Methods for preparing tumor-infiltrating lymphocytes

A method for preparing tumor-infiltrating lymphocytes (TILs) in vitro, the method comprising:

• (1) providing an initial cell population containing TILs from a sample, wherein the number of TILs in the initial cell population is n0 wherein n0 ranges from 1,000 to 50,000;
• (2) pre-treating the initial cell population, the pretreatment comprising culturing the initial cell population with IL7, IL15 and IL21 for 6 hours to 36 hours, to obtain a pretreated initial cell population, wherein each of IL7, IL15, and IL21 has a concentration ranging from 10 ng/ml to 100 μg/ml;
• (3) co-culturing the pretreated initial cell population, with first feeder cells in a first culture medium for a first period of 7 days to 16 days, to obtain a first expanded cell population, wherein the first culture medium comprises (i) one or more cytokines selected from the group consisting of IL2, IL7, IL15, and IL21, and (ii) one or more antibodies selected from the group consisting of an anti-CD3 antibody or fragments thereof, an anti-41BB antibody or fragments thereof, an anti-OX40 antibody or fragments thereof, and an anti-PD-1 antibody or fragments thereof, wherein the number of TILs in the first expanded cell population is n1, wherein n1/n0 ranges from 1000 to 10,000, and n1 is at least 1×107;
• and
• (4) co-culturing the first expanded cell population with second feeder cells in a second culture medium for a second period of 10 days to 16 days, to obtain an expanded TIL population, wherein the second culture medium comprises one or more antibodies selected from the group consisting of an anti-CD3 antibody or fragments thereof, and an anti-CD28 antibody or fragments thereof, wherein the number of TILs in the expanded TIL population is n2, wherein n2/n1 ranges from 4000 to 10,000, and n2 is at least 1×1010.