Lymphocytes are a type of white blood cells that help our body fight infections and diseases. We can distinguish two types of lymphocytes: B cells and T cells and despite the fact that they work together, both have different roles in the immune system. B cells originate from bone marrow and are responsible for producing small proteins (antibodies) directed against viruses, bacteria that block them from infecting healthy cells. T cells originate from bone marrow but, in contrast to B cells, they mature in thymus. Each T cell carries on its surface receptor (TCR) that can recognize specific foreign molecules called antigens. If a T cell recognizes an antigen that binds to its TCR, it will induce T cell activation which results in cell expansion and secretion of various proteins that can induce inflammation and eventually cell death. T cells that recognize antigens expressed by our own cells usually die during maturation in the thymus, thus T cells usually recognize and kill only foreign or infected cells. Tumor cells express antigens that are recognized by T cells as “self” and do not induce immune reaction.
Different flavors of T cell immunotherapy
The idea of using the immune system to fight cancer can be dated back to the late 19th century, when William Coley noticed spontaneous tumor regression in some patients after an unrelated bacterial, fungal and viral infection. But it was not until late 1950s, when the role of white blood cells in immunological surveillance as the main sentinels of the immune system, was recognized. Today due to an enormous development of genetic and cell engineering accompanied by increased understanding of how the immune system works, T cell adoptive cell therapy has become a viable treatment option for cancer patients.
Currently, we can distinguish two main approaches in T cell-based adoptive cell therapy. First one relies on the observation that some of the lymphocytes that penetrate tumor mass have antitumor reactivity. These tumor-infiltrating lymphocytes (in short- TILs) are collected from the tumor site by biopsy or surgery and further expanded to a large number in the laboratory before they are infused back into the patient. Since the first report of in-human trails1, TIL therapy has been successfully applied to patients with metastatic melanoma and other solid tumors. However, despite some unique advantages, TILs face a series of challenges, such as absence of effector T cells with anti-tumor activity or isolation and expansion process to obtain enough cells to treat patients. (Fig.1)
Currently, more efforts are being put into development of CAR (chimeric antigen receptor)-based therapy. The name refers to genetically engineered surface receptors recognizing molecules expressed by cancer cells which will trigger T cells and kill the tumor cells (Fig.2). T cells are isolated from patient blood, genetically modified to express tumor-specific CARs, expanded to large numbers and re-introduced into the same patient (Fig.1). Thanks to this design, CAR T cells can extremely precisely target any molecule on the surface of cells.
Critical choice of tumor antigen
CARs can be designed to recognize any cell surface antigen, however there is one requirement. In order to develop a successful CAR therapy, the target has to be associated only with the tumor cells and not being expressed by healthy tissues. In 2017 first CAR therapy targeting CD19 molecules expressed by B cells was approved by the FDA in the USA to treat patients with blood tumors. The fact that CD19 is restricted exclusively to the B cell lineage and that patients can live without B cells has made CD19 an extremely promising target for CAR T cell immunotherapy. To date more than 80% of patients with lymphoma that were refractory to first-line treatment showed a response after single infusions of CD19-CAR products, with two times more of patients with complete response as compared to the patients that underwent a standard care2.
However, as many remarkable results are obtained with CARs in the context of blood cancers, their use in patients with solid tumors has very limited efficacy. One of the obstacles for CAR T cells therapy in the solid tumors is lack of antigens that would be restricted only to tumor and non-vital tissues and as such a potential of killing healthy cells (called on-target/off-tumor toxicity) must be considered.
Unfortunately, even identification of a cancer-specific target does not always guarantee a successful CAR treatment. For example, in some cases CD19-CAR -treated patients who had recurrent disease after treatment, developed CD19 negative malignant cells4. This phenomenon has been widely described as antigen escape. Initially discovered in hematological malignancies, this mechanism of resistance has been observed in solid tumors as well. In order to address antigen, CARs are designed to target multiple antigens at the same time.
