Value of large scale expansion of tumor infiltrating lymphocytes in a compartmentalised gas-permeable bag: interests for adoptive immunotherapy

Abstract

Background: Adoptive cell therapy (ACT) has emerged as an effective treatment for patients with metastatic melanoma. However, there are several logistical and safety concerns associated with large-scale ex vivo expansion of tumour-specific T lymphocytes for widespread availability of ACT for cancer patients. To address these problems we developed a specific compartmentalised bag allowing efficient expansion of tumour-specific T lymphocytes in an easy handling, closed system.

Methods: Starting from lymph nodes from eight melanoma patients, we performed a side-by-side comparison of Tumour-Infiltrating Lymphocytes (TIL) produced after expansion in the compartmentalised bag versus TIL produced using the standard process in plates. Proliferation yield, viability, phenotype and IFNγ secretion were comparatively studied.

Results: We found no differences in proliferation yield and cell viability between both TIL production systems. Moreover, each of the cell products complied with our defined release criteria before being administered to the patient. The phenotype analysis indicated that the compartmentalised bag favours the expansion of CD8+ cells. Finally, we found that TIL stimulated in bags were enriched in reactive CD8+ T cells when co-cultured with the autologous melanoma cell line.

Conclusions: The stimulation of TIL with feeder cells in the specifically designed compartmentalised bag can advantageously replace the conventional protocol using plates. In particular, the higher expansion rate of reactive CD8+ T cells could have a significant impact for ACT.

Figure 1

TIL amplification after stimulation in multiple 96-wells plates or in bags. A, Illustration of the TIL production process for ACT. Briefly, lymph nodes are excised from patients and sectioned for cryopreservation. If the patient is included in the protocol, node fragments are thawed and TIL cultured in 12-well plates for 10 to 14 days. Then, TIL together with feeder cells (irradiated PBMC and irradiated LAZ cells) are plated in sixty 96-well plates for stimulation. Ten days later, TIL are pooled and expanded in culture bags before being administered to patients (see "Materials and Methods" for details). Even though 96-well plates have the advantage of enhancing TIL stimulation through close contact between TIL and feeders cells, they represents a time- and labour-intensive step in the TIL production process. Furthermore, as it requires multiple containers (i.e. sixty 96-well plates), this method is a source of potential contamination and variability of the cell therapy product. In order to make TIL stimulation with feeder cells safer and more straightforward, we developed a specific compartmentalised bag. B, Schematic representation of the compartmentalized bag. This bag is composed of a size-reduced lower compartment (L, 135 mm × H, 17 mm; Vmax 15 ml) that allows close contact between cells. This compartment has been thermoformed to avoid plastic sticking and facilitate cell injection. TIL and feeder cells are injected into this compartment through the bottom injection site. Then culture medium is added in the larger upper compartment (L, 135 mm × H, 93 mm; Vmax 195 ml) via the upper injection site. The two compartments are separated by a discontinuous welding (forteen 2 mm weldings separated by 5 mm) that allows exchanges between both compartments but prevents the passage of cells from the lower to the upper compartment during culture medium renewal.

Figure 2

Comparison of TIL proliferation in plates and bags. A, Total quantity of TIL produced in plates and bags during the culture period. B, Cell amplification of TIL produced in bags and plates between day 0 and day 20. C, Viability of TIL produced in bags and in plates at the end of the process (day 20). The data are the means of experiments carried out on triplicate from 8 donors. p-values are shown.

Figure 3

CD4 and CD8 expression by TIL produced in plates vs bags. A, representative cytometry histograms of TIL produced in plates (upper panels) and bags (lower panels) stained for CD4 (left panels) and CD8 (right panels) from patient 06. White histograms correspond to isotypic controls. B, graphical representation of the percentage of CD8 (left) and CD4 (right) positive cells produced in plates vs bags. Circles correspond to the mean of the percentage of CD8 and CD4 positive cells obtained for each donors. For both graphics, left and right circles correspond to TIL expanded in plates and bags respectively. For each line the horizontal bar corresponds to the mean of the 8 donors. The difference between means is indicated by the p-value.

Figure 4

IFNγ expression by TIL produced in plates vs bags. A, representative cytometry dot plots of cells produced in plates (upper panel) and bags (lower panel) double stained for CD8 and IFNγ (see "Materials and Methods") after 6 hours in contact (right panel) or not (left panel) with the autologous melanoma cell line. Representative data obtained from patient 06. Percentages of CD8+/IFNγ+ cells are shown. B, graphical representation of the percentage of CD8+ cells expressing IFNγ in plates (black histograms) and in bags (white histograms) for all donors. C, Total number of CD8+/IFNγ expressing cells produced in plates versus bags. Circles correspond to the mean of CD8+/IFNγ expressing cells obtained for each donors. Left and right circle lines correspond to TIL expanded in plates and bags, respectively. For each line the horizontal bar corresponds to the mean of the 8 donors. The difference between these means is indicated by the p-value.