Innovation in medical research offers hope to millions of patients and drives the passion of dedicated researchers. The approval of CAR T-cell (Chimeric Antigen Receptor T-cell) therapy for certain cancers was a thrilling milestone for scientists globally. It marked the beginning of efforts to enhance what had already been achieved, paving the way for innovative treatments.
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Indeed, CAR T-cell therapy has provided a cure for millions of patients suffering from cancer, particularly blood cancers such as leukaemia and lymphoma. This therapy utilises genetically modified T-cells, a type of immune cell, to recognise and eliminate cancer cells. Currently, first- and second-generation CAR T-cell therapies are available to patients, and have shown remarkable success in clinical trials and received FDA approval.
However, limitations persist, such as treatment-associated toxicities, relapse, and poor persistence of CAR T-cells in some patients. The development of third- and fourth-generation CAR T-cell therapies offers the promise of overcoming these challenges and providing long-term, safer, and more effective treatments.
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The first-generation CAR T-cells featured a simple structure comprising an extracellular domain for antigen recognition, a hinge, a trans-membrane domain, and an intracellular signalling domain. These CARs exhibited limited clinical success, primarily due to inadequate signalling for T-cell activation and expansion, resulting in poor persistence and function of the engineered T-cells.
Second-generation CAR T-cells improved on this design by adding co-stimulatory domains like CD28 or 4-1BB. These signals boost the activation, expansion, and long-term persistence of CAR T-cells in the body. Second-generation CAR T-cell therapies have demonstrated significant success in treating certain haematologic malignancies, leading to FDA approval for therapies targeting CD19-positive B-cell malignancies.
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Despite their success, second-generation CAR T-cells have limitations. They can cause severe side effects like cytokine release syndrome (CRS) and neuro-toxicity when the T-cells become over-active and release too many inflammatory signals. Additionally, tumour cells can stop expressing the targeted antigen, leading to cancer relapse.
One key disadvantage of mono-specific CAR-T cells, as seen in first- and second-generation CARs, is their limited ability to prevent relapse if the cancer returns through different antigens. For example, patients treated with CD19-targeted CAR-T cells may experience relapse if the tumour cells express alternate antigens such as CD20 or CD22. In these cases, the original CAR-T cells are ineffective against the new antigen.
This risk can be significantly reduced by using bi-specific CAR-T cells. Bi-specific CARs target two antigens simultaneously, such as CD19/CD20, CD20/CD22, or CD19/CD22. This dual targeting reduces the likelihood of antigen escape, where the cancer evades immune detection by down-regulating a single antigen. Third-generation CARs, which incorporate these bi-specific designs, not only lower the risk of antigen escape-mediated relapse but also address the poor persistence seen in second-generation CARs, improving long-term efficacy.
Third-generation CAR T-cells build upon their predecessors by incorporating multiple co-stimulatory domains into their structure. These designs typically combine CD28, 4-1BB, or other signalling molecules like OX40 or ICOS. The inclusion of multiple co-stimulatory domains helps to activate T-cells more effectively, improving their persistence, expansion, and ability to destroy cancer cells over time.
This generation of CAR T-cells is better at staying active and fighting cancer for longer, while also reducing the chances of tumours escaping. Improved signalling helps CAR T-cells survive and work effectively, even in tough conditions like solid tumours. They are also being designed to release fewer inflammatory signals, which could help lower the risk of severe side effects like CRS and neuro-toxicity.
Fourth-generation CAR T-cells are even more efficacious. Often referred to as T-cells Redirected for Universal Cytokine Killing (TRUCKs), these cells incorporate genetic elements that enable them to produce pro-inflammatory cytokines (such as IL-12) directly at the tumour site, further amplifying their anti-tumour activity and overcoming the suppressive tumour micro-environment.
By producing cytokines like IL-12 in the vicinity of the tumour, TRUCKs create a more hostile environment for the tumour cells while simultaneously recruiting and activating other immune cells, such as macrophages and natural killer cells. This transformation of the tumour micro-environment from immuno-suppressive to immuno-stimulatory facilitates more effective tumour eradication.
Besides, fourth-generation CAR T-cells allow for more refined control over T-cell activation. In some designs, the production of cytokines is triggered only when the CAR T-cell engages with the tumour antigen, preventing systemic cytokine release and reducing the risk of toxic side effects like CRS.
Moreover, advances in gene editing and fourth-generation CAR T-cell design allow for more precise targeting of tumour antigens. Some strategies target multiple antigens simultaneously (dual or bi-specific CARs), reducing the risk of off-tumour toxicity, where CAR T-cells mistakenly attack healthy tissues that share antigens with tumour cells.
Tumours are often highly heterogeneous, meaning that different tumour cells can express varying sets of antigens. Fourth-generation CAR T-cells tackle this by either targeting multiple antigens or releasing cytokines that boost the immune response in the tumour area, helping destroy a wider range of tumour cells.
While CAR T-cell therapies are highly promising, their long-term safety has been a concern. The third and fourth generations of CAR T-cell therapies incorporate several strategies to minimise the risk of toxicities and improve overall safety.
First, some advanced CAR T-cell designs include safety switches or suicide genes that allow doctors to rapidly eliminate the CAR T-cells in the event of severe side effects. This approach helps manage life-threatening toxicities, such as cytokine release syndrome (CRS), by enabling the controlled shutdown of the engineered T-cells.
Second, some CAR T-cells are designed with drug-inducible systems that can turn the CAR T-cells "on" or "off" using a small-molecule drug. This allows clinicians to fine-tune the activity of the CAR T-cells, reducing the risk of runaway immune responses.
To improve long-term persistence and reduce the risk of immune rejection, researchers are exploring ways to diminish the immuno-genicity of CAR T-cells. This involves making the CAR T-cells less recognisable to the patient’s immune system, allowing them to persist for longer periods without being attacked by the body's defences.
New advances in gene-editing technologies, such as CRISPR and TALENs, are being used to fine-tune CAR T-cell genomes, enhancing their efficacy while minimizing the risk of off-target effects or unintended consequences. For example, gene editing can disrupt immune checkpoint pathways (such as PD-1) that tumours use to deactivate T-cells, making CAR T-cells more resistant to suppression.
The third and fourth generations of CAR T-cell therapy represent a significant leap forward in the fight against cancer. As researchers continue to refine these therapies, there is hope that CAR T-cell treatments will become not only more effective but also safer and more widely applicable, particularly for patients with solid tumours and those at risk for treatment-related toxicities. The future of CAR T-cell therapy lies in combining innovation with precision, ensuring that these powerful treatments can be delivered safely and effectively to the patients who need them most.
The author is a Paediatric Haematologist and Oncologist with an active interest in Cell and Gene therapy.