Large scale ex vivo expansion of clinical-grade effector cells for adoptive immunotherapy

Abstract

Cell-based adoptive immunotherapy for the treatment of various cancer types has attracted the attention of scientists. However, due to the absence of unitary standard protocols to produce large quantities of clinical-grade effector cells, it remains challenging to translate the experimental findings into clinical applications. The present study used methods complying with good manufacturing practice to induce effector cells from human peripheral blood mononuclear cells (PBMCs) of healthy donors by interleukin-2 and anti-Her-2 antibody with or without anti-CD3 antibodies (OKT3). The results indicated that the addition of OKT3 resulted in a greater expansion of the total cells and CD8⁺ T cells, and primarily induced the PBMCs to differentiate into CD3⁺ T cells. Regardless of the presence of OKT3, the expression of activating receptor of natural killer (NK) group 2, member D, and the inhibitory receptors of CD158a and CD158b on NK cells and NKT cells was increased, while the expression of NKp46 was inhibited on NK cells, but not on NKT cells. Furthermore, OKT3 did not affect the toxicity of the effector cells. Subgroup analysis indicated that although a variation of the composition of effector cells was present in different individuals under identical culture conditions, consistent marker expression on effector cells and target cell-killing effects were observed in different subgroups treated with or without OKT3. Furthermore, western blot analysis indicated that OKT3, apart from its involvement in cell cycle regulation, affects transcription and protein translation during processes of proliferation and differentiation. The present study provided experimental data regarding the production of effector cells for adoptive immunotherapy as a clinical application.

Keywords: Her-2; adoptive immunotherapy; effector cells.

Figure 1.

Comparison of cell expansion in the different ex vivo culture systems. Fold expansion of total cells, CD8⁺ T cells and NK cells was measured via comparing the number of cells harvested on day 14 with the number of cells initially seeded on day 0 in system A (light gray bar) and B (dark gray bar). Values are expressed as the mean ± standard error of the mean (n=22). **P<0.01, system B vs. system A, paired-samples t-test. NK, natural killer; System A/B, induction in the absence/presence of anti-CD3.

Figure 2.

Cell-surface receptor expression on NK and NKT cells in different culture systems. Three-color fluorescence flow cytometry was used to analyze the percentage of associated cell-surface receptor-positive (A) NK and (B) NKT cells in isolated peripheral blood mononuclear cells on day 0 (light gray bar) or expanded cells on day 14 (dark gray bar). Values are expressed as the mean ± standard error of the mean (n=22). *P<0.05, **P<0.01 (paired-samples t-test). System A/B, induction in the absence/presence of anti-CD3; NKG2D, natural killer group 2, member D.

Figure 3.

Cytotoxicity in vitro. Cytotoxicity of K562 in expanded cells from system A and B. a lactate dehydrogenase assay was performed on day 14 after harvesting the cells cultured at the indicated E:T ratio. K526 cells were used as the target cells. The results are expressed as mean ± standard error of the mean from three independently performed experiments. System A/B, induction in the absence/presence of anti-CD3; E:T ratio, effector vs. target cell ratio.

Figure 4.

Effects of IL-2 and anti-CD3 antibody on JAK/STAT and PI3K/AKT/p70S6K1 signaling pathways and cell cycle regulation in CD8⁺ T and NK cells. Cultures enriched in CD8⁺ T and NK cells were harvested at day 9 and stimulated with either IL-2 only or anti-CD3/IL-2 for 24 h prior to protein extraction. Western blot analysis was performed to identify the variations in the expression of relevant proteins and their phosphorylation products. (A) JAK/STAT, (B) cell cycle regulatory proteins and (C) PI3K/AKT/p70S6K1/S6 axis in CD8⁺ T and NK cells. PI3K, phosphoinositide-3 kinase; IL, interleukin; JAK, Janus kinase; p-STAT, phosphorylated signal transducer and activator of transcription; CDK, cyclin D kinase; mAb, monoclonal antibody; IL, interleukin; NK, natural killer.

Figure 5.

Subgroup analysis of expanded cell subsets. The 22 healthy donors were divided into three groups based on the results of cell phenotype analysis of the final product in system A: NKPG, proportion of NK cells ≥45%; CD8PG, proportion of CD3⁺/CD8⁺ T cells ≥50%; and NK + CD8BG, proportion of NK cells <45% and proportion of CD3⁺/CD8⁺ T cells <50%. Subsets of effector cells, including (A) CD3⁺, (B) CD3⁺/CD4⁺, (C) CD3⁺/CD8⁺, (D) CD3⁻/CD56⁺ and (E) CD3⁺/CD56⁺ expanded in peripheral blood mononuclear cells from different subgroups in systems A and B were evaluated. Values are expressed as the mean ± standard error of the mean. *P<0.05 and **P<0.01. NK, natural killer; System A/B, induction in the absence/presence of anti-CD3.

Figure 6.

Subgroup analysis of cytotoxicity on expanded cells. The cytotoxicity of K562 in expanded cells from the NKPG, CD8PG and NK+CD8BG groups in systems A and B was assessed. A lactate dehydrogenase assay was performed on day 14 after harvesting the cells at the indicated ratios of effector cells vs. target cells. K526 cells were used as the target cells. Results are expressed as mean ± standard error of the mean from three independently performed experiments. Groups: NKPG, proportion of NK cells ≥45%; CD8PG, proportion of CD3⁺/CD8⁺ T cells ≥50%; and NK+CD8BG, proportion of NK cells <45% and proportion of CD3⁺/CD8⁺ T cells <50%. NK, natural killer; system A/B, induction in the absence/presence of anti-CD3.