A major obstacle to cancer immunotherapy is overcoming tolerance to ‘self-antigens’. While the immune responses against foreign antigens are not susceptible to self-tolerance, those mounted against tumour-associated self-antigens are subject to suppression by central or peripheral tolerance mechanisms (1). In humans, T-cell receptors (TCRs) that recognize self-tumour antigens have a lower affinity for the major histocompatibility complex (MHC) peptide complexes compared to their virus-specific TCR counterparts, due to the thymic elimination of high-affinity TCRs. Thus, endogenous T-cell activity is inadequate for the control of cancer (1).
Tumours are frequently invaded by immune cells as part of the body’s response to malignant growth. Among these immune infiltrates, tumour-infiltrating lymphocytes (TILs) can be isolated from a patient’s tumour tissues, expanded ex-vivo to high numbers, and adopted into the patient to elicit a durable anti-tumour response (2). This has certain drawbacks: TILs need to be harvested during surgery, they may not be available in sufficient numbers for all cancers, and they expand poorly in many cases (3).
Using tumour-specific TCRs enables the targeting of both membrane and cytoplasmic proteins. Unfortunately, cancers can evade this endogenous recognition-and-response program by downregulating MHC proteins or other factors required for antigen processing and presentation and thus become invisible to the patrolling T cells. To overcome this, T-cell specificity can be directed to tumour-associated antigens (TAAs) in an MHC-independent manner. This can be achieved by employing chimeric antigen receptors (CARs) (2).
Evolution of the CAR Design
CARs combine the specificity of a high-affinity recognition domain with the cytolytic properties of T cells (2). First-generation CARs had a CD3ζ signalling domain that contained three immunoreceptor tyrosine-based activation motifs (ITAMs), which are important for signal transduction. Second-generation and third-generation CARs incorporated one or more costimulatory domains (costim), respectively (2). The fourth generation of CARs is based on second-generation CARs (containing 1–3 ITAMs) paired with a constitutively or inducibly expressed chemokine (e.g., IL-12). These T cells are also referred to as ‘T-cell redirected for universal cytokine-mediated killing’ (TRUCKs). The fifth, or ‘next generation’, is currently being explored; they are also based on the second generation of CARs, with the addition of intracellular domains of cytokine receptors (e.g., IL-2Rβ chain fragment) (4).
Currently, there are 5 FDA-approved CAR T-cell therapies only for Acute lymphoblastic leukaemia (ALL), B-cell lymphoma, Follicular lymphoma (FL), Mantle cell lymphoma, and Multiple myeloma (5). In total, over 500 clinical trials analysing CAR-T cells for the treatment of cancer are currently being conducted around the world of which the majority are being performed in East Asia, followed by the US, and then Europe (6). Most notably, a study was conducted in which two people with chronic lymphocytic leukaemia (CLL) underwent CAR T-cell therapy as part of a treatment trial and achieved a complete remission in 2010. A decade later, they were still in remission with the persistence of CD4+ CAR T cells (7).
Success in Haematological Cancers
The use of anti-CD19 CAR-T cells has demonstrated consistently high anti-tumour efficacy in children and adults affected by relapsed B-cell acute lymphoblastic leukaemia (B-ALL), chronic lymphocytic leukaemia, and B-cell non-Hodgkin lymphoma, with the percentage of complete remissions ranging from 70 to 94% in the different trials (8).
In particular, CAR-T cell therapy has been found to be most effective for the treatment of fatal relapsed or refractory B-ALL. The most suitable CAR is anti-CD19, an essential biomarker of B cell lineage showing higher expression in B-ALL, while anti-CD20 and immunoglobulin light chains are also potential targets (9).
In one clinical study, following conditioning therapy (cyclophosphamide), CD19 CAR-T cells were infused, and the CR (complete response) rate was 88%. Meanwhile, because CLL pathogenesis leads to early immune deficiency, the efficacy of CAR-T cell therapy is limited by difficulties in the expansion of T cells ex vivo in CLL patients and their proliferative response in vivo (9).
Two parallel phase I/II studies (ClinicalTrials.gov identifiers: NCT02690545 and NCT02917083) at two independent centres were carried out for patients with relapsed or refractory (r/r) Hodgkin’s lymphoma, where they were administered CD30 CAR-Ts after fludarabine-based lymphodepletion. CAR-Ts had a high rate of durable responses with an excellent safety profile, thus highlighting the feasibility of extending CAR-T cell therapies beyond B-cell malignancies (15).
