An Introduction to T Cell Manufacturing and Expansion

T cells are an essential component of the human immune system, playing a critical role in recognizing and eliminating various foreign entities, such as viruses, bacteria, and cancer cells. However, in certain conditions, such as cancer or immunodeficiency diseases, the body’s natural T cell response is insufficient to combat the disease.

In such cases, T cell based therapies, including chimeric antigen receptor (CAR) T cell therapy and T cell receptor (TCR) therapy, have emerged as promising treatment options. To manufacture these T cell based therapies, it is necessary to obtain a sufficient number of functional T cells, which can be achieved through T cell culture and expansion techniques.

This article explores the methods and challenges of T cell manufacturing and expansion and the impact of these technologies on clinical outcomes.

What Is T Cell Culturing?

T cell culturing is a process that involves growing T cells in a laboratory setting to produce a large number of functional T cells for reintroduction into a patient’s body. This is typically achieved using a combination of growth factors and cytokines that stimulate T cell activation and proliferation.

Once sufficient T cells are obtained, they are genetically modified using CAR or TCR technology to enhance their tumor-targeting abilities. The modified T cells are then expanded further and tested for quality before being reintroduced to the patient.

Why Do T Cells Need to Be Expanded?

T cells must be expanded to obtain a sufficient number of functional cells for use in therapeutic applications like CAR-T or TCR-T cell adoptive immunotherapy. Typically, the number of T cells in a patient’s body is insufficient to provide a therapeutic benefit, especially in cases of cancer or immunodeficiency diseases.

Expansion allows for the production of a large number of T cells that are genetically modified to enhance their ability to recognize and fight disease before being reintroduced to the patient. Several combinations of activation and expansion protocols can be used to increase T cell numbers in culture.

Cell Culture Types

Adoptive immune cell culture protocols for activation and expansion can be broadly categorized into two main types:

1. Autologous cell culture and expansion involve collecting immune cells, such as T cells, from the patient. Their cells are then modified and expanded in vitro before being reintroduced into the same patient. This approach is commonly used in CAR-T cell therapy.
2. Allogeneic cell culture involves collecting cells from a healthy donor other than the patient. If necessary, the donor cells are modified and expanded in culture to enhance their therapeutic potential before being infused into the patient. This approach is used in natural killer (NK) cell therapy and CAR-NK therapy because NK cells do not elicit the same types of autoimmune response as T cell therapies do, although genetic engineering efforts are being made to solve this problem to allow for allogeneic T cell therapies.

How to Culture T Cells

In the body, mature T cells, such as cytotoxic CD8+ and helper CD4+, proliferate in large numbers when activated by dendritic cells or other antigen-presenting cells (APCs). However, this typical pathway is impractical for in vitro activation of T cells grown in culture.

Simplified activation and expansion strategies have been developed to avoid using APCs as natural endogenous activators for therapeutic T cell expansion. These protocols may involve antigen-conjugated beads, cytokines, or functionalized bioreactor-based methods. Each of these approaches has advantages and disadvantages and is used depending on the specific application and goals of the T cell culture. 

Once the initial population of T cells is isolated from the patient’s blood or tumor biopsy sample, they are grown in a lab for around two weeks in incubated and carefully monitored liquid flask cultures. The growth medium typically contains a buffer base system with proteins, inorganic salts, vitamins, energy sources, and other trace elements to promote growth. The cultures are also divided every couple of days to prevent the cells from becoming completely confluent, meaning covering the entire surface of the growth flask, which can affect cell behavior and cause irregular growth. During this time, the cultured T cells are activated using anti-CD3/CD28 pathways. 

If the T cells are being used for engineered TCR or CAR-T therapy, the genomic constructs are introduced to the culture at this stage via viral vector transduction or transfection methods. Which media is used and how the T cells are activated directly affects which T cell subtypes are more likely to successfully take up and integrate the engineered receptor genes and at what efficiency. 

Optimal activation should lead to sufficient T cell expansion without causing significant T cell differentiation or activation-induced cell death (AICD). Therefore, choosing the correct growth environment and activator reagents is vitally important to the downstream success of clinical therapeutic T cell applications. 

After the engineered genetic materials are integrated, antigens, cytokines, and other growth factors are added to encourage the rapid expansion of selected T cell populations. Naive and stem cell memory (T_SCM) T cells are specifically targeted for enrichment because of their enhanced clinical safety and self-renewing attributes. Once the T cells have expanded to sufficient numbers, they are removed from culture, purified, and preserved before being sent back to the patient’s medical center for treatment. 

Finding a T Cell Expansion Protocol That Works

Scientists use a combination of methods to evaluate T cell expansion protocols for safety and efficacy. These include:

• Cell counting and viability assays: T cells are counted and evaluated for viability using flow cytometry or other cell-based assays. This ensures the T cells remain healthy and viable.
• T cell receptor (TCR) analysis: TCR analysis ensures that the expanded T cells maintain their specificity and do not become activated against non-target cells, which could cause serious side effects.
• Quality control testing: The final product is tested to ensure it meets strict quality control standards for sterility, purity, and potency.
• Animal studies: Animal studies may be performed to evaluate the safety and efficacy of new expansion protocols. In these studies, the T cells are infused into animals to evaluate their ability to target and destroy cancer cells or other disease targets.
• Clinical trials: Before any immunotherapy treatment is approved by the Food and Drug Administration, the therapy must go through rigorous clinical trials in human patients. These trials are designed to evaluate the safety and efficacy of T cell therapy and to optimize the dosing and delivery of the expanded cells.

Nanotein Expands CAR-T Cells for Cancer Treatment

Many current methods of activation and expansion are inefficient and non-specific to stemlike T cells. They introduce clinical risks to the process by using synthetic ingredients and magnetic beads that may contaminate the therapy if not painstakingly removed from the expanded culture.

To address this, Nanotein Technologies developed novel protein-based CAR-T technology in a soluble format that requires less time, fewer T cells, and no magnetic components or synthetic contaminants. Nanotein’s NanoSpark™ STEM-T Soluble T Cell Activator focuses on T_SCM CAR-T expansion, generating large quantities of clinically potent T cells in a less differentiated state for increased self-renewal and clinical safety during treatment. Contact us today to learn more.