Single-pass, closed-system rapid expansion of lymphocyte cultures for adoptive cell therapy

Abstract

Adoptive cell therapy (ACT) for metastatic melanoma involves the ex vivo expansion and reinfusion of tumor infiltrating lymphocytes (TIL) obtained from resected specimens. With an overall objective response rate of 56%, this T-cell immunotherapy provides an appealing alternative to other therapies, including conventional therapies with lower response rates. However, there are significant regulatory and logistical concerns associated with the ex vivo activation and large-scale expansion of these cells. The best current practice uses a rapid expansion protocol (REP) consisting of an ex vivo process that occurs in tissue culture flasks (T-flasks) and gas-permeable bags, utilizes OKT3 (anti-CD3 monoclonal antibody), recombinant human interleukin-2, and irradiated peripheral blood mononuclear cells to initiate rapid lymphocyte growth. A major limitation to the widespread delivery of therapy to large numbers of melanoma patients is the open system in which a REP is initiated. To address this problem, we have investigated the initiation, expansion and harvest at clinical scale of TIL in a closed-system continuous perfusion bioreactor. Each cell product met all safety criteria for patient treatment and by head-to-head comparison had a similar potency and phenotype as cells grown in control T-flasks and gas-permeable bags. However, the currently available bioreactor cassettes were limited in the total cell numbers that could be generated. This bioreactor may simplify the process of the rapid expansion of TIL under stringent regulatory conditions thereby enabling other institutions to pursue this form of ACT.

Figure 1

The closed-system bioreactor is essentially a single, circular chamber. Three compartments of the closed device are shown in cross-section consisting of: 1) gas compartment, 2) cell bed compartment, and 3) waste medium compartment. Single-pass perfusion occurs from the center port of the biochamber and continues radially over the cell bed. Uniform oxygen delivery is achieved independently of the medium exchange rate through a gas-permeable and liquid-impermeable membrane separating the gas and liquid sides of the biochamber.

Figure 2

TIL after expansion in the perfusion bioreactor demonstrate comparable function and phenotype to static T-flasks/bags. A. Flow cytometric analysis of TIL after harvest on day 14 of culture using MART-1 tetramers in combination with anti-CD8 monoclonal antibody. Percentage positive cells are shown in each quadrant. B. Co-culture assay of TIL after harvest from the bioreactor or T-flasks/bags. TIL were evaluated for IFN γ release in a co-culture assay against a panel of MART-1⁺, HLA-A2⁺ (526, 624) or MART-1⁺, HLA-A2⁻ (888, 928) melanoma tumor cell lines.

Figure 3

Optimization of culture parameters to provide a straightforward process for closed-system TIL expansion in the bioreactor. A. Serum concentration. Two different TIL isolates designated TIL A and TIL B were expanded in side-by-side experiments by continuous perfusion of medium supplemented with either 1% or 5% human AB serum. B. Inoculum Density. TIL were inoculated within each individual experiment at a lower density of 5 million verses a higher density of either 15 million (Experiments 1 and 2) or 20 million (Experiments 3 and 4) TIL per bioreactor cassette. C. Culture Duration. TIL were harvested from bioreactors on days 14 vs. 18 (Experiment 1), days 14 vs. 20 (Experiment 2) and days 16 vs. 19 (Experiment 3).

Figure 4

A. Average lactate levels for TIL grown in 1% and 5% serum was lower over the course of a 14-day expansion as compared to TIL grown in flasks/bags. B. Total lactate produced in an individual cassette correlates strongly with eventual cell yield at harvest. C. Total lactate produced from two similar TIL cultures accumulates steadily over the REP and predicts relative fold expansion.

Figure 5

Phenotypic characteristics of cell products grown in flasks/bags (triangles) vs. cassettes (circles)were comparable in terms of their expression of a panel of CD markers.. Expression of individual markers (indicated on each plot) varied widely, but no consistent difference between expansion methods was observed for any marker.