In the last chapter (click to review), we mentioned that chimeric antigen receptor (CAR)-T cell therapy has been extremely successful in treating relapsed/refractory B-cell malignancies expressing CD19, however, there are still many toxicities in clinical applications, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Toxic side effects are the major factors that limit the efficacy of CART therapy, additionally hindering the standard approach of enhancing anti-tumor effect by increasing dosage or improving effector activity.
So, how could cell therapy researchers break through this impasse? What are the strategies to improve CAR-T treatment effectiveness? To explore some of the strategies for mitigating CAR-T cell-related toxicity, we will start by discussing engineering of CAR-T cells, changing CAR molecule structure, reducing CAR immunogenicity, and "closing the switch".
In order to achieve an effective therapeutic response, the CAR-T cell antigen-binding domain must bind to its target site and reach the minimum threshold level for inducing CAR-T cell activation and cytokine secretion, at the meantime, should not exceed the maximum threshold level, otherwise, it will produce a toxic level of cytokines and immune activation. The degree of CAR-T cell activation and activation kinetics is influenced by many factors, including level of antigen expression on tumor cells, tumor burden, affinity of antigen-binding domain to its target site, and co-stimulatory domains of CAR. Therefore, it is necessary to optimize the modular structure of CAR to improve treatment efficacy and restrict toxicity.
Reducing affinity of CAR-T cell antigen binding domains is an important way to lower toxicity, which leads to increased demand for higher antigen density on tumor cells to achieve high-level activation, thereby avoiding impact on healthy tissues with relatively lower antigen content. Modifying the hinge and transmembrane region of activated CAR-T cells can also reduce toxicity by regulating cytokine secretion. CAR-T cell therapy is a complex system of multiple factors, and the optimal strategy needs further optimization of CAR-T cell structure and extensive clinical trials.
In addition, the co-stimulatory domain in CAR design provides another modifiable domain area that can be customized according to tumor type, tumor burden, antigen density, and target antigen-antigen binding domain. Specifically, CAR cells containing 4-1BB domain have lower toxicity risk, higher T cell endurance, and lower T cell expansion peak levels; while those containing CD28 demonstrate faster activation speed and higher intensity, but also faster exhaustion rate and insufficient persistence.
Regarding cytokine-related toxicity, recognition of CAR by the host immune system may play an important role. Therefore, human or humanized antibody fragments instead of mouse-derived ones can be used to reduce CAR immunogenicity. Additionally, CAR immunogenicity can be reduced by optimizing hinge/transmembrane domains, which may also improve CAR-T cell persistence.
Another potential approach to improve CAR-T cell toxicity is to implement "off-switch," safety switch, or suicide gene strategies. This could help selectively reduce the function of CAR-T cells in case of adverse events, by using a secondary inducer agent. Based on this concept, methods have been developed to control the function of CAR-T cells, such as inducible caspase-9 (iCas9), which has been shown to eliminate over 90% of CAR-T cells within 30 minutes in clinical trials.
The main limitation of suicide strategies or similar methods is that the use could abruptly halt treatment for rapidly progressing diseases. One potential solution is to use dasatinib, a tyrosine kinase inhibitor that could suppress T cell activation by inhibiting proximal TCR signaling. In preclinical models, dasatinib can rapidly and reversibly block CAR-T cell activation, and early administration of dasatinib following CAR-T cell infusion significantly reduced the mortality rate of other lethal CRS mice. This approach could temporarily suppress CAR-T cell function and salvage CAR-T cell therapy once the toxicity has subsided.
Despite the numerous challenges that currently face CAR-T cell therapy, such as toxic side effects, resistance, and concerns over drug safety, with the continuing optimization of CAR molecule design and the development of combination therapies, it is believed that the application of CAR-T cell therapy will ultimately reach a more efficient and safer level. With fine-tuning of CAR-T approaches, these cell therapies will quickly improve their potential benefits to many more cancer patients in the near future.
With years of research experience in the field of tumor immunology, Cyagen can provide comprehensive CRO support for researchers involved in CAR-T/-NK and other cell therapy studies; our services range from antibody development, CAR virus preparation, immune cell preparation and phenotypic detection, to construction of cell and animal models, in vitro/in vivo efficacy evaluation, etc. Please feel free to contact us to further discuss how we can support your research efforts.
 Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021 Apr 6;11(4):69. doi: 10.1038/s41408-021-00459-7. PMID: 33824268; PMCID: PMC8024391.