The Challenge of Manufacturing Adoptive Cell Therapies

Marwan Alsarraj - Biopharma Segment Manager, Bio-Rad

Recent data revealed that some of the first people to receive Chimeric Antigen Receptor (CAR) T-cell therapy in 2010 still carry the cells in their blood. Scientists call this news a term not often used in biomedical science: a cure.¹ This landmark finding is the culmination of decades of breakthrough discoveries in genetic engineering, virology, and cancer biology. But despite their clinical success, challenges remain in developing CAR T-cells and other Adoptive Cell Therapies (ACTs) for a broader population. Namely, biopharmaceutical companies are actively investigating how to manufacture these treatments reliably and affordably.

CAR T-cells are the only form of ACT on the market today. Yet manufacturers are investigating the utility of several other immune cells as cancer therapies, including T-cell Receptor (TCR) T-cells, Tumor-Infiltrating Lymphocytes (TILs), and Natural Killer (NK) cells. Here is a summary of the challenges ahead in developing effective and affordable ACTs for patients in need.

CAR T-cell Therapy

CAR T-cell Therapy CAR T-cells changed cancer treatment forever when they were introduced in 2017. But they are expensive. For example, one treatment course of Yescarta costs $373,000, while Kymriah costs $475,000.² Besides the economic and ethical challenge of pricing this “cure,” these treatments cost so much because the manufacturing process is slow and complicated.

All of the CAR T-cells used in clinics today are autologous, meaning they are manufactured on demand for each patient using their T-cells. First, a patient has their blood drawn; then, the blood is delivered to a laboratory where scientists extract the T-cells. The cells are then genetically engineered using a viral vector or transposon system to express the CAR gene. Finally, the cells are expanded and returned to the patient for treatment.

This costly, manual process is slow: it takes several weeks to deliver the therapy to the clinic. This delay can affect treatment outcomes for many patients, including those with rapidly progressing cancers such as acute leukemia. In these patients, the disease will progress before CAR T-cells have been prepared for their use. Furthermore, in patients with T-cell dysfunction, such as those who have undergone immunosuppression therapy, autologous CAR T-cells might not even be effective.

Scientists are looking to overcome these limitations by developing allogeneic or “off-the-shelf” CAR T-cells derived from healthy donors. Potentially, this approach could enable manufacturers to standardize CAR T-cell development and streamline the process, thereby reducing costs. A manufacturer could prepare these cells before a patient receives a diagnosis, reducing the time to treatment.

Despite their potential, allogeneic CAR T-cells still present several challenges.³ First, they are still far from widespread because scientists have not developed a reliable method to induce substantial CAR T-cell expansion once they are administered to a patient. Furthermore, since they are derived from donor T-cells, they might cause graft-vs-host disease and fail to persist in the blood long enough to be effective. Finally, in studies of CAR T-cell expansion and persistence in patients’ blood, the numbers vary widely from patient to patient, suggesting there is still work to do before these therapies become available for widespread use.

These challenges represent just a few of those inherent in working with living cells. As evidenced by the unpredictability of CAR T-cell behavior, the manufacturer cannot control all aspects of CAR T-cell development, hence the need for rigorous quality control. For example, a manufacturer cannot control how many copies of the CAR gene the cell integrates into the genome during the transduction step. An unsuccessful transduction step will lead to an ineffective T-cell, yet too many copies of the CAR gene can lead to toxicity. Furthermore, the CAR T-cell batch may harbor replication-competent viruses that could infect patients and make them even more ill—all these factors cause the safety and effectiveness of CAR T-cells to vary. Quality control tools that can detect and quantify CAR transgenes and contaminants such as replication-competent viruses can help solve some of these challenges.

Fortunately, scientists have access to the tools they need to ensure the safety and efficacy of CAR T-cells. For example, Droplet Digital PCR quantifies nucleic acids directly, a useful feature during several stages of CAR T quality control. For instance, it can quantify the CAR transgene the ensure the cells contain an acceptable copy number. It can detect replication-competent viruses and other biological contaminants to help manufacturers screen out potentially harmful batches. Finally, it can quantify CAR T-cell persistence in patients’ blood over time to predict treatment response and guide treatment decisions.

