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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 enriched T cells during expansion and transduction on day 1 with lentiviral vector encoding GFP. (A) CD62L-enriched T cells cultured in TexMACS™ GMP medium supplemented with IL-7 and IL-15 were activated using MACS GMP TransAct CD3/CD28 Kit (1:200 and 1:400). T cell clustering after TransAct stimulation. Pictures taken with the integrated microscope camera in the Prodigy system 24–72 h after start of stimulation. (B) Flow cytometric analysis of transduction efficacy on day 7 (MACS Quant Analyzer 10). (C) Fluorescence microscopy (Zeiss Axiovert 200 M, Cellomics ArrayScan, HCS System). Sample was taken from the Prodigy system and transferred to a 6-well culture plate. After resting the cells for 2 h at 37°C, 5% CO2 pictures were taken.
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f5: CD62L enriched T cells during expansion and transduction on day 1 with lentiviral vector encoding GFP. (A) CD62L-enriched T cells cultured in TexMACS™ GMP medium supplemented with IL-7 and IL-15 were activated using MACS GMP TransAct CD3/CD28 Kit (1:200 and 1:400). T cell clustering after TransAct stimulation. Pictures taken with the integrated microscope camera in the Prodigy system 24–72 h after start of stimulation. (B) Flow cytometric analysis of transduction efficacy on day 7 (MACS Quant Analyzer 10). (C) Fluorescence microscopy (Zeiss Axiovert 200 M, Cellomics ArrayScan, HCS System). Sample was taken from the Prodigy system and transferred to a 6-well culture plate. After resting the cells for 2 h at 37°C, 5% CO2 pictures were taken.

Mentions: The stimulation of T cells via CD3 (first signal) and CD28 (second signal) leads to the clustering of the cells, which could be monitored by taking pictures with the Prodigy's integrated camera (Fig. 5A) during static culture until day 3. After cultivation was switched to dynamic mode by start of agitation of the chamber, clusters were dispersed. Transduction performed over a period of 48 h (from day 1 to day 3) with lentiviral vector containing GFP resulted in a transduction efficiency of 83%, as shown by flow cytometric analysis and fluorescence microscopy (Fig 5B and C).


Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L + T Cells for Manufacturing of Gene Therapy Medicinal Products
CD62L enriched T cells during expansion and transduction on day 1 with lentiviral vector encoding GFP. (A) CD62L-enriched T cells cultured in TexMACS™ GMP medium supplemented with IL-7 and IL-15 were activated using MACS GMP TransAct CD3/CD28 Kit (1:200 and 1:400). T cell clustering after TransAct stimulation. Pictures taken with the integrated microscope camera in the Prodigy system 24–72 h after start of stimulation. (B) Flow cytometric analysis of transduction efficacy on day 7 (MACS Quant Analyzer 10). (C) Fluorescence microscopy (Zeiss Axiovert 200 M, Cellomics ArrayScan, HCS System). Sample was taken from the Prodigy system and transferred to a 6-well culture plate. After resting the cells for 2 h at 37°C, 5% CO2 pictures were taken.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: CD62L enriched T cells during expansion and transduction on day 1 with lentiviral vector encoding GFP. (A) CD62L-enriched T cells cultured in TexMACS™ GMP medium supplemented with IL-7 and IL-15 were activated using MACS GMP TransAct CD3/CD28 Kit (1:200 and 1:400). T cell clustering after TransAct stimulation. Pictures taken with the integrated microscope camera in the Prodigy system 24–72 h after start of stimulation. (B) Flow cytometric analysis of transduction efficacy on day 7 (MACS Quant Analyzer 10). (C) Fluorescence microscopy (Zeiss Axiovert 200 M, Cellomics ArrayScan, HCS System). Sample was taken from the Prodigy system and transferred to a 6-well culture plate. After resting the cells for 2 h at 37°C, 5% CO2 pictures were taken.
Mentions: The stimulation of T cells via CD3 (first signal) and CD28 (second signal) leads to the clustering of the cells, which could be monitored by taking pictures with the Prodigy's integrated camera (Fig. 5A) during static culture until day 3. After cultivation was switched to dynamic mode by start of agitation of the chamber, clusters were dispersed. Transduction performed over a period of 48 h (from day 1 to day 3) with lentiviral vector containing GFP resulted in a transduction efficiency of 83%, as shown by flow cytometric analysis and fluorescence microscopy (Fig 5B and C).

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.