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

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 malignancies^1,2,3,4,5. 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 production^6,7,8.

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-complexes^7,9. 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 Practice (GMP) guidelines and be performed in GMP-compliant clean rooms. Special safety precautions are necessary when working with viral vectors, and these procedures should be conducted within a dedicated biosafety cabinet. In this study, the described procedures were conducted within a validated H35 HEPA Hypoxystation (see Table of Materials) to ensure a controlled and monitored environment. Decontamination of all hazardous liquids should be carried out using 10% bleach solution.

1. PBMC isolation and T cell activation (Day 0)

1. Collect cells from the donor apheresis bag (leukopaks, see Table of Materials) into 50 mL tubes, depending on the initial volume.
2. Perform two washes of the cell bag by infusing 15 mL of complete hematopoietic media (+5% HSA, commercial serum-free hematopoietic media, see Table of Materials).
3. Conduct polysaccharide-based density gradient centrifugation at a 1:2 (reagent:sample) dilution. Centrifuge at 800 x g for 30 min at room temperature with breaks off (A/D, 9/0).
4. Using a micropipette, collect PBMCs into a clean 50 mL tube.
5. Wash the cells twice with 1x PBS, filling up to 50 mL for each wash (400 x g, 10 min, room temperature). Resuspend the cells in 5 mL of complete media.
6. Count the cells using trypan blue using either a standard hemocytometer or an automated cell counter (see Table of Materials).
7. Dilute the cells with complete media to achieve a concentration of 2 x 10⁶ cells/mL.
8. Activate 20 x 10⁶ PBMCs by adding 10 µL of T cell stimulation reagent (see Table of Materials) per 2 x 10⁶ starting cells.
   NOTE: This protocol is designed for the activation of 20 x 10⁶ PBMCs but can be adjusted for larger cell numbers as needed.
9. Add rhIL-2 (20 U/mL) (see Table of Materials), mix gently, and transfer the mixture into an appropriate vessel (e.g., 6-well plate).
10. Incubate the cells at 37 °C, 5% CO₂ for 72 h.

2. Lentiviral T cell transduction (Day 3)

1. Transfer the cell culture into a suitable vessel, such as a 50 mL conical tube. Mix well and centrifuge at 300 x g for 10 min at room temperature (RT) to remove the activation reagent. Carefully discard the supernatant completely and resuspend the pellet in 1 mL of complete medium.
   NOTE: At this stage, it is possible to thaw the virus on ice.
2. Count the cells using trypan blue.
3. Resuspend the cells in complete medium to achieve a concentration that allows for plating 0.5 x 10⁶ cells in a total volume of 400 µL per well in a 24-well plate. Add rhIL-7 (155 U/mL) and rhIL-15 (290 U/mL) (see Table of Materials).
   NOTE: Ensure to account for the volume of viruses to be added. Refer to step 2.4.
4. In a separate tube, add Vectofusin-1 (see Table of Materials) to Opti-MEM medium at a concentration of 10 µg/mL, with a volume equal to the viral volume to be used.
   NOTE: Consider the total cell culture volume when calculating the concentration of Vectofusin-1. See step 2.3. Alternatively, Vectofusin-1 GMP can be used as a GMP-grade reagent.
5. Combine the concentrated virus at a Multiplicity of Infection (MOI) of 40 with the Vectofusin-1/Opti-MEM mixture (1:1 ratio) and mix thoroughly. Add this mixture to the cell culture and adjust the total volume to 400 µL per well using complete medium, if necessary.
6. Place the lid on the plate, seal it with paraffin film, and centrifuge at 1000 x g for 2 h at 32 °C. Subsequently, incubate the plate at 37 °C, 5% CO₂ overnight.

