Application of activated T cell and blocking antibody in combined preparation of anti-tumor drug and anti-tumor drug
An anti-tumor drug and combined preparation technology, applied in the field of tumor immunotherapy, can solve the problems of high cost and difficulty in implementation, and achieve the effect of improving therapeutic effect, inducing tumor cell apoptosis, and promoting killing effect
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Embodiment 1
[0044] Example 1 Preparation of activated T cells
[0045] 1.1 Isolation and culture of lymphocytes
[0046] 1.1.1 Isolation of human peripheral blood mononuclear cells
[0047] Mononuclear cells (PBMC) were isolated from normal human peripheral blood by density gradient centrifugation. The human lymphocyte separation solution was equilibrated to room temperature in advance, and 4 mL of lymphocyte separation solution was spread to the bottom of a 15 mL centrifuge tube; After centrifuging the mL blood sample, the plasma was discarded, and the blood cell pellet was diluted to 8 mL with PBS (phosphate buffered saline, pH 7.2). The diluted blood cells were slowly added to the lymphocyte separation solution equilibrated to room temperature along the wall of the centrifuge tube. The key to this step is not to destroy the separation interface; room temperature, 2000 rpm, the acceleration and deceleration brakes are set to 0, and centrifugation is performed for 20 minutes. After cen...
Embodiment 2
[0076] Example 2 Verification of the anti-tumor effect of activated T cells combined with blocking antibodies (in vitro experiment)
[0077] 2.1 Establishment of a co-culture model using ovarian cancer ascites cells to show that activated T cells can kill tumor cells in the same individual
[0078] According to previous experimental results, T cells in ovarian cancer ascites expressed higher levels of Siglec molecules. And it contains a lot of tumor cells and immune cells, allowing us to get enough cells for our experiments. Therefore, we first selected ovarian cancer ascites, sorted T cells and tumor cells, and constructed an in vitro co-culture model.
[0079] Tumor cells were sorted from ovarian cancer ascites with anti-human EpCAM antibody, and tumor cells in sorted ovarian cancer were analyzed by flow staining using antibody-RP215 that specifically recognizes SIA-IgG; in addition, ascites tumor cells were analyzed After the slides were fixed, immunofluorescence staining...
Embodiment 3
[0121] Example 3 Validation of the anti-tumor effect of activated T cells combined with blocking antibodies (in vivo experiment)
[0122] 3.1 The combination of anti-Siglec-7 antibody and activated T cells can enhance the anti-tumor effect of T cells in mice
[0123]Mononuclear cells were separated from ascites fluid of ovarian cancer patients, and monoclonal was established to obtain ovarian cancer cell line OAC-E, and animal experiments were carried out in vivo.
[0124] Nu / Nu female mice aged 6-8 weeks were selected, and the mouse ovarian cancer cell line OAC-E cells were revived one week in advance, routinely cultured and passaged, digested with trypsin in the logarithmic growth phase, and pre-cooled with sterile 4°C. washed twice with PBS (pH 7.2), centrifuged at 600 rpm for 5 min, and counted. Finally at 5×10 7 Cell concentrations per mL were resuspended in PBS, and 1 mL per tube was dispensed into several 1.5 mL sterile EP tubes for later use. Nu / Nu mice were inocula...
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