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Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L + T Cells for Manufacturing of Gene Therapy Medicinal Products

View Article: PubMed Central - PubMed

ABSTRACT

Multiple clinical studies have demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products (GTMPs) leads to a manufacturing challenge regarding automation, process robustness, and cell storage. Therefore, this study addresses the proof of principle in clinical-scale selection, stimulation, transduction, and expansion of T cells using the automated closed CliniMACS® Prodigy system. Naïve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n = 3) or split for cryopreservation and processed after thawing (n = 1). Starting with 0.5 × 108 purified CD3+ T cells, three mock runs and one run including transduction with green fluorescent protein (GFP)-containing vector resulted in a median final cell product of 16 × 108 T cells (32-fold expansion) up to harvesting after 2 weeks. Expression of CD62L was downregulated on T cells after thawing, which led to the decision to purify CD62L+CD3+ T cells freshly with cryopreservation thereafter. Most important in the split product, a very similar expansion curve was reached comparing the overall freshly CD62L selected cells with those after thawing, which could be demonstrated in the T cell subpopulations as well by showing a nearly identical conversion of the CD4/CD8 ratio. In the GFP run, the transduction efficacy was 83%. In-process control also demonstrated sufficient glucose levels during automated feeding and medium removal. The robustness of the process and the constant quality of the final product in a closed and automated system give rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies.

No MeSH data available.


Clinical scale cell expansion of CD62L selected T cells in the Prodigy system (three mock runs and one green fluorescent protein [GFP] vector run). (A) Increase in absolute viable leucocytes on different days up to harvesting; CD45+ 7-AAD neg. cells are quantified with single platform flow cytometry analysis. WBC, white blood cells. (B) Absolute cell numbers of viable CD3+ lymphocytes on different days in culture. (C) Expansion rate from beginning of cultivation (day 0) up to harvesting (days 10–13) for both, viable WBC and viable CD3+ T cells. (D) Ratio of CD3+CD4+ and CD3+CD8+ lymphocytes at different days of culture of all mock runs (donor1, donor2fresh, and donor2cryo). (E) Removal of AB serum during T cell expansion caused by medium exchange as indicated (125 mL, 150 mL, and 180 mL = exchange of 125 mL, 150 mL, and 180 mL), shown for the mock run starting with cryopreserved CD62L-selected T cells (donor2cryo). (F) In-process control of glucose level during T cell expansion shown for the mock run (donor2cryo).
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f3: Clinical scale cell expansion of CD62L selected T cells in the Prodigy system (three mock runs and one green fluorescent protein [GFP] vector run). (A) Increase in absolute viable leucocytes on different days up to harvesting; CD45+ 7-AAD neg. cells are quantified with single platform flow cytometry analysis. WBC, white blood cells. (B) Absolute cell numbers of viable CD3+ lymphocytes on different days in culture. (C) Expansion rate from beginning of cultivation (day 0) up to harvesting (days 10–13) for both, viable WBC and viable CD3+ T cells. (D) Ratio of CD3+CD4+ and CD3+CD8+ lymphocytes at different days of culture of all mock runs (donor1, donor2fresh, and donor2cryo). (E) Removal of AB serum during T cell expansion caused by medium exchange as indicated (125 mL, 150 mL, and 180 mL = exchange of 125 mL, 150 mL, and 180 mL), shown for the mock run starting with cryopreserved CD62L-selected T cells (donor2cryo). (F) In-process control of glucose level during T cell expansion shown for the mock run (donor2cryo).

Mentions: CD62L selection of three unstimulated leukapheresis products led to an overall recovery of CD3+ cells between 37.3 and 79.9 (median 66.8%). The recovery was calculated as the ratio between CD3+ cells after CD62L selection and CD3+ CD62L+ before selection due to the masking of the CD62L antigen by the selection reagent. While more than four out of five of selected cells were cryopreserved, only 70–100 × 106 (median 91 × 106) viable leucocytes containing 44–54 × 106 (median 47 × 106) viable CD3+ cells were further processed in the Prodigy system, leading to a final product containing 1,340–2,091 × 106 (median 1,685 × 106) viable leucocytes (viability >85%) and 1,313–1,889 × 106 (median 1,547 × 106) viable CD3+ cells after a cultivation time of 10–13 days (Fig. 3A and B). Accordingly, the expansion rate was 13- to 23-fold (median 22-fold) for viable leucocytes, and 28- to 42-fold (median 32-fold) for CD3+ T cells (Fig. 3C). Notably, this low variance includes the expansion of both fresh and cryopreserved CD62L selected cells (Fig. 3A–D).


Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L + T Cells for Manufacturing of Gene Therapy Medicinal Products
Clinical scale cell expansion of CD62L selected T cells in the Prodigy system (three mock runs and one green fluorescent protein [GFP] vector run). (A) Increase in absolute viable leucocytes on different days up to harvesting; CD45+ 7-AAD neg. cells are quantified with single platform flow cytometry analysis. WBC, white blood cells. (B) Absolute cell numbers of viable CD3+ lymphocytes on different days in culture. (C) Expansion rate from beginning of cultivation (day 0) up to harvesting (days 10–13) for both, viable WBC and viable CD3+ T cells. (D) Ratio of CD3+CD4+ and CD3+CD8+ lymphocytes at different days of culture of all mock runs (donor1, donor2fresh, and donor2cryo). (E) Removal of AB serum during T cell expansion caused by medium exchange as indicated (125 mL, 150 mL, and 180 mL = exchange of 125 mL, 150 mL, and 180 mL), shown for the mock run starting with cryopreserved CD62L-selected T cells (donor2cryo). (F) In-process control of glucose level during T cell expansion shown for the mock run (donor2cryo).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5035932&req=5

f3: Clinical scale cell expansion of CD62L selected T cells in the Prodigy system (three mock runs and one green fluorescent protein [GFP] vector run). (A) Increase in absolute viable leucocytes on different days up to harvesting; CD45+ 7-AAD neg. cells are quantified with single platform flow cytometry analysis. WBC, white blood cells. (B) Absolute cell numbers of viable CD3+ lymphocytes on different days in culture. (C) Expansion rate from beginning of cultivation (day 0) up to harvesting (days 10–13) for both, viable WBC and viable CD3+ T cells. (D) Ratio of CD3+CD4+ and CD3+CD8+ lymphocytes at different days of culture of all mock runs (donor1, donor2fresh, and donor2cryo). (E) Removal of AB serum during T cell expansion caused by medium exchange as indicated (125 mL, 150 mL, and 180 mL = exchange of 125 mL, 150 mL, and 180 mL), shown for the mock run starting with cryopreserved CD62L-selected T cells (donor2cryo). (F) In-process control of glucose level during T cell expansion shown for the mock run (donor2cryo).
Mentions: CD62L selection of three unstimulated leukapheresis products led to an overall recovery of CD3+ cells between 37.3 and 79.9 (median 66.8%). The recovery was calculated as the ratio between CD3+ cells after CD62L selection and CD3+ CD62L+ before selection due to the masking of the CD62L antigen by the selection reagent. While more than four out of five of selected cells were cryopreserved, only 70–100 × 106 (median 91 × 106) viable leucocytes containing 44–54 × 106 (median 47 × 106) viable CD3+ cells were further processed in the Prodigy system, leading to a final product containing 1,340–2,091 × 106 (median 1,685 × 106) viable leucocytes (viability >85%) and 1,313–1,889 × 106 (median 1,547 × 106) viable CD3+ cells after a cultivation time of 10–13 days (Fig. 3A and B). Accordingly, the expansion rate was 13- to 23-fold (median 22-fold) for viable leucocytes, and 28- to 42-fold (median 32-fold) for CD3+ T cells (Fig. 3C). Notably, this low variance includes the expansion of both fresh and cryopreserved CD62L selected cells (Fig. 3A–D).

View Article: PubMed Central - PubMed

ABSTRACT

Multiple clinical studies have demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products (GTMPs) leads to a manufacturing challenge regarding automation, process robustness, and cell storage. Therefore, this study addresses the proof of principle in clinical-scale selection, stimulation, transduction, and expansion of T cells using the automated closed CliniMACS® Prodigy system. Naïve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n = 3) or split for cryopreservation and processed after thawing (n = 1). Starting with 0.5 × 108 purified CD3+ T cells, three mock runs and one run including transduction with green fluorescent protein (GFP)-containing vector resulted in a median final cell product of 16 × 108 T cells (32-fold expansion) up to harvesting after 2 weeks. Expression of CD62L was downregulated on T cells after thawing, which led to the decision to purify CD62L+CD3+ T cells freshly with cryopreservation thereafter. Most important in the split product, a very similar expansion curve was reached comparing the overall freshly CD62L selected cells with those after thawing, which could be demonstrated in the T cell subpopulations as well by showing a nearly identical conversion of the CD4/CD8 ratio. In the GFP run, the transduction efficacy was 83%. In-process control also demonstrated sufficient glucose levels during automated feeding and medium removal. The robustness of the process and the constant quality of the final product in a closed and automated system give rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies.

No MeSH data available.