GSM7640556: 20190311NC_TIL5746_Library_16_screen_Lib16_CRISPRn_TIL_Donor_5746_D4_Time_point_0_509038_S1; Homo sapiens; OTHER
Dissociated melanoma tumors from donor 110005746 were obtained from Conversant Bio and seeded at 1x56 cells/ml in TIL-CM (RPMI 1640 + 10% HI HS, 1X HEPES, GlutaMAX, 2-Mercaptoethanol, Pen/Strep, Gentamicin) containing 300ng/ml IL-2 in a 24 well tissue culture treated plate, and incubated at 37C.  Every other day, 300ng/ml IL-2 was added to cell culture assuming consumption.  On Day 5, suspension cells were harvested and re-seeded at 1.5x10e6/ml in TIL-CM combined 1:1 with XVIVO-15 and supplemented with 300ng/ml IL-2, with adherent cells observed to have disappeared from the wells.  Between Days 7 through Day 22, cells were expanded in IL-2 with a 1:1 ratio of TIL-CM:XVIVO-15 media supplemented with 300ng/ml IL-2 added every other day in order to maintain cell density at 1e6 cells/ml.  On Day 35, cellularity was evaluated, with composition found to be 66% CD8+, and 32% CD4+.  TIL5746 were frozen on Day 35 in CS10 freezing medium at 1x108 cells/ml.  On the first day of the screen (screen Day 0), 2x108 TIL were thawed, washed in 40ml XVIVO-15 media twice, and resuspended to 2x106 cells/ml in XVIVO-15 media containing 600ng/ml IL-2 and 1X DNase (Stemcell, Cat# 07900).  In parallel, non-tissue culture treated 6 well plates were coated with 20ug/ml retronectin in PBS and incubated overnight in preparation for transduction. On Day 1, TIL were transduced with pKSQ017 lentivirus driving expression of Lib16 under control of the human U6 promoter, as well as driving expression of tagRFP under control of a UBC promoter.  Semi-random barcodes were used to track individual clones. In parallel, an aliquot of TIL5746 were separately transduced with the KSQ041 lentivirus which drives expression of a sgRNA targeting the essential gene RPL10a under control of a human U6 promoter as well as mNeonGreen under control of a UBC promoter.  Transduced cells were incubated in 6 well plates at 37C in XVIVO-15 overnight.  On Day 2, TIL were combined from 6 well plates, spun at 300g for 5 minutes, supernatant removed, and TIL resuspended in complete XVIVO-15 media and rested overnight.  On Day 3, transduced TIL5746 were electroporated with Cas9 mRNA (Trilink #L-7206, lot# T1COL01A) using an Amaxa 4D-Nucleofector unit and pulse code CA137 with Buffer P3 according to the manufacturer's instructions.  Following electroporation, 80l of pre-warmed XVIVO-15 media was added to each well, mixed, and wells combined and washed. TIL were re-suspended to 1x106 in complete XVIVO-15 media, and incubated overnight at 37C. On Day 4, 5x107 transduced TIL5746 were frozen to determine the input sgRNA Library distribution. The TIL Rapid Expansion Phase (REP) was initiated on 25x10e6 transduced and edited TIL with a 1:200 ratio of TIL to irradiated PBMCs derived from 5 pooled donors, 600ng/ml IL-2 and 30ng/ml OKT3 using XVIVO-15 media in a 5L Grex.  Cells were incubated at 37C.  On Days 8, 11 and 15, 2.5L of media was aspirated from the Grex and replaced with 2.5L complete XVIVO-15 media, with 3mgs of IL-2 added to the 5L Grex.  On Day 17, TIL were counted, with 13.2x109 TIL5746 obtained, and analyzed for cellularity on a FACS, with 85.5% of the TIL CD8+, and 12.9% of the TIL CD4+. TIL were harvested for screen analysis by freezing three 5x10e7 cell pellets for gDNA preparations.