Heat inactivated human ab serum

High-affinity NK (haNK) cells are an NK cell line, NK-92, which has been engineered to express IL-2 and the high-affinity valine (V) CD16 allele as previously described [5 (link)–7 (link)]. haNK cells were supplied by NantBioScience (Culver City, CA) through a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute (NCI) and cultured in X-Vivo-10 medium (Lonza, Walkersville, MD) supplemented with 5% heat-inactivated human AB serum (Omega Scientific, Tarzana, CA) at a concentration of 5 × 10⁵ cells/ml.

In a T25 flask, 1.43 × 10⁵ pancreatic TIL were stimulated with 30 ng/mL human anti-CD3 (OKT3, Ortho Pharmaceutical, Raritan, NJ) in the presence of 2.9 × 10⁷ irradiated (5000 rad) allogenic PBMC feeder cells. TIL were cultured in REP Media I comprised of RPMI 1640, 2.05 mM L–glutamine (HyClone, Thermo Fisher Scientific), 10 % heat-inactivated human AB serum (Omega Scientific), 55 μM 2-mercaptoethanol (Invitrogen), and 10 mM HEPES Buffer (Mediatech). On day 5, 70 % of the media was replaced with REP Media II comprised of a 1:1 (v:v) mixture of REP Media I and AIM V (Invitrogen). Media was supplemented with 6000 I.U./mL rhIL-2 on days 2 and 5 of the 8 day REP. This protocol was also implemented in T75 flasks at exactly triple the aforementioned cell counts and reagents.

Pancreatic tumors were minced into ~1 mm³ fragments, placed in 24 well plates with 2 mL of TIL media containing IL-2, and pancreatic TIL were allowed to extravasate from the tissue. If available, excess tissue was physically and enzymatically digested as described below. Alternatively, 48 well plates were used following the same procedure, using 1 mL of TIL media and IL-2. TIL were expanded in vitro for 3–6 weeks in 6000 I.U./mL IL-2 (Proleukin, Novartis, Emeryville, CA) per mL of complete TIL media (TIL-CM) consisting of RPMI 1640, 2.05 mM L–glutamine (HyClone, Thermo Fisher Scientific, Waltham, MA), 10 % heat-inactivated human AB serum (Omega Scientific, Tarzana, CA), 55 μM 2-mercaptoethanol (Invitrogen), 50 μg/mL gentamicin (Invitogen), 100 I.U./mL penicillin, 100 μg/mL streptomycin, and 10 mM HEPES Buffer (Mediatech, Manassas, VA) in 24 or 48 well plates. Half of the media was replaced every 2 to 3 days or wells were split when 80 % confluent. For TIL generation in the presence of costimulatory antibodies, TIL were propagated as above with the addition of an antagonistic human PD-1 antibody (Nivolumab, 10 μg/mL) or with an agonistic human 4-1BB antibody (Urelumab, 10 μg/mL at initiation, followed by continued supplement with 1 μg/mL at each feeding or splitting as above) generously provided by Drs. Alan Korman and Maria Jure-Kunkel (Bristol Myers Squibb, Princeton, NJ).