This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.
The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution’s production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.
The emergence of Adoptive Cell Therapies (ACT) and Chimeric Antigen Receptor (CAR)-T cells have brought about a revolutionary shift in modern clinical practice, establishing a new paradigm of personalized medicine. Particularly, CAR-T cells targeting CD19 have demonstrated exceptional clinical responses and represent the most advanced T cell therapy for B cell malignancies1,2,3,4,5. However, the current commercialized gene-modified T cell therapies heavily rely on viral vectors for gene transfer. The implementation of viral vectors in ACT, especially under Good Manufacturing Practice (GMP) conditions within an academic setting, presents significant challenges due to scalability limitations, specialized equipment requirements, the need for highly skilled personnel, and the use of GMP-compliant reagents for plasmid-mediated viral particle production6,7,8.
Consequently, the manufacturing process for gene-modified T cell therapies is intricate and typically commences with the isolation of Peripheral Blood Mononuclear Cells (PBMCs) from a leukapheresis product. T cells are often enriched from the PBMC pool and activated using anti-CD3/CD28 micro-complexes7,9. Subsequently, T cells are genetically modified with either viral or non-viral vectors, which can be produced in situ or outsourced. To attain the required cell numbers for infusion, gene-modified cells are expanded before final formulation and/or cryopreservation. Finally, the cell product must satisfy various release criteria based on multiple quality control assays9.
This protocol presents a comprehensive methodology utilizing simple techniques for the ex vivo manipulation of healthy PBMCs to manufacture a gene-modified T cell product under GMP-compliant conditions. The protocol incorporates the use of a lentiviral vector, which can be outsourced to a viral production company providing all necessary quantitative/functional, purity, and safety tests (e.g., viral titer, detection of host cell DNA replication-competent lentivirus). For this study, the vector employed is a VSV-g (Vesicular Stomatitis Virus G Protein)-based second-generation lentiviral vector with Green Fluorescent Protein (GFP), as the gene of interest, in constitutive expression.
Lentiviral vectors as gene delivery vehicles are important tools in the field of cell and gene therapy because of their ability to transduce both dividing and non-dividing cells12. However, large-scale production of lentiviral vectors using GMP-compatible methods is still challenging due to various parameters, such as transfection methods and purification steps, which can result in variability in lentivirus batches. Academic centers often obtain viral stocks from viral production biotechnology com…
The authors have nothing to disclose.
This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH – CREATE – INNOVATE (project code: T2EDK – 00474). We would also like to express our gratitude to the Choose Life Foundation for providing continuous support to the GMP Lab "Dimitris Lois" of the Institute of Cell Therapy.
24-well TC-treated plate | Corning | 353047 | |
5 mL FACS tube | Corning | 352054 | |
50 mL Falcon tubes | Greiner biomedica | 227261 | |
6-well TC-treated plate | Corning | 353046 | |
7-AAD | BD Biosciences | 559925 | |
BD FACS CANTO II | BD Biosciences | V96300084 | |
BSA | Applichem | A1391 | |
Cell Counter | Corning Cell Cytosmart | J21E0081 | |
Cryovials | Greiner biomedica | 122263 | |
Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich | D2438 | |
Dulbecco’s Phosphate Buffered Saline (D-PBS) | Life Technologies | 14190 | |
EDTA | Invitrogen | 15575-038 | |
FACS Buffer (1x D-PBS, 0.5% BSA, 2 mM EDTA) | – | – | |
FlowJo Software v10.6.2 | TreeStar Inc | – | |
Gibco CTS Opti-MEM I Medium | ThermoFisher Scientific | A4124801 | |
GMP rhIL-15 | Miltenyi Biotec | 170-076-114 | |
GMP rhIL-2 | Miltenyi Biotec | 170-076-146 | |
GMP rhIL-7 | Miltenyi Biotec | 170-076-111 | |
Hanks’s Balanced Salt Solution (HBSS) | Life Technologies | 14175 | |
HEPA Whitley H35 Hypoxystation | Don Whitley Scientific | HA0315172H | |
Human Serum Albumin (HSA) | Baxter | B05AA01 | |
Junior LB 9509 Portable Luminometer | Berthold Technologies | 6506 | |
Lenti-X Provirus Quantitation Kit | Takara Bio | 631239 | |
Lymphoprep | Stemcell Technologies | 7811 | |
MACS GMP T cell TransAct | Miltenyi Biotec | 170-076-156 | |
Mouse Anti-Human CD3 (Clone: UCHT-1) | BD Biosciences | 557706 | |
MycoAlert Plus Detection kit | Lonza Bioscience | LT07-703 | |
Parafilm ultra-expandable closing film tape 5 cm x 75 m | D.Dutscher | 90261 | |
PeriStem PS-650-DB (apheresis bag) | Biomed Device | SC00816 | |
Planer Kryo10 Series III | Planer | ||
T75 flasks | Greiner biomedica | 658175 | |
Trypan blue, 0.4% solution | Invitrogen | T10282 | |
Vectofusin-1 GMP | Miltenyi Biotec | 170-076-165 | |
X-VIVOTM 15 Serum-free Hematopoietic Cell Medium | Lonza Bioscience | A1048501 |