A full GMP process to select and amplify epitope-specific T lymphocytes for adoptive immunotherapy of metastatic melanoma

Abstract

A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved. We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting. In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1. All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification. The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 10⁸ pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients. This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.

Figure 1

Peptide stimulation step. (a) 10⁷ PBMC from HLA-A2 healthy donors were stimulated in 96-well plated (containing 2 × 10⁵ cells/well) for 14 days with Melan-A or MELOE-1 peptides. (b) Percentages of Melan-A- or (c) MELOE-1-specific T cells detected by double labelling with tetramer and anti-CD8 antibody, in microcultures stimulated with 0.1 to 10 μM of Melan-A_A27L or MELOE-1_36-44. Each symbol corresponds to a microculture containing more than 0.5% of specific T cells.

Figure 2

Influence of IL-2 concentration on the efficiency of peptide stimulation. 10⁷ PBMC from HLA-A2 healthy donors were stimulated in 96-well plated (2 × 10⁵ cells/well) with either Melan-A_A27L peptide (1 μM) or the MELOE-1_36-44 peptide (10 μM). (a) Percentages of Melan-A- or (b) MELOE-1-specific T cells detected by double labelling with tetramer and anti-CD8 antibody, in microcultures stimulated in the presence of various IL-2 concentrations. Each symbol corresponds to a microculture containing more than 0.5% of specific T cells.

Figure 3

Influence of the culture medium on the efficiency of peptide stimulation. 10⁷ PBMC from HLA-A2 healthy donors (a and b) or from melanoma patients (c) were stimulated in 96-well plated (2 × 10⁵ cells/well) with either Melan-A_A27L peptide (a) at 1 μM or MELOE-1_36-44 peptide (b and c) at 10 μM, in either RPMI 8% HS or X-Vivo 15 in the presence of 50 IU/mL of IL-2. (a) Percentages of Melan-A- or (b and c) MELOE-1-specific T cells detected by double labelling with tetramer and anti-CD8 antibody, in microcultures stimulated in the two culture media. Each symbol corresponds to a microculture containing more than 0.5% of specific T cells.

Figure 4

Sorting procedure. (a) 10⁷ Chim-AvT dynabeads are coated with HLA-A2-peptide monomers, and coating efficiency is assessed on 10⁵ beads, by labelling with an PE-labelled anti-HLA-A2 mAb (5 μg/mL). (b) Peptide stimulated T cells, containing at least 1% of specific T cells are incubated with Clinimers (ratio 1/1) and recovered on a magnet. After washes in PBS, rosetted T cells are seeded in 96-well plates (10³ cells/well), containing irradiated allogeneic feeder cells, for amplification. After 14 days, the specificity of T cells is assessed by double labelling with an anti-CD8 mAb and each specific tetramer. (c) Influence of the number of washes on sorting yields. Sorting yields are calculated by dividing the number of rosetted T lymphocytes counted after the sort by the number of specific T cells in the sorted population estimated by tetramer staining. Blue bars represent Melan-A-specific T cells and red bars MELOE-1-specific ones. (d) Influence of the number of washes on purity of selected and amplified T cells. The purity of amplified sorted-T cells is assessed after the 14-day amplification period on feeder cells, by double staining with an anti-CD8 mAb and the specific tetramer. Blue bars represent Melan-A-specific T cells and red bars MELOE-1-specific ones.

Figure 5

Detection of residual beads in amplified T cells. (a) Limit of detection of magnetic beads among a T cell population. Variable numbers of beads were mixed with 10⁶ T cells in 1 mL. Beads were detected by their autofluorescence in the various channels of a Facs Canto II. (b) Absence of residual beads in amplified T cells. After 8 days of culture of sorted T cells, each T cell suspension is placed inside a magnet to remove magnetic beads. After two rounds of elimination, the absence of residual beads is checked by flow cytometry.

Figure 6

Purity and reactivity of sorted and amplified T cells. (a) Purity of sorted and amplified T cell populations is assessed by double staining with an anti-CD8 specific mAb and each specific HLA-p tetramers. (b) Activity of sorted T cells is measured by TNF production in response to the cognate peptide or to HLA-A2 melanoma cell lines expressing Melan-A and MELOE-1 antigens. After activation, T cells are stained with their specific HLA-p tetramer, and TNF producing cells are visualized by intracellular staining with an anti-TNF mAb. Blue histograms represent Melan-A- specific T cells and red ones MELOE-1-specific T cells.

Figure 7

Design of the whole GMP process. At day 0, 100 mL of blood is collected from HLA-A2 melanoma patients. Total PBMC are stimulated with the two antigenic peptides during 14 days. At day 14, antigen-specific T cells are sorted using Clinimers, and 10⁵ rosetted T cells are seeded for amplification on feeder cells. At day 28, purity of amplified cells is assessed by flow cytometry and between 10⁸ and 5 × 10⁸ T lymphocytes specific for each peptide will be infused to the autologous melanoma patient.