Summary

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

Published: October 06, 2023
doi:

Summary

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

Abstract

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.

Introduction

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.

Protocol

This study was approved by the Ethics Committee of the University of Patras and the Institutional Review Board of the University General Hospital of Patras. Prior to obtaining biological specimens, informed consent was obtained from healthy individuals. The eligibility assessment of participants was conducted according to institutional procedures and in compliance with JACIE standards for leukapheresis. If this protocol is to be implemented in a clinical ACT setting, all procedures must adhere to Good Manufacturing Pract…

Representative Results

Assessment of transduction efficiency The transduced cells were evaluated for the expression of the gene of interest (GFP) using flow cytometry. Representative results are depicted in Figure 1. On Day 14, more than 95% of the cells were CD3+, indicating successful T cell activation and expansion. The transduction efficiency within the CD3+ population was measured at 58.7% (TD, Figure 1C) compared to the non…

Discussion

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…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Savvopoulos, N., Stampolitis, K., Alexandropoulos, G., Kefala, D., Lysandrou, M., Zacharioudaki, V., Tsolakos, N., Spyridonidis, A. A GMP-Compliant Procedure for the Generation of Gene-Modified T cells. J. Vis. Exp. (200), e65097, doi:10.3791/65097 (2023).

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