On-target/off-tumor toxicity
Solid tumor antigens are often detected on normal tissues at various levels and this fact creates a potential of killing healthy cells by CAR T cells leading to on-target/off-tumor toxicity. One of the most revolutionary strategies to discriminate between tumor and healthy tissues is incorporation of logic gates. This effort was pioneered by development of a so-called synNotch circuit which works in “IF-THEN” manner (Fig.3A). In this system recognition of antigen A by a synthetic receptor triggers expression of CAR against tumor-associated antigen B and as a consequence- T cell activation. Recently an “AND” gate platform called LINK was developed by a team at Stanford University. In the LINK system only simultaneous engagement of two CARs targeting different antigens activates T cells (Fig.3B).
Alternatively, “AND-NOT” gate is as well in development, in which one CAR recognizes tumor-associated antigen but T cell activation can be inhibited by a second CAR that recognizes antigen expressed on healthy tissue but not on tumor tissue (Fig.3C).
CAR T cell- associated toxicities
There is a specific therapeutic window for CAR T cell activation level in order to be clinically effective. Only antigen-CAR binding interactions above the minimum threshold induce activation signal in T cells, however, if it is too strong, cells can become overactivated causing toxicity. A potential route to control the toxicity is through introduction of kill switches that in the case of severe toxicity allow for depletion of engineered cells. However, it is not an ideal solution, as this approach is associated with complete clearance of the infused patient product which would mean the need for another infusion. That is why, a lot of effort is being invested in development of regulatable CAR platforms that can serve as reversible safety switches and also tune CAR signaling, without the necessity of killing cells. Modulation of CAR T cell activity by providing patients with specific drugs shows a promise to improve the safety profile of CAR T cells and can be universally applied regardless of the disease indications3.
T cell manufacturing challenges
Manufacturing CAR T cells for the clinical scale is challenging on its own. T cells need to be collected from patients in hospitals specialized in collecting apheresis departments. Further, cells are genetically modified to express CAR using specific bioreactors and expanded to very high numbers. Formulated patient dose then undergo a rigorous quality and sterility control before infusion into patients. All those steps generate enormous costs per patient product especially if patients require multiple manufacture runs for multiple injections. Autologous character of CAR T cell product, meaning each patient can only be infused with CARs produced from his/her own T cells further limits applicability of the therapy. Thus, various labs are working on developing ways to improve the anti-tumor potency of CAR products during manufacturing process either by adding drugs5 or changing the cell culture media composition6. Another viable option is allogeneic CAR therapy. This approach is based on using T cells from healthy universal donors instead of the patient’s. Allogeneic CAR Ts have the advantage of being ready for the infusion at the moment of diagnosis. Unfortunately, allogeneic CAR T cells will attack patient’s normal cells causing graft vs host disease (GVHD) and rejection of the transferred cells by the host immune system. Some of the strategies to overcome GVHD include augmenting immune suppression of the host using conventional chemotherapy prior product infusion, knocking down molecules involved in recognition of donor cells or injection of immunosuppressive antibodies which the targets are edited from the universal donor cells7. Early response rates with this approach are promising, but long-term safety and efficacy have not been demonstrated.
Since its first approval in 2017, the development of CAR T cells has been almost a decades-long journey that created a multibillion-dollar industry. Unfortunately, despite an amazing success, the high cost (EUR 275,000/USD 312,500) per patient, need for infrastructure, extremely trained medical and scientific staff is making immunotherapy out of reach for many. Globally, USA and China are leading in terms of CAR T cell immunotherapy trials and applications, with several CAR T cell therapies going already beyond cancer application. In Europe, countries like Poland and Romania remain behind larger European countries like Spain, which is the leader with an approval for a CAR T-cell therapy developed entirely in Europe. In Poland, CAR-T cells were administered to patients for the first time in November 2019 and by this opening doors for adoptive cell therapy in Poland8.
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6. Klysz, D., Carley, F., Meena, M., Lucille, S., Katherine, A.F., Stefanie, M., Bence, D., Katalin, S., Peng, X., Jing, H., et al. (2023). Inosine Induces Stemness Features in CAR T cells and Enhances Potency. bioRxiv, 2023.2004.2021.537859. 10.1101/2023.04.21.537859.
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Dorota Klysz, Ph.D.
