The Manufacturing Journey of CAR-T Cellular Therapy—An Overview

Abstract

Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized immunotherapy, offering innovative solutions for cancer treatment. Allogeneic CAR-T therapies utilize T cells from healthy donors or such sources as umbilical cord blood and induced pluripotent stem cells, providing, scalable, off-the-shelf treatments with broader applicability and reduced waiting times, compared to autologous CAR-T therapies. The manufacturing process includes T-cell activation, genetic modification using viral vectors to express CARs, cell expansion, rigorous quality control testing, and cryopreservation for storage and shipment. These therapies demonstrate clinical efficacy in hematologic malignancies, making them cost-effective and accessible to patients unsuitable for autologous approaches. In contrast, autologous CAR-T therapies leverage a patient’s own T cells to create personalized, highly targeted treatments. Beginning with T-cell collection via apheresis, the process involves activation, genetic engineering to express CARs targeting specific cancer antigens, cell expansion, and thorough quality control. Despite challenges in T-cell quality and manufacturing complexity, autologous CAR-T therapies have shown remarkable success in treating acute lymphoblastic leukemia and non-Hodgkin lymphoma, offering hope to patients who have exhausted other treatment options. Together, both approaches highlight the transformative potential of CAR-T therapies in cancer treatment.

Introduction

Allogeneic chimericantigenreceptorT-cell (CAR-T) therapies represent a remarkable advancement in the field of immunotherapy, aiming to provide off-the-shelf cellular treatments for various forms of cancer (1). This innovative approach differs from autologous CAR-T therapies, which utilize a patient's own T cells in a process that can be time-consuming and is tailored to each patient. In the case of allogeneic CAR-T therapies, the process is designed for more extensive scalability and wider applicability.

Allogeneic CAR-T therapy manufacturing

The manufacturing journey of allogeneic CAR-T cellular therapies begins with the collection of healthy donor T cells (type of white blood cells). These T cells are typically isolated from peripheral blood mononuclear cells from healthy donors, umbilical cord blood, or derived from induced pluripotent stem cells (iPSCs), offering a significant advantage in terms of consistency and availability when compared to autologous approaches (1). Once collected, the T cells are then transported to a manufacturing facility, where they undergo a multi-step process to become potent CAR-T cells.

The first critical step is T-cell activation, which involves exposing the collected T-cells to specific stimulation, often utilizing activation beads, such as Dynabeads (for cell isolation and expansion) or other methods (2,3). Activation is a pivotal moment in the process, as it prompts the T cells to become highly functional and receptive to genetic modification. Activation effects the CAR-T transduction efficiency, rate of CAR-T cell expansion, and differentiation (3). The next step involves genetic engineering of the cells. During this stage, the T cells are manipulated (transduced with CAR genes) to express CARs (synthetic receptors that enable T cells to recognize and target cancer cells) on their surface (2). These CARs are typically designed to recognize specific antigens commonly found on the surface of cancer cells, such as CD19 for B-cell malignancies (2).

The introduction of CARs into the T cells is usually achieved using viral vectors, such as lentiviral or gamma retroviral vectors (the main two classes of retroviral vectors that are derived from enveloped RNA viruses of the retroviridae family), adenoviruses, and adeno-associated viruses (4,5). These vectors deliver the genetic load into the T cells, leading to the expression of CARs on the cell surface. This genetic modification provides the T cells with the ability to identify and destroy cancer cells, effectively turning them into CAR-T cells.

Following genetic engineering, the CAR-T cells undergo an expansion phase (2). This step is crucial for generating a sufficient number of CAR-T cells for therapeutic doses. T-cell production is stimulated through the addition of cytokines and other growth factors. This phase can take several days and may involve multiple rounds of cell division to achieve the desired cell density.

After expansion, the CAR-T cells are subjected to comprehensive quality control testing for safety and potency, to ensure the final product is both effective and free from contaminants. The cells are assessed for their CAR expression, viability, and functionality. Moreover, they are screened for the presence of any viral or microbial contamination. Upon passing quality control tests, the CAR-T cells are formulated into a final product for patient administration.

The formulated product is then cryopreserved to maintain cell viability and function during storage. This cryopreservation enables the cells to be shipped and stored until they are ready for infusion into patients. Once the CAR-T product is needed for a patient, it undergoes a thawing process and is infused intravenously. The CAR-T cells then circulate within the patient's body, targeting cancer cells and initiating their destruction. This process can lead to remarkable clinical responses in patients with certain haematological malignancies, often resulting in remissions that may be durable (1,2).

The manufacturing of allogeneic CAR-T cellular therapies offers several advantages over autologous CAR-T therapies:

• More cost-effective
• Allows for the creation of large batches that can treat multiple patients
• Significantly reduces the waiting time for patients who require treatment
• Off-the-shelf nature makes allogeneic CAR-T therapies a viable option for patients who may not be suitable candidates for autologous approaches.

Figure 1 represents the manufacturing process of an allogeneic CAR-T cellular therapy.

Autologous CAR-T therapy manufacturing

Autologous CAR-T therapies have advanced the response to cancer, offering personalized, highly targeted therapies for individuals suffering from specific hematological malignancies (1,2). Unlike allogeneic CAR-T therapies that use donor-derived T cells, autologous CAR-T therapies harness a patient's own immune cells, making it a truly individualised treatment approach (7).

The journey of manufacturing autologous CAR-T cellular therapies commences with a patient-specific process. It begins with the collection of the patient's own T cells through a procedure called apheresis. During apheresis, a patient's blood is drawn, and the T cells are separated from other blood components, such as red and white blood cells (7). A significant challenge is the quantity and quality of the starting autologous T cells, as patients often have low leukocyte counts due to undergoing lymphodepleting chemotherapy and/or radiotherapy (1,8).