T-Cell Receptor (TCR) T-Cells

Scientists are developing another T-cell therapy to treat cancer called T-cell receptor (TCR) T-cell therapy. Like the CAR T-cell development process, it takes genetic engineering to create TCR T-cells. But compared to the CARs, TCRs offer greater flexibility and personalization.

TCR T-cells’ flexibility comes from their simplicity. TCRs target major histocompatibility complexes (MHCs), a protein complex that tags a cell for destruction by the innate immune system. TCR T-cells promote this natural response. Furthermore, unlike CARs, which are synthetic, TCRs are naturally occurring. Therefore, they involve, at most, minimal modification to make them suitable for therapy. Also, CAR T-cells can only target tumor cells that express specific antigens on their surfaces, but TCR T-cells can target T-cells with any antigen—surface protein or intracellular.⁴ Since MHCs express more tumor-specific genetic sequences than tumor-specific proteins on the cell surface, this might make TCR T-cells effective against more cancer types. For example, since TCRs can access intracellular targets, they could be effective against solid tumors.

TCR T-cells and CAR T-cells present similar manufacturing challenges since they are produced using similar, personalized methods. As developers learn to streamline manufacturing for one type of engineered T-cell, it will likely lead to improvements in the other.

Tumor-Infiltrating Lymphocytes (TILs)

A T-cell or B-cell that successfully enters a tumor is considered a TIL. Since they can infiltrate tumors (unlike CAR T-cells), TILs offer greater flexibility than CAR T-cells in treating different types of cancer. And while their function is relatively intuitive, their development for clinical use is not.

By definition, TILs are naturally effective at recognizing tumor antigens, and manufacturers do not need to work as hard to transform them into a potent therapeutic. Instead, a manufacturer simply needs to extract the lymphocytes from a tumor sample, expand the cells, and turn them to the clinic for administration into the patient. This process amplifies innate immune system activity, providing the immune system with reinforcements to fight against a tumor.

Scientists have been working with TILs for decades, yet most attempts to bring these cells to the clinic have failed. While scientists have proven that TILs effectively treat cancer, they are hard to manufacture on a scale needed to meet demand. In 2021, the FDA granted an Orphan Drug Designation to a novel TIL therapy or advanced-stage melanoma based on data from just a handful of patients.⁵ The FDA will need to see clinical data from a much larger cohort of patients and proof that these cells can be produced on a mass scale for the therapy to achieve full approval.

TILs are hard to manufacture for several reasons. First, the cells must be collected directly from solid tumors, which means a patient must undergo an invasive tissue biopsy. Also, the extraction process is convoluted, requiring six to eight weeks of work – more than three times the length of the CAR T-cell development process.

TILs also received a breakthrough designation for the treatment of advanced cervical cancer, but again, the FDA based their decision on phase 2 clinical trial data from just a few patients.⁶ These cells’ regulatory journey so far demonstrates that a primary barrier to their widespread approval and adoption is the ability to manufacture the therapies on a larger scale and test them in more patients.

Natural Killer (NK) Cells

While many biopharmaceutical manufacturers are focused on developing T-cell therapies, scientists are examining another cell type as a potential candidate for therapeutic use to make the development process faster and cheaper. Natural killer cells, another lymphocyte, might provide a shortcut to cancer treatment because these cells do not need to identify a specific antigen to hunt down tumor cells. These cells detect abnormal surface protein expression and kill the T-cell to prevent further damage. Importantly, this mechanism of action means cells do not need to be genetically modified to identify cancer cells.

Stressed cells such as tumor cells express a unique array of cell surface proteins that make them highly attractive to NK cells. For instance, tumor cells do not express MHC class I molecules, which generally identify cells as part of the body. In addition, the cells express several other signals that indicate the cell should be killed. These signals activate NK cells, which leads the cells to attack the tumor and activate the adaptive immune system.⁷ This further enhances the immune response to cancer.

Since they are a natural part of the innate immune system, NK cells are generally safe.⁸ Furthermore, manufacturers could easily design “off-the-shelf” NK cells that physicians can bring to patients more quickly than cells that need to be genetically modified.

