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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.


Flow cytometric analyses of CD62L purified T cells on different days during manufacturing in the Prodigy system. (A) Frequency of CD62L+ cells gated on viable T lymphocytes (7-AAD−CD45+CD3+). Upper row: starting material (apheresis of donor2), after CD62L-selection (non-target and target fraction), on days 6 and 10 (harvesting) of cultivation. Lower row: first plot—donor1 cells on day 6, plots 2–5—donor2cryo on the day of thawing after 24 h recovery on days 4 and 12 (harvesting) of cultivation. NF, non-target fraction. (B) Cellular subpopulation during cultivation (donor2cryo). Upper row: forward (FSC) and side light-scatter (SSC) features of the cell suspension, plots gated on viable leucocytes (7-AAD−CD45+). Middle row: frequency of T cells (7-AAD−CD45+CD3+) and B cells (7-AAD−CD45+CD20+); plots gated on viable leucocytes (7-AAD−CD45+). Lower row: distribution (ratio) of CD4+ and CD8+ cells; plots gated on T lymphocytes. (C) Frequency of T cells (7-AAD−CD45+CD3+) in all four Prodigy products (at harvesting); plots gated on viable leucocytes (7-AAD−CD45+), including three mock runs and one run using GFP transduced T cells. (D) Phenotyping profile of the final product of donor2fresh and donor2cryo (flow cytometric analysis performed on cryopreserved/thawed product; cells analyzed 48 h after thawing). Distribution of naïve (TN CD45RA+CD62L+), central memory (TCM CD45RA−CD62L+), effector memory (TEM CD45RA−CD62L−), CD45RA+ effector memory cells (TEMRA CD45RA+CD62L−), and stem memory T cells (TSCM CD45RA+CD45RO+CD62L+). Left panel gated on CD3+ cells; right panel gated on CD3+CD62L+ cells.
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f4: Flow cytometric analyses of CD62L purified T cells on different days during manufacturing in the Prodigy system. (A) Frequency of CD62L+ cells gated on viable T lymphocytes (7-AAD−CD45+CD3+). Upper row: starting material (apheresis of donor2), after CD62L-selection (non-target and target fraction), on days 6 and 10 (harvesting) of cultivation. Lower row: first plot—donor1 cells on day 6, plots 2–5—donor2cryo on the day of thawing after 24 h recovery on days 4 and 12 (harvesting) of cultivation. NF, non-target fraction. (B) Cellular subpopulation during cultivation (donor2cryo). Upper row: forward (FSC) and side light-scatter (SSC) features of the cell suspension, plots gated on viable leucocytes (7-AAD−CD45+). Middle row: frequency of T cells (7-AAD−CD45+CD3+) and B cells (7-AAD−CD45+CD20+); plots gated on viable leucocytes (7-AAD−CD45+). Lower row: distribution (ratio) of CD4+ and CD8+ cells; plots gated on T lymphocytes. (C) Frequency of T cells (7-AAD−CD45+CD3+) in all four Prodigy products (at harvesting); plots gated on viable leucocytes (7-AAD−CD45+), including three mock runs and one run using GFP transduced T cells. (D) Phenotyping profile of the final product of donor2fresh and donor2cryo (flow cytometric analysis performed on cryopreserved/thawed product; cells analyzed 48 h after thawing). Distribution of naïve (TN CD45RA+CD62L+), central memory (TCM CD45RA−CD62L+), effector memory (TEM CD45RA−CD62L−), CD45RA+ effector memory cells (TEMRA CD45RA+CD62L−), and stem memory T cells (TSCM CD45RA+CD45RO+CD62L+). Left panel gated on CD3+ cells; right panel gated on CD3+CD62L+ cells.

Mentions: While approximately two thirds of the unmanipulated apheresis consisted of CD62L+ cells, the target fraction after CD62L enrichment contained >80% CD62L+ cells. The percentage of CD62L+ rose to >95% of the final product at day 10–13, as shown in Fig. 4A for donor2fresh and donor2cryo. In this respect, it has to be noted that due to the masking of the CD62L receptor during immunomagnetic labeling, the quantification of enriched CD62L+CD3+ cells is impaired (Fig. 4A). Additionally, the downregulation of CD62L expression after cryopreservation/thawing adversely affects the precision of flow cytometric analysis of CD62L+ cells after selection/thawing (donor2cryo) in the first days of cultivation. Both effects disappeared during early cultivation, and the frequency of CD62L+ rose to 99% for donor1 (day 6), 98% for donor2fresh (day 6), and 99% for donor2cryo (day 4).


Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L + T Cells for Manufacturing of Gene Therapy Medicinal Products
Flow cytometric analyses of CD62L purified T cells on different days during manufacturing in the Prodigy system. (A) Frequency of CD62L+ cells gated on viable T lymphocytes (7-AAD−CD45+CD3+). Upper row: starting material (apheresis of donor2), after CD62L-selection (non-target and target fraction), on days 6 and 10 (harvesting) of cultivation. Lower row: first plot—donor1 cells on day 6, plots 2–5—donor2cryo on the day of thawing after 24 h recovery on days 4 and 12 (harvesting) of cultivation. NF, non-target fraction. (B) Cellular subpopulation during cultivation (donor2cryo). Upper row: forward (FSC) and side light-scatter (SSC) features of the cell suspension, plots gated on viable leucocytes (7-AAD−CD45+). Middle row: frequency of T cells (7-AAD−CD45+CD3+) and B cells (7-AAD−CD45+CD20+); plots gated on viable leucocytes (7-AAD−CD45+). Lower row: distribution (ratio) of CD4+ and CD8+ cells; plots gated on T lymphocytes. (C) Frequency of T cells (7-AAD−CD45+CD3+) in all four Prodigy products (at harvesting); plots gated on viable leucocytes (7-AAD−CD45+), including three mock runs and one run using GFP transduced T cells. (D) Phenotyping profile of the final product of donor2fresh and donor2cryo (flow cytometric analysis performed on cryopreserved/thawed product; cells analyzed 48 h after thawing). Distribution of naïve (TN CD45RA+CD62L+), central memory (TCM CD45RA−CD62L+), effector memory (TEM CD45RA−CD62L−), CD45RA+ effector memory cells (TEMRA CD45RA+CD62L−), and stem memory T cells (TSCM CD45RA+CD45RO+CD62L+). Left panel gated on CD3+ cells; right panel gated on CD3+CD62L+ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f4: Flow cytometric analyses of CD62L purified T cells on different days during manufacturing in the Prodigy system. (A) Frequency of CD62L+ cells gated on viable T lymphocytes (7-AAD−CD45+CD3+). Upper row: starting material (apheresis of donor2), after CD62L-selection (non-target and target fraction), on days 6 and 10 (harvesting) of cultivation. Lower row: first plot—donor1 cells on day 6, plots 2–5—donor2cryo on the day of thawing after 24 h recovery on days 4 and 12 (harvesting) of cultivation. NF, non-target fraction. (B) Cellular subpopulation during cultivation (donor2cryo). Upper row: forward (FSC) and side light-scatter (SSC) features of the cell suspension, plots gated on viable leucocytes (7-AAD−CD45+). Middle row: frequency of T cells (7-AAD−CD45+CD3+) and B cells (7-AAD−CD45+CD20+); plots gated on viable leucocytes (7-AAD−CD45+). Lower row: distribution (ratio) of CD4+ and CD8+ cells; plots gated on T lymphocytes. (C) Frequency of T cells (7-AAD−CD45+CD3+) in all four Prodigy products (at harvesting); plots gated on viable leucocytes (7-AAD−CD45+), including three mock runs and one run using GFP transduced T cells. (D) Phenotyping profile of the final product of donor2fresh and donor2cryo (flow cytometric analysis performed on cryopreserved/thawed product; cells analyzed 48 h after thawing). Distribution of naïve (TN CD45RA+CD62L+), central memory (TCM CD45RA−CD62L+), effector memory (TEM CD45RA−CD62L−), CD45RA+ effector memory cells (TEMRA CD45RA+CD62L−), and stem memory T cells (TSCM CD45RA+CD45RO+CD62L+). Left panel gated on CD3+ cells; right panel gated on CD3+CD62L+ cells.
Mentions: While approximately two thirds of the unmanipulated apheresis consisted of CD62L+ cells, the target fraction after CD62L enrichment contained >80% CD62L+ cells. The percentage of CD62L+ rose to >95% of the final product at day 10–13, as shown in Fig. 4A for donor2fresh and donor2cryo. In this respect, it has to be noted that due to the masking of the CD62L receptor during immunomagnetic labeling, the quantification of enriched CD62L+CD3+ cells is impaired (Fig. 4A). Additionally, the downregulation of CD62L expression after cryopreservation/thawing adversely affects the precision of flow cytometric analysis of CD62L+ cells after selection/thawing (donor2cryo) in the first days of cultivation. Both effects disappeared during early cultivation, and the frequency of CD62L+ rose to 99% for donor1 (day 6), 98% for donor2fresh (day 6), and 99% for donor2cryo (day 4).

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.