Rapid manufacturing process allows CAR T cells to be produced in less than a day
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One of the major complaints of clinicians who treat patients with chimeric antigen receptor T-cell therapies is the time required to produce the highly individualized treatment.
Many patients with advanced disease develop unsurmountable progression or die before CAR-T infusion, and cell therapy investigators have conducted extensive research into how the production process can be accelerated to overcome this challenge.
In a study published in Nature Biomedical Engineering, researchers described a new CAR-T manufacturing process that eliminates the need for viral transduction with activated T cells and instead delivers a genetic payload to insert chimeric antigen receptors into nonactivated T cells.
The result is a more potent therapy manufactured from the patient’s fresh blood cells in less than a day, according to Michael C. Milone, MD, PhD, associate professor of pathology and laboratory medicine and associate director of the toxicology laboratory at University of Pennsylvania Perelman School of Medicine.
“We used this more efficient transduction process and demonstrated its ability in a well-established preclinical model of acute lymphoblastic leukemia via treatment with a CD19-directed CAR-T,” Milone told Healio. “CAR T cells made in less than 24 hours using this process can be infused into a preclinical animal model and generate a response that is actually superior with lower doses of cells required to achieve the same anti-leukemic effect.”
Milone spoke with Healio about the manufacturing process, what makes it unique and the potential it may offer as a treatment approach for people with cancer.
Healio: Can you explain the rationale for your study and summarize your findings?
Milone: A major goal of this investigation was to determine whether we really need to activate T cells prior to infusion to produce a functional CAR-T product. To accomplish this, we had to figure out a way to make the gene transfer process more efficient.
Lentiviral vectors derived from HIV are used to produce FDA-approved CAR T-cell therapies. These therapies traditionally comprise activated T cells derived from a process that can take a few days or even weeks. Lentiviral vectors require T-cell activation for efficient gene transfer, but these vectors are capable of transferring genes into cells that are nondividing.
Our preclinical work focused on exploring mechanisms for how HIV lentiviral vectors get into cells and their ability to carry out a critical step in the viral life cycle known as reverse transcription, which is necessary for integration into the genome. The reverse transcription process is well known to be inefficient and slow in quiescent, nonactivated T cells, and we capitalized on new science around this process that has developed over the past couple of decades.
Our research also looked at ways to enhance the internalization and perhaps increase the receptor expression at the surface, which we hypothesized was being reduced by serum. A simple manipulation of removing serum from the transduction system for a couple hours before viral transduction turned out to be a very important way to enhance the gene transfer process.
An investigator at Penn previously demonstrated that one of the limiting factors for the speed of the reverse transcription process in quiescent T cells is nucleoside concentrations. By simply increasing the concentration of nucleosides in the medium, we increased the efficiency of the process. Additionally, we needed to address a slow diffusion process because the viral particles must find the T cells in the medium. Therefore, we changed the configuration of the transduction system to enhance the probability of these cells and viral particles finding each other.
These three simple adjustments provided us with more than a 10-fold increase in the efficiency of transduction.
Healio: Does this process reduce the cost of manufacturing?
Milone: Every CAR-T process is different, but most still require lentiviral vectors. There is much work to be done to improve transduction efficiency. One of my lab’s research interests is how to remove things from HIV to improve the safety and efficiency of producing these products. For example, we removed a lot of accessory genes that are not necessary for infection of activated T cells, but some of those genes might be important and useful for enhancing efficiency of CAR-T products.
The field needs more virus to produce CAR-T, which is a bottleneck in the manufacturing process. If we scaled up our new manufacturing process to large numbers, it likely would require the same amount of virus and, therefore, it doesn't really save costs. However, we would save in terms of time in culture and volume of media required for CAR-T production.
I think cost savings will come in time, regardless of the process. If we get to a point where CAR T cells do not have to be cultured at large, centralized manufacturing facilities and require minimal manipulation, it is possible the process could be done onsite by the CAR-T provider. One of my goals is to develop the ability to substantially outscale the CAR-T process. Culturing of these cells changes them in ways that we are still trying to understand. This was a proof-of-concept study and this novel process is not ready to jump into manufacturing. We have to adapt this principle of minimal manipulation to individual manufacturing processes.
Healio: Would this process work for any CAR-T or for treatment of diseases beyond B-cell ALL?
Milone: I believe it could work for other targets, and it certainly is relevant to T-cell therapies. Our focus was on T-cell engineering, so I don't think it is restricted to CARs. It could be applied to T-cell receptor gene transfer or for any gene being transferred into a T cell. It potentially could be used for other therapeutic cell systems that require a culture process, such as hematopoietic stem cells.
Healio: Are there plans for further study or to translate what you learned into clinical research?
Milone: There still is a lot of work to be done to understand this new process because most of the work in this field has been with activated T cells. Further research should establish whether we are using the appropriate costimulatory domains and CARs, and questions remain about CAR design for a quiescent T cell.
Further investigation on vector engineering is needed, as well. We have optimized currently used viral vectors for activated T cells, and now we are considering exploring whether some of the things we removed from the vector might be important for quiescent T cells and should be added back to the manufacturing process.
We are re-exploring how to make the overall process more efficient, but we are still considering ways we can move this shortened manufacturing method into a clinical study. No decision has been made yet about how we can translate this into the clinic, but we are looking for the appropriate opportunity to do so.
Healio: What is the potential impact if this accelerated manufacturing process can be used to develop therapies for human use?
Milone: This would shorten the time needed to get this therapy into patients and could positively affect the potency of these products. Perhaps the largest challenge we face is providing these therapies to patients as quickly and as efficiently as possible. Removing the complex manufacturing and expansion that is necessary to produce CAR T cells makes this process far simpler. The simpler it is to produce, the more likely it will be provided in more places. This could move the CAR-T process outside medical centers that provide highly complex therapies, potentially allowing it to be done anywhere in the world with a clean space to work with the materials.
The goal is to get as close to direct gene transfer with minimal manipulation so that CAR-T manufacturing can be done at any facility, not just academic medical centers or centralized commercial labs. Our research could be impactful because it helps dramatically simplify the CAR-T manufacturing process.
Reference:
Ghassemi S, et al. Nat Biomed Eng. 2022;doi:10.1038/s41551-021-00842-6.
For more information:
Michael C. Milone, MD, PhD, can be reached at Perelman School of Medicine at University of Pennsylvania, 3400 Spruce St., 7103 Founders Pavilion, Philadelphia, PA 19104; email: milone@pennmedicine.upenn.edu.
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