Adoptive therapies based on CAR/TCR T cells—that is, T cells with an engineered chimeric antigen receptor (CAR) or a T-cell receptor (TCR)—have shown promise for a range of cancers, offering hope to patients whose cancers have become refractory to conventional therapeutic options. However, as several scientists observed at the recent CAR-TCR Summit Europe in London, CAR/TCR T-cell therapies face challenges that can result in high costs and limited accessibility.

“Investment in advanced therapies is dropping, so drug developers are restricting their pipeline development to save costs,” noted Qian Liu, PhD, head of plasmid engineering and lentiviral vectors, WuXi Advanced Therapies. “This means many therapies are slower to reach regulatory approval and commercialization and are usually expensively priced, which restricts market and patient access.”

“With cell therapy, many of the cost and time issues are related to manufacturing complexity,” added Victor Vinci, PhD, global vice president, product development, Catalent Biologics. “There is variability in the initial quality of the patient’s T cells, as well as the reagents, growth media, and range of equipment and automation available for the different production stages, which means there is currently no one-size-fits-all solution for CAR T-cell manufacturing.”

Optimizing the process

Enhancing manufacturing efficiency is crucial for scaling up production, reducing costs, and ultimately making CAR/TCR T-cell therapies more accessible to patients. However, the manufacturing process for CAR/TCR T-cell therapies is complex, involving multiple steps, including steps for apheresis, T-cell selection, genetic modification, transduction, expansion, purification, and fill/finish.

Ali Mohamed, PhD, senior vice president, CMC, Immatics, discussed how evaluating different steps in the process had enhanced manufacturing of the company’s ACTengine IMA203 and IMA203CD8 TCR-engineered T cells (TCR-T cells) for targeting PRAME (PReferentially expressed Antigen in MElanoma). “ACTengine is our personalized cell therapy approach for patients with advanced solid tumors,” he said.

According to Mohamed, Immatics’ scientists have made several alterations to the standard method of producing TCR-T cells to enhance the manufacturing process. For example, they have moved to a serum-free transduction stage, where serum is not added during transduction. This, Mohamed says, has “significantly increased the numbers of T cells transfected without affecting cell viability, cell expansion, or the cell’s phenotype.”

Another process change that Immatics has made is to remove monocytes and adherent cells by resting T cells in plasticware, such as a CellSTACK, for a few hours. “Monocytes can make up as much as 50% of the T cells we collect during apheresis,” Mohamed explained. “They sometimes recognize viral vectors as foreign and destroy them, which can result in their rapid clearance and lower T-cell transduction rates. By removing them, we have seen our transduction rates increase significantly.”

The current manufacturing process implements enrichment of CD4 and CD8 T cells using specific antibodies, thereby replacing the adherent cells that have been depleted. “By selecting CD8 and CD4 cells, we can use a defined T-cell population at the start of the manufacturing process,” Mohamed explained. “This can increase the chances of manufacturing TCR-T cells in sufficient numbers to reach the required cell dose.

“In using these three process optimization steps, we can produce TCR-T cells at the recommended Phase II dose (RP2D, 1–10 × 109 total TCR-T cells) in just 14 days with a 7-day manufacturing process plus 7-day quality control release testing. Using our optimized process, we have increased our seeding density and use fewer vessels. All these features help us reduce costs, shorten the turnaround time, and provide the cell products to patients faster while maintaining a manufacturing success rate of over 95%.”

Catalent’s Vinci also emphasized that process optimization is key for de-risking and streamlining a manufacturing pathway. He added, “We have used a quality-by-design approach for process optimization and have developed our UpTempo CAR T-cell therapy platform for manufacturing autologous cell therapy.”

According to Vinci, the Catalent platform provides a modular, flexible CAR T-cell cGMP workflow that utilizes aseptically connected, closed systems—including the G-Rex, Xuri, and CliniMACS Prodigy—to automate, evaluate, and optimize the manufacturing process. “We produce T-cell therapies that typically have around 90% cell viability at harvest,” Vinci noted. “This ensures our manufacturing is efficient, which reduces costs.”

Improving viral gene delivery

To reduce some of the costs involved with manufacturing CAR T-cell therapies, WuXi Advanced Therapies is developing technologies such as the XOFLX packaging and producer cell lines. These cell lines are designed to reduce the cost of producing lentiviral vectors (LVVs), which are commonly used for delivering therapeutic genes in cell therapy because they can efficiently modify T cells in a permanent manner and have a reliable safety profile for this application.