To make an NK cell therapy, a manufacturer needs to activate and expand someone’s NK cells. Although the process seems simple, it is challenging to expand NK cells in vitro, making it difficult to scale up production. The cells also present clinical limitations: they do not infiltrate solid tumors or persist in the body for very long.⁸

However, genetic modification may make NK cells even stronger. NK cells are not strong enough to defeat cancer on their own, but data shows that an NK cell that expresses the CAR protein will more easily overcome immune suppression.⁹

The Future of Adoptive Cell Therapy

Physicians and scientists can barely contain their enthusiasm for ACTs. Recent short- and long-term successes with CAR T-cell therapies, combined with promising clinical trial results and technical achievements involving other forms of treatment, demonstrate the impact of this approach on cancer therapy. Yet one common factor that limits all these therapies is the complexity of the manufacturing process. For example, the most clinically advanced ACT, CAR T-cell therapy, still takes three weeks to develop for each patient, mainly because each dose needs to be made from scratch following the decision to pursue such treatment. In some cases, future ACTs will solve some of the limitations of CAR T-cells but will introduce their own, which manufacturers will need to consider as they seek to mass-produce these therapies.

Some of these improvements will manifest as scientists develop a greater understanding of immune cell activity on a fundamental level. Others will derive from industrial ingenuity on the part of the manufacturer. As experts improve extraction, genetic engineering, and expansion protocols, it will become easier to generate effective therapies for more people.

ACT development is ongoing. The FDA receives hundreds of Investigational New Drug applications for cell therapies yearly.¹⁰ They recognize the unique manufacturing challenges associated with these therapies, and to help manufacturers, they have received several guidances on the subject.¹¹ With demand from physicians and patients, significant financial investment, and regulatory backing, the biopharmaceutical industry is primed to deliver safer, more effective ACTs to more people, potentially saving thousands of lives.

References

1. Melenhorst JJ, Chen GM, Wang M, et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T-cells. Nature. 2022;602(7897):503-509.
2. Anderson LA, ed. What is the cost of kymriah? Drugs.com. https://www.drugs.com/ medical-answers/cost-kymriah-3331548/. Published May 20, 2021. Accessed March 4, 2022.
3. Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. ‘Off-the-shelf’ allogeneic CAR T-cells: development and challenges. Nat Rev Drug Discov. 2020;19(3):185-199.
4. Arnaud M, Bobisse S, Chiffelle J, Harari A. The promise of personalized tcr-based cellular immunotherapy for cancer patients. Front Immunol. 2021;12:701636.
5. Tucker, N. FDA Grants Orphan Drug Designation to Novel TIL Therapy for Advanced-Stage Melanoma. Targeted Oncology. https://www.targetedonc.com/view/fda-grants-orphan-drug-designation-to-novel-til-therapy-for-advanced-stage-melanoma. Published April 27, 2021. Accessed March 4, 2022.
6. National Cancer Institute Center for Cancer Research. FDA grants breakthrough therapy designation of new TIL therapy for advanced cervical cancer. https://ccr.cancer.gov/ news/article/fda-grants-breakthrough-therapy-designation-of-new-til-therapy-for-advanced-cervical-cancer. Published August 16, 2019. Accessed March 4, 20
7. Paul S, Lal G. The molecular mechanism of natural killer cells function and its importance in cancer immunotherapy. Front Immunol. 2017;8:1124.
8. Liu S, Galat V, Galat4 Y, Lee YKA, Wainwright D, Wu J. NK cell-based cancer immunotherapy: from basic biology to clinical development. J Hematol Oncol. 2021;14(1):7.
9. Albinger N, Hartmann J, Ullrich E. Current status and perspective of CAR T and CAR NK cell therapy trials in Germany. Gene Ther. 2021;28(9):513-527.
10. US Food and Drug Administration. Statement from FDA Commissioner Scott Gottlieb, M.D. and Peter Marks, M.D., Ph.D., Director of the Center for Biologics Evaluation and Research on new policies to advance development of safe and effective cell and gene therapies. https://www.fda.gov/news-events/press-announcements/statement-fda-commissioner-scott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics. Published January 15, 2019. Accessed March 4, 2022.
11. US Food and Drug Administration. Cellular & Gene Therapy Guidances. https://www.fda. gov/vaccines-blood-biologics/biologics-guidances/cellular-gene-therapy-guidances. Published December 20, 2021. Accessed March 4, 2022.

Marwan Alsarraj is the Biopharma Segment Manager at Bio-Rad. He has been at the forefront of developing, marketing, and commercializing technologies in the past 15 years in the life science research industry. Marwan obtained his M.S. in Biology at the University of Texas, El Paso.

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