Name: a gmp-compliant procedure for the generation of gene-modified t cells
Uploaded: 2023-10-06T14:40:00
Description: Watch this Scientific Journal Video about A GMP-Compliant Procedure for the Generation of Gene-Modified T cells at JoVE.com

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 &#34;Dimitris Lois&#34; of the Institute of Cell Therapy.

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...

<ol>
	<li>Maude, S. L., Teachey, D. T., Porter, D. L., Grupp, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD19-targeted+chimeric+antigen+receptor+T-cell+therapy+for+acute+lymphoblastic+leukemia.">CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia.</a> Blood. 125 (26), 4017-4023 (2015).</li><li>Brentjens, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD19-targeted+T+cells+rapidly+induce+molecular+remissions+in+adults+with+chemotherapy-refractory+acute+lymphoblastic+leukemia.">CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.</a> Science Translational Medicine. 5 (177), 177ra38 (2013).</li><li>Maude, S. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1+spinoculation+enhances+infection+through+virus+binding.">Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding.</a> Journal of Virology. 74 (21), 10074-10080 (2000).</li><li>Cieri, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+and+IL-15+instruct+the+generation+of+human+memory+stem+T+cells+from+naive+precursors.">IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.</a> Blood. 121 (4), 573-584 (2013).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+(CAR-T)+cells+expanded+with+IL-7/IL-15+mediate+superior+antitumor+effects.">Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects.</a> Protein Cell. 10 (10), 764-769 (2019).</li><li>Ghassemi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+manufacturing+of+non-activated+potent+CAR+T+cells.">Rapid manufacturing of non-activated potent CAR T cells.</a> Nature Biomedical Engineering. 6 (2), 118-128 (2022).</li><li>Wang, R. -. 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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 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well TC-treated plate</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL FACS tube&#160;</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tubes</td>
			<td>Greiner biomedica</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well TC-treated plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>7-AAD</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS CANTO II</td>
			<td>BD Biosciences</td>
			<td>V96300084</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Applichem</td>
			<td>A1391</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter&#160;</td>
			<td>Corning Cell Cytosmart</td>
			<td>J21E0081</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Greiner biomedica</td>
			<td>122263</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (D-PBS)</td>
			<td>Life Technologies</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Buffer (1x D-PBS, 0.5% BSA, 2 mM EDTA)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software v10.6.2&#160;</td>
			<td>TreeStar Inc</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco CTS Opti-MEM I Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A4124801</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP rhIL-15</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-114</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP rhIL-2</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-146</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP rhIL-7</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Life Technologies</td>
			<td>14175</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPA Whitley H35 Hypoxystation</td>
			<td>Don Whitley Scientific</td>
			<td>HA0315172H</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin (HSA)</td>
			<td>Baxter</td>
			<td>B05AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Junior LB 9509 Portable Luminometer</td>
			<td>Berthold Technologies</td>
			<td>6506</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Provirus Quantitation Kit</td>
			<td>Takara Bio</td>
			<td>631239</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stemcell Technologies</td>
			<td>7811</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS GMP T cell TransAct</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-156</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human&#160; CD3 (Clone: UCHT-1)</td>
			<td>BD Biosciences</td>
			<td>557706</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoAlert Plus Detection kit</td>
			<td>Lonza Bioscience</td>
			<td>LT07-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm ultra-expandable closing film tape 5 cm x 75 m</td>
			<td>D.Dutscher</td>
			<td>90261</td>
			<td></td>
		</tr>
		<tr>
			<td>PeriStem PS-650-DB (apheresis bag)</td>
			<td>Biomed Device</td>
			<td>SC00816</td>
			<td></td>
		</tr>
		<tr>
			<td>Planer Kryo10 Series III</td>
			<td>Planer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Greiner biomedica</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4% solution</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectofusin-1 GMP</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-165</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVOTM 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Lonza Bioscience</td>
			<td>A1048501</td>
			<td></td>
		</tr>
	</tbody>
</table>

a gmp-compliant procedure for the generation of gene-modified t cells

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.

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...

Watch this Scientific Journal Video about A GMP-Compliant Procedure for the Generation of Gene-Modified T cells at JoVE.com

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

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 ...

