Protocol detail

Enriched T-cell Manufacture for CAR-T Therapy

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Healthy donor-derived enriched CD4+/8+ T cells were isolated from leukocyte cones using SepMate, RosetteSep reagent (Stemcell Technologies) and Ficoll Paque density grade centrifugation. Excess cryopreserved leukapheresis from B-ALL patients on the ALLCAR19 trial (NCT02935257) were thawed, rested overnight in TexMACS (Miltenyi Biotec) supplemented with 3% human serum (Sigma Aldrich, Life Science Production) and enriched for CD4+/8+T cells using a microbead-based pan T-cell isolation kit (Miltenyi Biotec). Activation/transduction at small-scale was designed to mimic the cGMP Miltenyi CliniMACS Prodigy-based CAR-T manufacture process on ALLCAR19.20 (link) Selected T-cells were maintained in TexMACS supplemented with 3% human serum and 10 ng/mL IL7/IL15 (cTexMACS). Cells were activated with TransAct (Miltenyi Biotec) and transduced with concentrated lentivirus on retronectin-coated plates at a multiplicity of infection of 5, cells were maintained for a further 4 days for a total manufacturing time of 8 days. AKT inhibitor VIII (Merck Millipore/Ardena) was added at T-cell activation at a concentration of 1–5 µM and maintained throughout the culture period, with a media change every 48 hours.

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Mehra V., Agliardi G., Dias Alves Pinto J., Shafat M.S., Garai A.C., Green L., Hotblack A., Arce Vargas F., Peggs K.S., van der Waart A.B., Dolstra H., Pule M.A, & Roddie C. (2023). AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival. Journal for Immunotherapy of Cancer, 11(9), e007002.

Publication 2023

RosetteSep reagent TexMACS human serum AKT inhibitor VIII

Akt inhibitor viii Cd4 t cells Cell isolation Cells Centrifugation Cgmp Cones Donor Ficoll Human Il15 Infection Lentivirus Leukapheresis Leukocyte Microbead Patients Prodigy Retronectin Serum Stemcell T cell Transact

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Variable analysis

independent variables

AKT inhibitor VIII (Merck Millipore/Ardena) concentration (1–5 µM)

dependent variables

T-cell activation and transduction efficiency
T-cell proliferation and viability

control variables

Activation and transduction conditions (e.g., TransAct, lentivirus MOI, retronectin-coated plates)
Cell culture conditions (TexMACS supplemented with 3% human serum and 10 ng/mL IL7/IL15)

controls

Positive control: T-cells from healthy donors
Negative control: Not explicitly mentioned

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