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Abstract

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Medicine

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Published: August 18th, 2023

DOI:

10.3791/65488

1Cell and Gene Therapy Laboratory, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, 2Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, 3Perelman School of Medicine at the University of Pennsylvania, 4Division of Transfusion Medicine, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus minimizes human involvement in the process, which saves time and reduces variability and errors.

This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice (cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate the feasibility and discuss the advantages and disadvantages of using a semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

Adoptive transfer of T cells engineered to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating patients with refractory B-cell malignancies1,2,3,4,5. However, the traditional manufacturing methods for CAR-T cells are labor-intensive, time-consuming, and require highly trained technicians to carry out highly specialized steps. For example, the traditional manufacturing process of an autologous CAR-T cell product involves density gradient centrifugation, elutriation or mag....

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All research was performed in compliance with institutional guidelines with approval by the hospital's Institutional Review Board (IRB), and all subjects have provided informed consent for publication of the data collected within the context of the trial.
NOTE: The first section of the Protocol provides a high-level overview of the CAR-T manufacturing process. The remaining sections provide the step-by-step instructions. The protocol describes the workflow using TCT software version 1.4, which is the .......

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Results from the initial three CAR-T manufacturing runs of the NCT05480449 trial are presented below in Table 3. The starting material, vector, culture cytokines, and AB serum concentrations were kept consistent for each run. Products were harvested on day 7 or 8. The average daily cell growth was 46% (increase in total cell count), indicating that the TCT process was effective in promoting cell expansion. These results suggest that the processor can produce consistent and reproducible CAR-T cell product.......

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CAR-T cell therapy has emerged as a promising treatment approach for B-cell and other malignancies. However, traditional CAR-T cell manufacturing methods have several limitations, such as high cost, labor-intensive production, and open steps that increase the risk of contamination. Recently, several semi-automated platforms, including the Miltenyi CliniMACS Prodigy (the "processor"), have emerged to address these limitations. The T cell transduction (TCT) process, integrated into the processor described in this m.......

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The authors would like to acknowledge the contributions of several individuals and organizations to this work. The Cell and Gene Therapy Laboratory and the Penn Translational and Correlative Studies Laboratory provided valuable assistance with process development and preparation for IND submissions. Melissa Varghese and Amanda DiNofia contributed to the process development and preparation for IND submissions that underly this manuscript. This work was supported by an Acceleration Grant of the Cell and Gene Therapy Collaborative of the Children's Hospital of Philadelphia. The authors would also like to thank Miltenyi Biotec for their technical and research sup....

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Name Company Catalog Number Comments
12 x 75 borosilicate tubes Charles River TL1000
20 mL Reagent Bag Miltenyi Biotec 170-076-631
50 mL Conical Tube Fisher 05-539-10
150 mL Transfer Set Fenwal 4R2001
2,000 mL Transfer Set Fenwal 4R2041
7AAD Fisher Scientific BDB559925
Alcohol Prep Tyco/Healthcare
Bag Access Medline 2300E-0500
CD19 APC-Vio770 REAfinity Miltenyi Biotec 130-113-643
CD19 CAR Detection Reagent Biotin Miltenyi Biotec 130-129-550
CD19 PE BD 555413
CD3 APC BD 340440
CD4 VioBright FITC REAfinity Miltenyi Biotec 130-113-229
CD45 VioBlue REAfinity Miltenyi Biotec 130-110-637
CD8 APC-Vio770 REAfinity Miltenyi Biotec 130-110-681
Cellometer Reference Beads 10um Nexcelom B10-02-020
Cellometer Reference Beads 15um Nexcelom B15-02-010
Cellometer Reference Beads 5um Nexcelom B05-02-050
Cellometer Slides Nexcelom CHT4-SD100-002
CliniMACS CD4 GMP MicroBeads Miltenyi Biotec 276-01 The CD4 reagent
CliniMACS CD8 GMP MicroBeads Miltenyi Biotec 275-01 The CD8 reagent
CliniMACS PBS/EDTA Buffer Miltenyi Biotec 130-021-201 The buffer
DMSO Origen CP-10
Freezing Bag 50 mL Miltenyi Biotec 200-074-400
Freezing Vial, 1.8 mL Nunc 12565171N
Freezing Vial, 4.5 mL Nunc 12565161N
Human AB serum Valley Biomedical Sterile filtered, heat inactivated
Human Serum Albumin 25% Grifols 68516-5216-1
Human Serum Albumin 5% Grifols 68516-5214-1
MACS GMP Recombinant Human IL-2 Miltenyi Biotec 170-076-148 The cytokines
MACS GMP T Cell TransAct Miltenyi Biotec 200-076-202 The activation reagent
MycoSeq Mycoplasma Detection Kit Life Technologies 4460623
Needles, Hypodermic 14G Medline SWD200573
Needles, SlideSafe 18G BD B-D305918
Pipet tips, 2-200 μL, individually wrapped Eppendorf 022492209
Pipet tips, 50-1000 μL, individually wrapped Eppendorf 022492225
Pipets 10 mL Fisher 13-678-27F
Pipets 25 mL Fisher 13-675-30
Pipets 5 mL Fisher 13-678-27E
Plasmalyte-A Baxter 2B2544X The electrolyte solution
Prodigy TS520 Tubing Set Miltenyi Biotec 170-076- 600 The tubing set
Sterile Field Medline NON21001
Streptavidin PE-Vio770 Miltenyi Biotec 130-106-793
Syringe 1 mL BD 309628
Syringe 10 mL BD 302995
Syringe 3 mL BD 309657
Syringe 30 mL BD 302832
Syringe 50 mL BD 309653
TexMACS GMP Medium Miltenyi Biotec 170-076-306 The medium
Triple Sampling Adapter Miltenyi Biotec 170-076-609
Viral Vector CHOP Clinical Vector Core huCART19
Equipment
Biological Safety Cabinet The Baker Co
Cellometer Auto 2000 Nexcelom
CliniMACS Prodigy Miltenyi Biotec 200-075-301 The processor
Controlled Rate Freezer Planer/Kryosave
Endosafe nexgen-PTS150K Charles River
Mettler Balance Mettler
Refrigerated Centrifuge Thermo Fisher
Refrigerated Centrifuge Fisher Sci
SCD Sterile Tubing Welder Terumo
Sebra Tube Sealer Sebra
Varitherm Barkey The dry thaw device
XN-330 Hematology Analyzer Sysmex

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