This protocol describes a simplified and GNP compliant process of transducing human primary T-cells with gene of interest. This particular technique has been designed to be cost-effective and GNP compliant without the use of expensive closed system manufacturing platforms currently available in many academic centers. This method could potentially be applied to the generation of gene-modified cell therapies, including but not limited to CAR T-cells, which have been a paradigm shift to tackle hematologic malignancies.To isolate PBMC from the cells collected after leukapheresis of donor blood, perform a polysaccharide-based density gradient centrifugation of the sample, maintaining a reagent to sample ratio of 1:2, by centrifuging the mixture at 800 G for 30 minutes at room temperature with the brakes off. Then using a micropipette, collect the PBMCs into a clean 50 milliliter tube. Wash the cells twice with PBS by centrifugation.Before resuspending the washed cells in complete hematopoietic media. Activate about 20 million of the obtained cells by adding 10 microliters of T-cell stimulation reagent for every 2 million cells. Next, after adding recombinant human interleukin-2 at 20 units per milliliter, mix the solution gently and transfer it into a six-well plate.Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 72 hours before performing viral transduction. On day three, transfer the cell culture to a 50 milliliter conical tube and mix it thoroughly. Centrifuge the tube at 300G for 10 minutes at room temperature to remove the activation reagent.Discard the supernatant completely and resuspend the pellet in one milliliter of complete medium before counting the cells. Adjust the cell suspension volume using complete medium to achieve a concentration that allows for plating 0.5 million cells per well in a 24 well plate. Add recombinant human interleukin-7 and recombinant human interleukin-15 to the cells at appropriate concentrations.In a separate tube, add the desired amount of vectofusin-1 to Opti-MEM medium, considering the final viral volume to be used. Combine this mixture with the concentrated virus at a one-to-one ratio, ensuring thorough mixing. Next, add the resultant mixture containing the virus to the cells.Adjust the total volume of each well to 400 microliters using complete medium if necessary. After covering the plate with a lid, seal it using a paraffin film before centrifuging it at 1000G for two hours at 32 degrees Celsius. Incubate the plate overnight at 37 degrees Celsius and 5%carbon dioxide.On the next day, collect and pull the cells from each well into a 50 milliliter tube. Next, centrifuge the cells at 400G and room temperature for five minutes. After carefully removing the supernatant, fully resuspend the pellet in one milliliter of complete medium before counting the cells.Then adjust the cell concentration with the media to achieve 1 million cells per milliliter and add human recombinant interleukin-7 and human recombinant interleukin-15 at 155 and 290 units per milliliter respectively before culturing the cells. Once the desired cell number is reached, harvest and transfer the cells into 50 milliliter tubes and centrifuge them. After removing the supernatant, resuspend the pellet in 10 milliliters of media to count the cells.After centrifuging again, discard the supernatant completely and resuspend the pellet in a cryopreservation solution consisting of 50%HSA, and 40%HBSS, at a concentration of 10 million cells per milliliter per cryo vial. Transfer 0.9 milliliters of cell suspension each to the required numbers of cryo vials, and add 10%dimethyl sulfoxide to each cryo vial. Place the cryo vials on ice and quickly shift them into a controlled-rate freezer for cryo-preservation.Then transfer the cryopreserved samples to a monitored liquid nitrogen tank for long-term storage. To evaluate transduction efficiency, add 500 microliters of FACS buffer to the sample in a fluorescence activated cell sorting or FACS tube. After centrifuging the sample at 400G and room temperature for five minutes, discard the supernatant completely using a pipette.Resuspend the pellet in a one to five diluted solution of CD3 in FACS buffer. Vortex the sample and incubate for 30 minutes on ice in the dark before centrifuging as demonstrated previously. Next, after discarding the supernatant entirely, resuspend the pellet in a one to 20 diluted solution of 7-AAD in FACS buffer.Vortex this mixture before incubating it for 10 minutes on ice in a dark setting. Finally, add 200 microliters of FACS buffer to it And proceed to analyze the sample using a flow cytometer. Viability analysis of the transduced cells revealed that on day 14, more than 95%of the cells were CD3 positive and alive after excluding 7-AAD positive cells, indicating successful T-cell activation and expansion.The transduction efficiency within the CD3 positive population was measured to be 58.7%compared to the non-transduced cells, which exhibited a transduction efficiency of 0.51%When the transduced cells were expanded from day four until day 14, with sub-culturing every two days, the cells underwent a 25 fold expansion. The product underwent various quality control tests and met all the set criteria, indicating its suitability for further use. The whole procedure should be performed with strictly aseptic techniques.And the most tricky step is the transduction process where one needs to take into account all the reagents to be added in order to maintain a specific total volume.

Research

JoVE Journal

Bioengineering

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