Another use of r/r Hodkin’s lymphoma for post-transplant patients as a maintenance or consolidation strategy is currently under investigation. A total of 15 patients received CAR-T cells on this Phase I trial at a median of 22 days post-transplant, with the treatment being well tolerated (no grade 2 or higher cytokine release syndrome (CRS), no neurotoxicity), although hematologic toxicity was seen (grade 3/4 cytopenias in 40% of patients). The PFS (progression-free survival) at 1 year was 79%, with an OS (overall survival) of 100% (16).
Status in Solid Tumours
Contrary to haematologic tumours, the majority of treatment for solid tumours is unsuccessful due to insufficient and atypical molecular targets for CAR-T cells to attack and control the microenvironment of tumour (9).
A key factor responsible for the poor specificity and efficacy of CAR-T against malignant epithelial cells is the lack of specific, targetable antigens. An ideal target is the signalling active splice variant of EGFR (EGFRvIII) because it is specifically expressed in glioma cells and indispensable for cell survival. However, encouraging results from early-phase trials have only been obtained in neuroblastoma patients treated with anti-GD2 CAR-T cells and in ErbB2-positive sarcomas treatment (10).
The ACT in melanoma requires more cells, more profound lymphodepletion, and the use of IL-2 support to obtain optimal results. Furthermore, to exploit their cytotoxic function, CAR-T lymphocytes need to overcome the limitations imposed by the physical and functional barriers preserving epithelial and mesenchymal compartments. Thus, in perspective, T cell extravasation, tumour homing, and persistence in a hostile microenvironment are goals to be accomplished to increase the chances of treating solid tumours with CAR-T cell immunotherapy (10).
Scope in Pancreatic Cancer
A study by Watanabe et al. (11) found that although Meso-CAR T cells (Mesothelin-redirected chimeric antigen receptor T cells) failed to work effectively as monotherapy, potentially novel combination therapy of oncolytic adenovirus expressing TNF-α and IL-2 with Meso-CAR T cells in the treatment of pancreatic ductal adenocarcinoma by modulating the immunosuppressive tumour microenvironment and inducing CAR-dependent and CAR-independent host immunities is a promising approach.
What are the Toxicities associated with CAR-T Cell Therapy?
Recent clinical trials have reported CAR-T-associated toxicities such as cytokine-release syndrome (CRS), neurotoxicity, off-tumour effects, and acute respiratory distress syndrome, all of which are potentially fatal (12,13).
Neurotoxicity caused by cerebral edema has led to fatal outcomes in a few clinical trials. In addition, reversible symptoms of neurotoxicity, including confusion, delirium, expressive aphasia, encephalopathy, and seizures, were reported in several other studies. In some patients, CD19-CAR T cells have been found in cerebrospinal fluid (14).
Tumour lysis syndrome (TLS) has been reported as well. Dai et al. reported that one ALL patient died from acute TLS 12 hours after receiving a second CD19-CAR T cell infusion. Reducing tumour size before treatment and/or controlling the extent of tumour lysis by adapting the amount of infused CAR T cells can be applied to control TLS (14).
On-target, off-tumour recognition has become a relevant concern since many targeted tumour antigens are also expressed in normal tissue. Among these, B-cell aplasia is a common adverse event in CAR T cell trials targeting B-cell malignancies (14).
Therapy Failure
A mechanism of CAR T-cell failure in paediatric acute lymphoblastic leukaemia is the loss of the CD19 antigen or epitope, despite otherwise adequate persistence of transferred cells. Antigen-negative tumour escape variants likely emerge through the selection of sub-clonal leukaemia cells that possess a survival advantage in the face of a powerful antigen-specific assault mediated by CAR T cells (1).
An emerging threat to CAR-T immunotherapy is the antigen escape that makes CAR-T cells inefficient against cancer cells. CAR-T cell tumour sculpting exerts a selective pressure involving the selection of antigen-negative cells over time. This phenomenon has been described in many clinical studies, including a glioblastoma trial with anti-ErbB2-CAR (10).
Conclusion
While CAR-T cells have shown successful clinical outcomes in haematological malignancies such as relapsing and refractory B-ALL, modest progress has been made in solid tumours. While their usage has been limited due to their adverse effects and toxicity, these results illustrate the scope of immunotherapy for cancer treatments. The success of CAR-T cells has also paved the way for CAR-NK cells, which have shown promising results in clinical trials and can potentially serve as alternatives for cancer treatment. With emerging technology and research, the use of CAR cells for wider therapeutic applications holds great promise for the future.
References
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