T cell Isolation, Engineering, Activation, and Expansion

T cells, also known as T-lymphocytes, are one of several cells operating within the adaptive immune system to protect the body from pathogens. T cells arise from immature T stem cells in the bone marrow and migrate to the thymus, where, in response to the hormone thymosin and other factors, they mature and differentiate into several subtypes. Every T cell that survives migration and maturation develops a receptor targeting a specific antigen.

From the thymus, T cells enter peripheral lymphoid circulation where they encounter pathogens through action of major histocompatibility (MHC) molecules. Once primed through exposure to an antigen, T cells activate, proliferate, and differentiate further into effector T cells.

Because the thymus atrophies as we age, it produces fewer naïve T lymphocytes—T cells that have yet to encounter their antigen—over time.

Complex cellular lineages

When naïve T cells are presented with antigens by MHC Class I molecules, the predominant products are cytotoxic lymphocytes, which destroy their targets by releasing cytotoxic granules—essential vesicles or organelle-like structures—into their target. Cytoxic T cells require several signals for activation, for example from T helper cells or dendritic cells.

T helper cells, often referred to as "the most important cells in adaptive immunity," are required for nearly all functions of adaptive immunity. In addition to helping to activate cytotoxic lymphocytes, T helper cells signal B cells to release antibodies and macrophages to destroy invading species.

Long-lived memory T lymphocytes do what their name suggests. For years the origins of these cells were unknown, but their lineage was recently traced back to effector T cells. Memory T cells carry the CD8 receptor for the cognate antigen and persist for years or even decades. A 2008 study found that immunity to the 1918 influenza pandemic persisted in survivors 90 years later.

Adoptive cellular immune therapies seek to exploit the complex processes involved naturally in the acquisition of antigen specificity, activation, and expansion. They achieve this through a multi-step process involving isolation of naïve T cells from patients, engineering those cells to recognize specific antigens, activating and expanding the cells ex vivo, and re-introducing them into the patient.

Where activation and expansion are natural processes that developers of immune therapies seek to emulate, antigen specificity must be genetically engineered into T cells to confer specificity to specific targets. More often than not the targets are cancer cells bearing molecular signatures that the T cells were programmed to recognize.

But first, one must obtain cells in sufficient numbers to permit expansion into much larger populations suitable for immunotherapy.

Isolation, engineering, expansion

The first hurdle is isolating naïve T cells within the background of tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs). A recent paper explains current strategies for identifying relevant cells from TILs on the basis of surface markers, for example CD137 members of the tumor necrosis factor family. The drawback to this method is that CD137-positive cells are highly activated and differentiated, with limited potential for differentiation.

Another approach uses T cell receptor frequency, or abundance, to identify suitable cells. This laborious technique has been streamlined through the availability of low-cost next-generation sequencing. When naïve T cells are undetectable among TILs, investigators turn to PBLs, a reliable and relatively plentiful source of neoantigen-specific T cells. PBLs are attractive because they may be engineered to target almost any tumor antigen through the genetic transfer of an antigen-specific receptor.

Gene transfer techniques vary according to the type of receptor desired on the therapy cell, e.g., antigen-restricted, heterodimeric T cell antigen receptor, or chimeric antigen receptors (CARs).

T cells are susceptible to a variety of genetic manipulations but engineering entails much more than simply plopping in a gene. Among factors requiring precise control are receptor coverage on the cells' surfaces, the inclusion of co-stimulatory or activating moieties, toxicities, antigen escape, and overcoming immunosuppressive tumor environments.

For example: CAR T

CAR T cell manufacturing, which is illustrative of adoptive cell therapy, involves T cell isolation, cell activation, gene transfer of the CAR vector, and CAR T cell expansion followed by re-infusion into the patient.

CARs consist of a hinge and a separate antigen binding domain, transmembrane domain, and an intracellular signaling domain, each with distinct function. Improving a CAR therefore involves optimizing the four units independently, and, through combinatorial methods, together. Cell-level engineering is also applied to mitigate toxicities and to broaden the range of antigens the CAR treatment can handle.

T cell activation, an indispensable step in CAR T manufacturing, affects the cellular composition and phenotype of the cell therapy product. Activation further supports robust T cell expansion without causing differentiation or activation-induced cell death. Antigen-presenting cells, such as dendritic cells, are the main mediators of T cell activation in nature but they are impractical to use in a production setting. Manufacturers therefore turn to simpler ex vivo activation strategies. Examples include anti-CD3 or anti-CD28 antibodies, a recombinant human fibronectin component, and artificial antigen-presenting cells. These methods undergo constant revision and improvement. The most popular methods are anti-CD28 and -CD3-coated magnetic beads and monoclonal antibodies.

Adoptive T cell therapies require the administration of billions of engineered T cells. Like most cell culture scaleups, successful T cell expansion requires optimizing the culture media, feeds, growth factors, cell density, hormones, cultureware, vitamins, and physical conditions such as temperature, pH, culture time, etc. Obtaining clinically relevant quantities of cells at the highest quality requires optimizing conditions to favor the desired phenotype, and of course live cells to dead cells.

In T cell expansion, protocols predicting a priori the effects of culture conditions on quality attributes and yields is more an art than a science. Cell density is an attribute where, generally speaking, more is better. Not so with T cell expansions, where a recent study demonstrated the superiority of low cell density in expanding therapeutic cells. This protocol involves standard culture optimization but at a somewhat lower cell density than normal, resulting in 800-fold expansion of human T cells at greater than 85% viability over 10–14 days in static culture.

Conclusion

Adoptive T cell therapy, which is sometimes called a living drug, shows great promise in fighting both blood and solid tumors. Because of their $300,000 to $400,000 price tag, these treatments are reserved for serious cases of relapse, aggressive, or refractory cancers. Industrializing CAR T methodology through the application of optimized isolation, activation, engineering, and expansion protocols will be key to wider adoption of these technologies, and perhaps their expansion beyond cancer indications.