CAR-T cell development platform

What are chimeric antigen receptors (CARs)?

Chimeric antigen receptors (CARs) are synthetic structures designed to modulate natural T cell receptors (TCRs). They consist of four main components:

1. An extracellular antigen-binding domain: either a single-chain variable fragment (scFv), single-domain antibody (sdAb), or nanobody (VHH) designed to target tumor-associated antigens
2. A hinge or spacer region: the length and composition of this region dictates the flexibility of the extracellular portion of CARs, in turn, this flexibility determines what type of epitopes (sterically inaccessible or otherwise) can be targeted by the receptor
3. A transmembrane domain: this region anchors the CAR to the cell membrane, moreover, it has also been shown to influence the stability and expression levels of CARs
4. One or several intracellular signaling domains or endodomains: typically containing a CD3ζ chain, this region of the CAR plays a vital role in triggering and sustaining the signaling cascade that initiates a plethora of different mechanisms leading to the apoptosis of cancer cells

CAR structures differ from TCR in the way they interact with the antigen. TCRs only bind peptides that are bound and presented by major histocompatibility molecules (MHC). In contrast, CARs bind antigens that are directly bound to the membrane of cancer cells.
This small difference in terms of structure and recognition has a huge impact in terms of function. MHC loss or downregulation is a common mechanism employed by cancer cells to evade the organism’s immune response. For this reason, most cancer antigens are displayed on the surface of the cell membrane not bound by MHCs. By forgoing MHC-mediated antigen recognition, CARs become invaluable for treating immune escape refractory cancers (that do not respond to any other treatment).
To elicit a specific anti-cancer response, CARs are introduced into T cells where they will redirect their cytotoxic activity towards tumor cells.

How many generations of CAR-T cells are there?

The first T cells engineered to express CARs were developed in the late 80s and early 90s. Reports of an effective application of this immunotherapy was published as early as 2010. Only seven years later, the first of these “living drugs” received marketing approval by the Food and Drug Administration (FDA).
Despite their recency, the structure of CARs has evolved tremendously since their inception. From the first to the latest generation of CARs, these constructs have kept their canonical structure (extracellular, transmembrane, and intracellular) but evolved significantly in terms of composition and additional domains to increase signal strength and sustain the cytotoxic response.
To date, four generations of CAR-T cells have been described and a fifth in underway. In each new generation of CARs, the signaling domains are modified while the other domains remain constant:

• The first generation is the most similar to endogenous TCRs containing a single CD3ζ chain. Despite their ability ability to bind to tumor-associated antigens with great specificity, they were unable to generate a sustainable cytotoxic response and showed limited proliferation.
• The second generation included costimulatory domains such as CD28 bound to CD3ζ in an attempt to amplify the signal and trigger a strong cytotoxic response. Clinical studies showed the extra domain significantly enhanced proliferation, cytotoxicity, and more durable responses. However, many reported early exhaustion as the most significant problem of this generation of CAR-T cell therapies.
• The third generation attempted to solve this problem by adding another costimulatory domain such as 4-1BB or OX40, but the change led to no significant improvement of efficacy.
• The fourth generation of CAR-T cells (also called TRUCKS) was based on second generation constructs. It generally contains a NFAT domain designed to induce the overexpression of cytokines (e.g., IL-12) which enhances both the cytotoxicity and the ability to modulate the immune response (i.e., recruitment of innate immune cells)
• The fifth generation is also based on second-generation CARs. Besides the costimulatory domains, these CAR constructs are designed with an additional fragment of the IL-2 receptor which induces the production of Janus kinases (JAKs) and signal transducer and activators of transcription (STAT). The efficacy and safety of these constructs is still under investigation.

How do CAR-T cells kill cancer cells?

CAR-T cells are a form of adoptive T cell therapy (ACT), a type of immunotherapy that relies on the therapeutic use of T cells. Due to their complex intracellular domains, CAR-T cells are known to leverage different mechanisms to eliminate tumor cells. These mechanisms are triggered in response to a specific tumor-associated antigen. Once the antigen-binding domain (scFv, sdA, or VHH) binds the antigen, an immune synapse forms in the interface between the two cell types.
Lysis subsequently occurs via three known pathways:

1. Degranulation: the primary mechanism of cancer elimination relies on the formation of cytotoxic granules containing perforin and granzyme. Once the granules reach the interface in the immune synapse, perforins are released resulting in the formation of pores on the cancer cell membrane. These pores grant access to granzymes which, in turn, trigger apoptosis once they reach the cytoplasm. Degranulation targets only antigen-positive cells.
2. Fas-FasL pathway: this pathway also triggers apoptosis in a caspase-dependent way. It serves as an alternative to degranulation because it triggers this response mostly in antigen-negative cells found in the tumor milieu.
3. Cytokine production: in early generations CAR-T cells, cytokine production was mostly considered a secondary mechanism of action. But the introduction of cytokine-associated domains (ligands and receptors) in the most recent generations has led them to gain a greater importance in tumor elimination. Cytokines are known modulators of the immune response and can act as enhancers of cytotoxicity, activators and recruiters of innate immune cells, and reprogramming agents of stroma cells which act as physical barriers and block access to tumors.

How are CAR-T cells manufactured?

The process of manufacturing CAR-T cells requires two components: a well-developed CAR and a source of T cells. All CAR-T cell therapies approved to date are manufactured by sourcing T cells from the patients who will receive the treatment – also named autologous T cells. These cells are harvested by leukapheresis, a form of apheresis designed to extract lymphocytes from the blood of patients. The system does so by centrifugation or membrane filtration followed by reinfusion of the remaining components together with replacement fluid.

These T cells are subsequently activated using antibodies or interleukins. Activated cells are subsequently modified by delivery via transduction (viral vectors) or transfection (plasmids) of CAR components. CAR-positive cells are subsequently expanded in vitro and reinfused back into the patient.

CAR-T cells are currently considered a last-line treatment reserved to patients with refractory tumors, unresponsive to conventional chemotherapy or immunotherapy. Most patients eligible for CAR-T cell therapies have previously received aggressive immune-depleting treatments. For this reason, the development of cell therapies based on allogeneic T cells (sourced from healthy individuals) remains one of the most important trends in CAR-T cell manufacturing. Allogeneic cell therapies alongside the construction of cell banks may propel these therapies to the first-line of anti-cancer treatments.

Challenges and opportunities in CAR-T cell engineering

CAR-T cell therapies have demonstrated their effectiveness across multiple clinical trials. However, from a technical maturity viewpoint, the technology is still considered new. For this reason, many technical challenges remain to be solved until its widespread use becomes possible. Most challenges are tied to unspecific toxicity, limited activity, difficulties in penetrating solid tumors and in targeting antigen escapes.
Much effort has been put into CAR-T cell engineering in an attempt to solve these limitations. In the table below, you can find a summary of the most promising approaches.