3. Virus removal and T cell expansion (Day 4)

1. Collect the cells and pool them into a suitable vessel, such as a 50 mL tube.
2. Centrifuge the cells at 400 x g at room temperature (RT) for 5 min. Carefully remove the supernatant completely and resuspend the pellet in 1 mL of complete media.
3. Count the cells using trypan blue. Resuspend the cells in complete media at a concentration of 1 x 10⁶ cells/mL. Add rhIL-7 (155 U/mL) and rhIL-15 (290 U/mL) (see Table of Materials).
4. Transfer the cells to an appropriate vessel, such as a T25 flask, at a concentration of 0.3-0.5 x 10⁶ cells/cm² and incubate them at 37 °C, 5% CO₂, for 2 days.
   ​NOTE: If a larger cell population is required, cells can be subcultured (1:2) in the presence of rhIL7 and rhIL-15 every 2 days, following the same incubation conditions mentioned above, until the desired cell numbers are achieved.

4. Cryopreservation (Day 14)

NOTE: Ice will be required to transfer cryovials to the storage location during this step. The timing of cryopreservation depends on the desired cell numbers. For this protocol, cryopreservation is performed on Day 14 after achieving a 25-fold expansion.

1. Once the desired cell number is reached, harvest the cells and transfer them to 50 mL tubes (adjust the number of tubes based on the total volume).
2. Centrifuge the tubes at 400 x g at room temperature (RT) for 5 min. Carefully remove the supernatant completely and resuspend the pellet in 10 mL of media. If necessary, pool the cells from multiple tubes.
3. Count the cells using trypan blue. Keep an aliquot of 1 x 10⁶ cells for each quality control (QC) testing (refer to Section 5) to assess Vector Copy Number and Transduction Efficiency.
4. Centrifuge the tubes at 400 x g for 10 min at RT. At this point, label the cryovials accordingly.
5. Completely remove the supernatant and resuspend the pellet in a cryopreservation solution consisting of 50% HSA and 40% HBSS at a concentration of 1 x 10⁷ cells per mL per cryovial. Transfer the cell suspension into the appropriate number of cryovials and add 10% dimethyl sulfoxide (DMSO) to each cryovial.
6. Place the cryovials on ice and promptly transfer them to a controlled-rate freezer for cryopreservation. After cryopreservation, transfer the cryovials to a monitored liquid nitrogen tank for long-term storage.

5. Quality control (Day 14)

NOTE: For release testing, the product underwent several tests, including sterility tests for microbial growth, and endotoxin level assessment. These tests were conducted in certified laboratories at the University General Hospital of Patras and the University of Patras. Mycoplasma presence was determined in-house using a biochemical detection assay and measured on a luminometer following the manufacturer's instructions (see Table of Materials). Cell phenotype (Figure 1) as well as cell number and viability were assessed in-house by flow cytometry (see section 5.1) and an automated cell counter (see Table of Materials), respectively. Release criteria were established based on published data and recommendations^10,11for GMP-produced cell products (Table 1).

1. Perform transduction efficiency-flow cytometry staining (Day 14)
   NOTE: An aliquot of 1 x 10⁶ cells will be stained for viability (7-AAD) and CD3 marker² to evaluate the transduction efficiency (see Table of Materials). If the gene of interest, such as in the case of CAR T cells, needs to be detected, the specific antibody should be in a different color from that of the CD3 marker and 7-AAD to avoid compensation. Appropriate control samples should be taken into account.
   1. Place the sample in a FACS tube and add 500 µL of FACS buffer.
   2. Centrifuge the tube at 400 x g at RT for 5 min.
   3. Completely discard the supernatant with a pipette and resuspend the pellet in a 1:5 diluted solution of CD3:FACS buffer (see Table of Materials).
   4. Vortex the sample and incubate for 30 min on ice in the dark.
   5. Add 500 µL of FACS buffer. Centrifuge at 400 x g for 5 min (RT).
   6. Completely discard the supernatant and resuspend the pellet in a 1:20 diluted solution of 7-AAD:FACS buffer (see Table of Materials).
   7. Vortex the sample and incubate for 10 min on ice in the dark.
   8. Add 200 µL of FACS buffer and analyze the sample using a flow cytometer (see Table of Materials).

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...

Materials

Name	Company	Catalog Number	Comments
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