Approximately 50 mL of peripheral blood from the patient. Collect the plasma in the supernatant in a 50-mL conical tube. Add an equal volume of saline to the blood cells in the bottom and resuspend the cells for lymphocyte separation. Transfer the plasma tube to a 56 °C water bath for 30 min, centrifuge at 400×g for 10 min, and transfer the supernatant into a new 50-mL conical tube and store at 4 °C for further applications. Carefully lay equal volumes of the blood cell suspension onto the lymphocyte separation solution in two 50-mL conical tubes, centrifuge at 600×g for 15 min, transfer the lymphocytes into the middle layer into a 50-mL conical tube, and wash twice with saline.
The NK cells were cultured as follows. On day 1, mix 4×107 lymphocytes, 50 mL NK cell culture medium, and one tube of recovered HK-001 in a T175 culture flask and incubate at 37 °C with 5% CO2. On day 3, add 30 mL NK cell culture medium to the T175 flask. On day 5, add 60 mL NK cell culture medium to the T175 flask and adjust the cell concentration to 1×106/mL. On day 6, add 60 mL NK cell culture medium to the T175 flask and, adjust the cell concentration to 1×106/mL. On day 7, add 50 mL NK cell culture medium (1–2% plasma concentration) to the T175 flask. On day 8, transfer the total culture from the T175 flask to the 2-L cell culture bag. Add 200 mL NK cell culture medium to the cell culture bag. On days 9, 10, and 11, add 150 mL NK cell culture medium to the cell culture bag each day. On day 12, add 350 mL NK cell culture medium to the cell culture bag. On day 13, harvest the HANK cells (should be around 1×1010 cells). Collect the cultures into 450-mL conical centrifuge tubes. Adjust the cell concentration to 2×107/mL with cell infusion solution (400 mL saline with 1% human serum albumin and 6 mL HK-003). A sample of 3–5×109 HANK cells was harvested into blood transfusion bags each day for infusion at a concentration of 2×107/mL.
All patients received at least one course of treatment (each course included three infusions, the length of time for each infusion was within 30 min). No more than three courses of treatment were received monthly. Each course with three infusions was delivered consecutively over 3 days.
During all infusions, the patients did not report a cold chill, fever, or any other discomfort. There was no case of queasiness or vomiting. After 14 days of HANK cell infusion, the white blood cell count and the aspartate transaminase, alanine aminotransferase, hemoglobin, and creatinine levels were not significantly different from those before treatment. It was demonstrated that blood function, liver function, and renal function remained at a normal level.
A total of 13 patients received different courses of autologous HANK cell infusion. After 3 months, 11 patients (84.6%) experienced stable disease and 2 patients (15.4%) experienced progressive disease according to the RECIST guidelines.
This is the first study to describe the efficacy of NK cell therapy of patients with advanced lung cancer. These clinical observations demonstrated that NK cell is safe and efficient for advanced lung cancer therapy.
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Scientific article publishing date : 7/1/2019
Immucura identifier BSC21_182EN