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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.


CD62L expression on naïve and central memory T cells is downregulated due to freezing and thawing. (A) Characterization of T cell phenotype in apheresis product of healthy donor using CD62L and CD45RO expression among all viable CD45+CD3+ cells. Naïve T cells were defined as CD3+CD62L+CD45RO−, central memory T cells as CD3+CD62L+CD45RO−, and effector memory T cells as CD3+CD62L−CD45RO−. (B) Apheresis product of a healthy donor was stored at –70°C, and thawed and recovered in TexMACS™ medium without supplements (37°C, 5% CO2). T cells were analyzed directly as well as 24, 48, and 72 h after thawing. Characterization of T cell phenotype was done via CD62L and CD45RO re-expression among all viable CD45+CD3+ cells.
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f2: CD62L expression on naïve and central memory T cells is downregulated due to freezing and thawing. (A) Characterization of T cell phenotype in apheresis product of healthy donor using CD62L and CD45RO expression among all viable CD45+CD3+ cells. Naïve T cells were defined as CD3+CD62L+CD45RO−, central memory T cells as CD3+CD62L+CD45RO−, and effector memory T cells as CD3+CD62L−CD45RO−. (B) Apheresis product of a healthy donor was stored at –70°C, and thawed and recovered in TexMACS™ medium without supplements (37°C, 5% CO2). T cells were analyzed directly as well as 24, 48, and 72 h after thawing. Characterization of T cell phenotype was done via CD62L and CD45RO re-expression among all viable CD45+CD3+ cells.

Mentions: In order to evaluate whether part of the apheresis or CD62L+ selected T cells can be cryopreserved for later cell processing including TCT, CD62L antigen expression on T cells was analyzed. The fresh apheresis product contains CD45RO−CD62L+ naïve T cells, CD45RO+CD62L+ central memory T cells, as well as CD45RO+CD62L− effector memory and CD45RO−CD62L− effector T cells (Fig. 2A). Freezing and thawing of the apheresis resulted in a clear downregulation of CD62L expression, which was restored during cultivation (gated on CD45+CD3+). The percentage of CD62L+ cells increased from 15% immediately after thawing to 50% after 72 h of cultivation (Fig. 2B). These experiments led to the decision to purify naïve and central memory T cells from fresh apheresis products and consequent cryopreservation.


Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L + T Cells for Manufacturing of Gene Therapy Medicinal Products
CD62L expression on naïve and central memory T cells is downregulated due to freezing and thawing. (A) Characterization of T cell phenotype in apheresis product of healthy donor using CD62L and CD45RO expression among all viable CD45+CD3+ cells. Naïve T cells were defined as CD3+CD62L+CD45RO−, central memory T cells as CD3+CD62L+CD45RO−, and effector memory T cells as CD3+CD62L−CD45RO−. (B) Apheresis product of a healthy donor was stored at –70°C, and thawed and recovered in TexMACS™ medium without supplements (37°C, 5% CO2). T cells were analyzed directly as well as 24, 48, and 72 h after thawing. Characterization of T cell phenotype was done via CD62L and CD45RO re-expression among all viable CD45+CD3+ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035932&req=5

f2: CD62L expression on naïve and central memory T cells is downregulated due to freezing and thawing. (A) Characterization of T cell phenotype in apheresis product of healthy donor using CD62L and CD45RO expression among all viable CD45+CD3+ cells. Naïve T cells were defined as CD3+CD62L+CD45RO−, central memory T cells as CD3+CD62L+CD45RO−, and effector memory T cells as CD3+CD62L−CD45RO−. (B) Apheresis product of a healthy donor was stored at –70°C, and thawed and recovered in TexMACS™ medium without supplements (37°C, 5% CO2). T cells were analyzed directly as well as 24, 48, and 72 h after thawing. Characterization of T cell phenotype was done via CD62L and CD45RO re-expression among all viable CD45+CD3+ cells.
Mentions: In order to evaluate whether part of the apheresis or CD62L+ selected T cells can be cryopreserved for later cell processing including TCT, CD62L antigen expression on T cells was analyzed. The fresh apheresis product contains CD45RO−CD62L+ naïve T cells, CD45RO+CD62L+ central memory T cells, as well as CD45RO+CD62L− effector memory and CD45RO−CD62L− effector T cells (Fig. 2A). Freezing and thawing of the apheresis resulted in a clear downregulation of CD62L expression, which was restored during cultivation (gated on CD45+CD3+). The percentage of CD62L+ cells increased from 15% immediately after thawing to 50% after 72 h of cultivation (Fig. 2B). These experiments led to the decision to purify naïve and central memory T cells from fresh apheresis products and consequent cryopreservation.

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.