“The industry standard for LVV manufacture is to use four plasmids—a transfer vector containing the gene of interest, two packaging plasmids, and an envelope plasmid,” Liu pointed out. “What we have done with our XOFLX system is to first develop a Packaging Cell Line, which has all the LVV packaging elements stably integrated into the cells’ genome and requires transfection of only one transfer plasmid for LVV production. Additionally, we developed XOFLX Producer Cell Lines, which have also integrated the LVV genomes containing the therapeutic genes and allow scalable transfection-free LVV production.”

WuXi Biologics’ XOFLX packaging and producer cell lines
WuXi Biologics’ XOFLX packaging and producer cell lines are designed to offer a simplified and high-yielding lentiviral vector (LVV) manufacturing system. The system is engineered for low homology between expression cassettes for an improved safety profile.

Liu presented data to show that at 10 L scale the XOFLX Packaging Cell Line produced comparable LVV titers when compared to WuXi Advanced Therapies’ conventional LVV production system. A research cell bank and a master cell bank have been created for the Packaging Cell Line. She also showed 1 L LVV production data from XOFLX Producer Cell Lines encoding enhanced green fluorescent protein or a therapeutic transgene. The data suggested that production could be easily scaled up from shake flasks due to the simplified, transfection-free process.

Liu concluded, “As our XOFLX system only uses one transfer plasmid or no plasmid at all for LVV production, this reduces the costs of plasmid use and the complexity of LVV manufacturing, which provides cost and quality benefits for drug developers and ultimately for patients.”

Taking the road less travelled—nonviral delivery

According to Ting-Wan Lin, PhD, director, business development, GenomeFrontier Therapeutics, the firm is focusing on making advanced, affordable cell therapies but is choosing the less well-trodden path of using nonviral cell engineering. “Despite advances in viral vector design, there are some challenges and/or disadvantages associated with virus-based vectors for gene therapy, such as their intrinsic safety concerns, costly vector manufacturing, and limited payload capacity,” she noted. “A nonviral approach for cell engineering can overcome these drawbacks.”

Lin added, however, that the nonviral approach poses other challenges. These include poor gene delivery rate, ineffective gene integration, and low cell expansion capacity caused by electroporation-based gene delivery.

To overcome the challenges currently encountered using either viral or nonviral cell engineering technologies, GenomeFrontier Therapeutics has developed Quantum Engine, a technology for facilitating development and manufacturing of high-quality, clinical-scale, and virus-free cell and gene therapy products. This system integrates four platforms: G-Tailor, Quantum pBac, Quantum Nufect, and iCellar, for candidate gene design, therapeutic gene integration, gene delivery, and cell expansion, respectively.

GenomeFrontier Therapeutics four platforms
GenomeFrontier Therapeutics has integrated four platforms—G-Tailor, Quantum pBac, Quantum Nufect, and iCellar—to improve gene design, therapeutic gene integration, gene delivery, and cell expansion capacity, respectively. Together, the platforms form the Quantum Engine, which is designed to produce virus-free cell and gene therapy products.

“Quantum pBac, the key platform of Quantum Engine, is our proprietary piggyBac-based transposon, which is potentially safer and much more effective for integrating larger sized gene compared to hyperactive piggyBac, the commercially available piggyBac vector,” Lin stated. “By finely tuning Quantum pBac along with the other three platforms, we have recently developed a robust Quantum engine, named Quantum CART (qCART), for development and manufacturing of multiplex CAR T cells.”

Lin presented data demonstrating that qCART produced CAR T cells that yielded higher percentages of CAR+ stem cell–like memory T cells with both CD4 and CD8 plus low expression of senescence/exhaustion markers and good expansion capacity. Furthermore, these CAR T cells also demonstrated robust antitumor efficacy in both lymphoma and gastric solid tumor mice models.

“Our qCART system not only enables us to produce high-quality and clinical scale CAR T cells with great product consistency in a time- and cost-effective manner, but also is capable of rejuvenating aged and exhausted T-cells in refractory patients,” Lin noted. “Quantum Engine is a powerful technology, enabling us to build cell and gene therapy pipelines by using piggyback and thus riding on the shoulders of giants. Our lead candidate, GF-CART01, a CD20/CD19-targeting CAR T-cell therapy to treat B-cell malignancies, has shown promising results in preclinical studies of mice, and we are looking for partners to work with.”

Enhancing manufacturing efficiency is critical for scaling up production, driving down costs, and increasing the accessibility of CAR/TCR T-cell therapies. Speakers at the CAR-TCR Summit Europe agreed that by embracing closed automated systems and adopting standardization and optimization strategies, manufacturers could overcome existing challenges and realize the transformative potential of CAR T-cell therapies.

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