pleural and peritoneal fluid to kill tumor cells in malignant pleural and peritoneal fluid to further control the volume of malignant pleural and peritoneal fluid.~Our study indicated that UV-oHSV2 potently activated human peripheral blood mononuclear cells leading to increased antitumor activity in vitro and in vivo. We also found that UV-oHSV2 could induce NK cells (isolated from healthy donors' PBMC or patient pleural effusion) proliferation, secretion of IFN-γ. We further found that UV-oHSV2 could enhance NK cells (isolated from healthy PBMC or patient pleural effusion) antitumor ability in vitro and in vivo.~Based on the above research, we carried out an open, single center, prospective clinical trial to evaluate the safety and effectiveness of VAK cells in the treatment of malignant pleural and peritoneal effusions.~The detailed protocol as follow:~Isolation of lymphocytes from the patient's pleural effusions or ascites:~Add pleural fluid into a 50ml centrifuge tube, centrifuge at 400g, and discard the supernatant.~Resuspend the precipitate with 40ml of normal saline and pass through 70 μ M U.M and then rinse the cell filter once with 5ml physiological saline.~Discard the cell filter, and centrifuge the filtered cell suspension in the centrifuge tube at 400g for 5min. After centrifugation, discard the supernatant and resuspend the cell pellet with 5ml of normal saline.~Mix Ficoll upside down before use, take a new 15ml centrifuge tube and add 5ml Ficoll (suck with syringe).~Gently add 5-6ml cell suspension onto Ficoll layer.~Centrifuge 400g, 30-40min, 18 ℃ - 20 ℃, the centrifuge needs to be slowly raised and lowered, and the sudden stop braking setting should be closed.~Take the middle white membrane layer (lymphocytes) into a new centrifuge tube.~Add 3 times the volume of normal saline to the absorbed lymphocytes, resuspend the cells, centrifuge 400-500g, 10-15min, 18 ℃ - 20 ℃.~Repeat washing once, and the final precipitate is resuspended with 5ml serum-free medium (RPMI1640).~Take 20 μ L of cell suspension in EP tube, add equal volume of trypan blue staining solution, gently blow and count with cell counter.~In the process of planting the isolated immune cells, the cell culture density was controlled within the range of 106, and 10% autologous plasma was added.~Take 4ml of cell suspension for separate culture, and add the corresponding amount of inactivated virus to the remaining cell suspension with the infection number MOI = 1. The calculation formula is as follows: volume of uv-oh2 added = (1 × Number of cells added per well plate or bottle / oh2 virus titer)~After the immune cells are planted, they are placed in a 5% CO2 incubator for 36h-48h.~After cell culture, 30-50ML of prepared lymphocytes (qualified by PCR sterility test and endotoxin test) were infused into the chest and abdominal cavity of the patient.
Theory of VAK：~Immune cells (T cells for example) of cancer subjects may be domesticated by the tumor microenvironment, and have low efficacy to kill cancer cells. They could be restimulated by virus antigen, and play a powerful tumor killing role while intrapleural to subjects.~Releasing of tumor-associated antigen could induce specific anti-tumor immune response.~Preparation of VAK：~Separate the immune cells and tumor cells from Malignant Pleural and Peritoneal Effusion.~Incubate the immune cells with inactivated viruses and tumor cells.~Wash to remove impurities.~Intrapleural the immune cells to patients