The collected T cells are then transported to a dedicated manufacturing facility, where the isolated T cells are activated and expanded. This activation phase is crucial for priming the T cells, rendering them more receptive to genetic modification and enhancing their cytotoxic capabilities. As mentioned earlier, activation is typically achieved by exposing the T cells to specific activation beads or molecules, a crucial step in the CAR-T production process (2,3).
The next essential step in the manufacturing process is genetic engineering. Here, the isolated and activated T-cells are modified to express CARs on their surface (7). As with allogeneic CAR-T therapies, the choice of antigen targeted by the CAR is carefully selected based on the type of cancer being treated. For example, CD19 is often the target for B-cell malignancies.

The introduction of CARs into the patient's T cells is typically achieved using viral vectors, most commonly lentiviral or retroviral systems. These vectors carry the genetic load, which integrates into the T cells, ensuring constant CAR expression on the cell surface. This genetic engineering step provides the T cells with the ability to specifically recognize and attack cancer cells. Following genetic modification, the CAR-T cells undergo an expansion phase (7). This stage is crucial for producing a sufficient number of cells to create the therapeutic dose. T-cell production is stimulated through the addition of cytokines and growth factors. It often takes several days and multiple rounds of cell division to achieve the desired cell quantity.

Quality control testing is a pivotal aspect of the manufacturing process. As with allogeneic CAR-T therapies, rigorous screening is also performed to detect any potential contaminants, including viruses and microbes. As with the allogeneic CAR-T manufacturing process, once the CAR-T cells successfully pass the quality control tests, they are formulated into a final product suitable for patient administration. The product is then delivered to the patient or cryopreserved until suitable timing for the patient. In the latter, when the time comes for treatment, the cryopreserved CAR-T product is thawed and administered to the patient intravenously (6).

The individualized treatment strategy of autologous CAR-T therapy has shown remarkable success in treating such conditions as acute lymphoblastic leukemia and non-Hodgkin lymphoma (Figure 2), offering renewed hope to patients who may have exhausted other treatment options.

Cost-effective vs personalized

Allogeneic CAR-T therapy is a major advancement in immunotherapy, offering scalable, off-the-shelf treatments for various cancers. Using T cells from healthy donors or such sources as umbilical cord blood and iPSCs, this approach ensures consistency, broader applicability, and reduced waiting times. The manufacturing process involves T-cell activation, genetic modification with viral vectors, expansion, and rigorous quality control before cryopreservation and patient infusion. These therapies are cost-effective, enable large-batch production, and are suitable for patients ineligible for autologous treatments.

In contrast, autologous CAR-T therapy uses a patient's own T cells to provide personalized treatments for hematologic malignancies. After collecting T cells via apheresis, they are activated, genetically modified to target cancer antigens (e.g., CD19), expanded, and quality tested. Despite such challenges as limited T-cell quality due to prior treatments, autologous CAR-T has shown significant success in treating such conditions as acute lymphoblastic leukemia and non-Hodgkin lymphoma, offering hope to those who have exhausted other options.

References

1. Bedoya, D. M.; Dutoit, V.; Migliorini, D. Allogeneic CAR T Cells: An Alternative to Overcome Challenges of CAR T Cell Therapy in Glioblastoma. Front. Immunol. 2021, 12–2021. DOI: 10.3389/fimmu.2021.640082
2. Wang, H.; Tsao, ST.; Gu, M.; et al. A Simple and Effective Method to Purify and Activate T Cells for Successful Generation of Chimeric Antigen Receptor T (CAR-T) Cells from Patients with High Monocyte Count. J. Transl. Med. 2022, 20, 608. DOI: 10.1186/s12967-022-03833-6
3. Zhang, D. K .Y.; Adu-Berchie, K., Iyer, S.; et al. Enhancing CAR-T Cell Functionality in a Patient-Specific Manner. Nat. Commun. 2023, 14, 506. DOI: 10.1038/s41467-023-36126-7
4. Ghosh, S.; Brown, A. M.; Jenkins, C.; Campbell, K. Viral Vector Systems for Gene Therapy: A Comprehensive Literature Review of Progress and Biosafety Challenges. Appl. Biosaf. 2020, 25 (1), 7–18. DOI: 10.1177/1535676019899502
5. Labbé, R. P.; Vessillier, S.; Rafiq, Q. A. Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives. Viruses 2021, 13 (8), 1528. DOI: 10.3390/v13081528
6. Harrison, R. P.; Zylberberg, E.; Ellison, S.; Levine, B. L. Chimeric Antigen Receptor–T Cell Therapy Manufacturing: Modelling the Effect of Offshore Production on Aggregate Cost of Goods. Cytotherapy 2019, 21 (2), 224–233. DOI: 10.1016/j.jcyt.2019.01.003
7. Palmer, R.; Evans, N. Medication Restrictions for Patients Receiving CAR-T Therapy. pharmaceutical-journal.com, July 5, 2022.
8. Ceppi, F.; Rivers, J.; Annesley, C.; Pinto, N.; et al. Lymphocyte Apheresis for Chimeric Antigen Receptor T‐Cell Manufacturing in Children and Young Adults with Leukemia and Neuroblastoma. Transfusion 2018, 58 (6), 1414–1420. DOI: 10.1111/trf.14569

About the author

Shada Warreth is Global Partnerships Implementation senior manger at NIBRT and PhD Candidate at TU Dublin, Ireland.