Faculty Collaboration Database

Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center. Cytotherapy 2018 Mar;20(3):394-406

Date

12/31/2017

Pubmed ID

29287970

DOI

10.1016/j.jcyt.2017.09.005

Scopus ID

2-s2.0-85039050230 (requires institutional sign-in at Scopus site)   76 Citations

Abstract

BACKGROUND AIMS: Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients.

METHODS: The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days.

RESULTS: Enrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3⁺ T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5-10 resulted in transduction averaging 37%. An average 30-fold expansion of 10⁸ CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82-100% CD3⁺ with a mix of CD4⁺ and CD8⁺ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays.

DISCUSSION: The CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure.

Author List

Zhu F, Shah N, Xu H, Schneider D, Orentas R, Dropulic B, Hari P, Keever-Taylor CA

Authors

Parameswaran Hari MD Adjunct Professor in the Medicine department at Medical College of Wisconsin
Nirav N. Shah MD Associate Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Academic Medical Centers
Antigens, CD19
Antigens, CD20
B-Lymphocytes
CD28 Antigens
CD4-Positive T-Lymphocytes
CD8-Positive T-Lymphocytes
Cell Line
Cytological Techniques
Humans
Immunophenotyping
Receptors, Antigen, T-Cell
T-Lymphocytes
T-Lymphocytes, Cytotoxic
Transduction, Genetic