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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.


Related in: MedlinePlus

Automated manufacturing of both expanded and genetically modified naïve and central memory T cells with the Prodigy system. The manufacturing process of genetically modified T cells involves the immunomagnetic enrichment of CD62L+ cells from unstimulated leukapheresis products followed by polyclonal activation (CD28 and CD3), lentiviral transduction, and subsequent expansion of the target fraction: day 0, immunomagnetic selection and activation; day 1, start of transduction; day 3, removal of TransAct CD3/CD28 Reagent and vector, start of dynamic culture; day 5, feed by addition of medium with AB serum; days 6, 8 (10–12), feed by exchange of increasing volumes of cell-free supernatant by medium without AB serum; between days 10 and 13, harvest and final formulation of cells. The implementation of additional feeding steps depends on the duration of the expansion phase. The fading of the red color depicts depletion of AB serum and vector. The integrated process is conducted in a sterile functionally closed disposable tubing system. Sample and waste receptacles as well as inoculum, reagent, and media reservoirs are aseptically dis/connected by heat sealing/welding. IPC, in-process control.
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f1: Automated manufacturing of both expanded and genetically modified naïve and central memory T cells with the Prodigy system. The manufacturing process of genetically modified T cells involves the immunomagnetic enrichment of CD62L+ cells from unstimulated leukapheresis products followed by polyclonal activation (CD28 and CD3), lentiviral transduction, and subsequent expansion of the target fraction: day 0, immunomagnetic selection and activation; day 1, start of transduction; day 3, removal of TransAct CD3/CD28 Reagent and vector, start of dynamic culture; day 5, feed by addition of medium with AB serum; days 6, 8 (10–12), feed by exchange of increasing volumes of cell-free supernatant by medium without AB serum; between days 10 and 13, harvest and final formulation of cells. The implementation of additional feeding steps depends on the duration of the expansion phase. The fading of the red color depicts depletion of AB serum and vector. The integrated process is conducted in a sterile functionally closed disposable tubing system. Sample and waste receptacles as well as inoculum, reagent, and media reservoirs are aseptically dis/connected by heat sealing/welding. IPC, in-process control.

Mentions: First, CliniMACS® phosphate-buffered saline (PBS)/ethylene-diamine-tetraacetic acid (EDTA) buffer containing 0.5% (w/v) human serum albumin (HSA; 200 g/L; Baxter, Unterschleissheim, Germany) and TexMACS™ GMP medium supplemented with interleukin-7 (IL-7) and IL-15 (both Miltenyi Biotec) and 3% (v/v) of heat-inactivated AB serum (Biochrom, Berlin, Germany) were prepared. According to the instructions on the guided user interface of the Prodigy system, the TS520 tubing set was installed, and reagents, buffer, media, and max. 3 × 109 CD62L+ cells (aliquot of the unstimulated apheresis product) were connected to the appropriate ports of the tubing set. The processing was controlled by the Prodigy system's software v1.1.4 and process version v0.8. CliniMACS® CD62L Reagent (Miltenyi Biotec) was used for enrichment, and MACS GMP TransAct CD3 Reagent (1:200) and MACS GMP TransAct CD28 Reagent (1:400) were used for stimulation of 0.5–2 × 108 WBCs according to the manufacturer's instructions (Miltenyi Biotec). Transduction took place on day 1 of cultivation by adding 10 mL of a GFP-containing SIN, VSV-G pseudotyped lentiviral vector with an SFFV promotor (donor 3GFP run) or 10 mL of supplemented TexMACS™ GMP medium in case of mock runs (donor1, donor2fresh/cryo). The lentiviral vector was produced using HEK293T cells (ATCC CRL-11268). The supernatant was concentrated and stored at <–60°C until transduction. The vector titer was determined on HT1080 cells (ATCC CCL-121). A multiplicity of infection (MOI) of 1 was used for transduction. After day 5 of cultivation, the concentration of AB serum was reduced using TexMACS™ GMP medium containing IL-7 and IL-15, respectively, but without AB serum for further medium exchange. On day of harvest, cells were formulated in Composol® PS (Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany) containing 2.86% (w/v) HSA (200 g/L, Baxter). A summary of the process is shown in Fig. 1. After CD62L selection, a part of the positive fraction not being used for cell stimulation and expansion was cryopreserved in Composol® PS containing 2.86% (w/v) HSA (as above) with 10% (v/v) DMSO (Cryosure; WAK-Chemie Medical GmbH, Steinbach/Ts., Germany). After cryopreservation in a <–60°C freezer overnight, the cells were stored in the vapor phase above liquid nitrogen at <–140°C. Cryopreserved CD62L selected cells were thawed, washed, and resuspended in TexMACS™ GMP medium with 3% (v/v) heat-inactivated human AB serum (Biochrom), 10 ng/mL MACS® GMP recombinant human IL-7 and 10 ng/mL MACS® GMP recombinant human IL-15 for cultivation in a CO2 incubator (37°C, 5% CO2) overnight. The T cell transduction (TCT) process was started with stimulation, mock transduction, and expansion (as described above).


Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L + T Cells for Manufacturing of Gene Therapy Medicinal Products
Automated manufacturing of both expanded and genetically modified naïve and central memory T cells with the Prodigy system. The manufacturing process of genetically modified T cells involves the immunomagnetic enrichment of CD62L+ cells from unstimulated leukapheresis products followed by polyclonal activation (CD28 and CD3), lentiviral transduction, and subsequent expansion of the target fraction: day 0, immunomagnetic selection and activation; day 1, start of transduction; day 3, removal of TransAct CD3/CD28 Reagent and vector, start of dynamic culture; day 5, feed by addition of medium with AB serum; days 6, 8 (10–12), feed by exchange of increasing volumes of cell-free supernatant by medium without AB serum; between days 10 and 13, harvest and final formulation of cells. The implementation of additional feeding steps depends on the duration of the expansion phase. The fading of the red color depicts depletion of AB serum and vector. The integrated process is conducted in a sterile functionally closed disposable tubing system. Sample and waste receptacles as well as inoculum, reagent, and media reservoirs are aseptically dis/connected by heat sealing/welding. IPC, in-process control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Automated manufacturing of both expanded and genetically modified naïve and central memory T cells with the Prodigy system. The manufacturing process of genetically modified T cells involves the immunomagnetic enrichment of CD62L+ cells from unstimulated leukapheresis products followed by polyclonal activation (CD28 and CD3), lentiviral transduction, and subsequent expansion of the target fraction: day 0, immunomagnetic selection and activation; day 1, start of transduction; day 3, removal of TransAct CD3/CD28 Reagent and vector, start of dynamic culture; day 5, feed by addition of medium with AB serum; days 6, 8 (10–12), feed by exchange of increasing volumes of cell-free supernatant by medium without AB serum; between days 10 and 13, harvest and final formulation of cells. The implementation of additional feeding steps depends on the duration of the expansion phase. The fading of the red color depicts depletion of AB serum and vector. The integrated process is conducted in a sterile functionally closed disposable tubing system. Sample and waste receptacles as well as inoculum, reagent, and media reservoirs are aseptically dis/connected by heat sealing/welding. IPC, in-process control.
Mentions: First, CliniMACS® phosphate-buffered saline (PBS)/ethylene-diamine-tetraacetic acid (EDTA) buffer containing 0.5% (w/v) human serum albumin (HSA; 200 g/L; Baxter, Unterschleissheim, Germany) and TexMACS™ GMP medium supplemented with interleukin-7 (IL-7) and IL-15 (both Miltenyi Biotec) and 3% (v/v) of heat-inactivated AB serum (Biochrom, Berlin, Germany) were prepared. According to the instructions on the guided user interface of the Prodigy system, the TS520 tubing set was installed, and reagents, buffer, media, and max. 3 × 109 CD62L+ cells (aliquot of the unstimulated apheresis product) were connected to the appropriate ports of the tubing set. The processing was controlled by the Prodigy system's software v1.1.4 and process version v0.8. CliniMACS® CD62L Reagent (Miltenyi Biotec) was used for enrichment, and MACS GMP TransAct CD3 Reagent (1:200) and MACS GMP TransAct CD28 Reagent (1:400) were used for stimulation of 0.5–2 × 108 WBCs according to the manufacturer's instructions (Miltenyi Biotec). Transduction took place on day 1 of cultivation by adding 10 mL of a GFP-containing SIN, VSV-G pseudotyped lentiviral vector with an SFFV promotor (donor 3GFP run) or 10 mL of supplemented TexMACS™ GMP medium in case of mock runs (donor1, donor2fresh/cryo). The lentiviral vector was produced using HEK293T cells (ATCC CRL-11268). The supernatant was concentrated and stored at <–60°C until transduction. The vector titer was determined on HT1080 cells (ATCC CCL-121). A multiplicity of infection (MOI) of 1 was used for transduction. After day 5 of cultivation, the concentration of AB serum was reduced using TexMACS™ GMP medium containing IL-7 and IL-15, respectively, but without AB serum for further medium exchange. On day of harvest, cells were formulated in Composol® PS (Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany) containing 2.86% (w/v) HSA (200 g/L, Baxter). A summary of the process is shown in Fig. 1. After CD62L selection, a part of the positive fraction not being used for cell stimulation and expansion was cryopreserved in Composol® PS containing 2.86% (w/v) HSA (as above) with 10% (v/v) DMSO (Cryosure; WAK-Chemie Medical GmbH, Steinbach/Ts., Germany). After cryopreservation in a <–60°C freezer overnight, the cells were stored in the vapor phase above liquid nitrogen at <–140°C. Cryopreserved CD62L selected cells were thawed, washed, and resuspended in TexMACS™ GMP medium with 3% (v/v) heat-inactivated human AB serum (Biochrom), 10 ng/mL MACS® GMP recombinant human IL-7 and 10 ng/mL MACS® GMP recombinant human IL-15 for cultivation in a CO2 incubator (37°C, 5% CO2) overnight. The T cell transduction (TCT) process was started with stimulation, mock transduction, and expansion (as described above).

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&reg; Prodigy system. Na&iuml;ve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n&thinsp;=&thinsp;3) or split for cryopreservation and processed after thawing (n&thinsp;=&thinsp;1). Starting with 0.5&thinsp;&times;&thinsp;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&thinsp;&times;&thinsp;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.


Related in: MedlinePlus