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Why is the durability of CAR-T cell therapy limited?
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Why is the durability of CAR-T cell therapy limited? What are the improvement strategies?
Limited durability is still a major obstacle to the development of CAR-T therapy, which is a problem encountered from the beginning of the cell manufacturing step.
Including CAR design and in vitro operations and culture conditions, which may play a key role.
1. Factors affecting the persistence of CAR-T cells
1.1 CAR design
The type of extracellular CAR domain, the type of spacer that connects the single-chain antibody and the transmembrane domain, and the costimulatory domain that constitute the CAR have been shown to have a profound impact on T cell function and persistence.
Most CARs used in clinical trials are derived from mice, which may cause an immune response mediated by body fluids and CD8+ T cells, leading to immune rejection.
Re-infusion of CAR-T cells containing mouse components into patients with CD19+ relapse after the first treatment is basically ineffective.
In addition, the mouse CAR receptor clusters on the cell surface can generate a tonic signal, leading to depletion of T cells.
In 2016, CAR-T with humanized scFv entered clinical trials. The humanized CAR showed similar cytotoxic activity to mouse CAR, but enhanced durability due to lower immunogenicity.
The affinity of CAR affects the tonic signal, and too strong affinity for target cells may lead to T cell depletion.
A study developed a CAT-CAR ( CD19 single-chain antibody ) whose affinity to CD19 is 40 times lower than that of traditional CARs.
This structure was tested in patients with relapsed or refractory BLL in children, and clinical studies found that 11 of 14 patients with increased CAR-T cell expansion showed persistence.
In addition, the costimulatory domain also plays a key role in the persistence and effectiveness of CAR-T cells.
The costimulatory molecules include CD28, ICOS, CD27, 4-1BB, OX40 and CD40L. Several studies have found that CAR structures equipped with a 4-1BB domain instead of a CD28 domain have enhanced T cell persistence.
Other studies reported that 4-1BB contains a CAR-T cells exhibit enhanced memory T cells ( T the EM ) phenotype, which may delay the CAR-T cell depletion.
ICOS belongs to the family of CD28 costimulatory molecules.
Studies have shown that the combination of ICOS and 4-1BB costimulatory domains in the CAR structure significantly increases the persistence of T cells.
Despite conflicting evidence, it is recognized that CD28-CAR is associated with high effector function and limited T cell persistence, while 4-1BB-CAR and ICOS-CAR are less effective but have a longer duration.
Therefore, these observations indicate that the structure of the costimulatory domain is an important factor affecting the persistence of CAR-T cells.
1.2 Extracorporeal manipulation and lymphatic clearance
The process of making T cells requires obtaining a sufficient number of healthy T cells from the patient.
However, after chemotherapy treatment, especially those containing clofarabine or doxorubicin, the resulting lymphopenia will make the final CAR-T cell quality unsatisfactory, and the type of T cell used for infusion will seriously affect the effect of the treatment.
In fact, by normalizing the CD4/CD8 ratio or using naive or memory cell subpopulations, better persistence is obtained in preclinical models.
Several studies have shown that the use of T cell populations enriched with early lineage cells can better expand and improve the durability and efficacy of CAR-T cell therapy.
In addition, multiple studies have shown that adding antioxidants, such as N-acetylcysteine, to cell culture during the manufacturing process can also inhibit effector differentiation and promote TSCM cell expansion.
A few days before the CAR T cell infusion, the patient received a lymphatic clearance chemotherapy regimen, the most common being cyclophosphamide ( cy ), fludarabine ( flu ) and bendamustine ( ben ).
Lymphatic clearance therapy eradicates regulatory T cells ( Treg ) and other immunosuppressive cells, increases the expansion of CAR-T cells and prolongs their persistence.
As observed in clinical trials of B-ALL and B-NHL in adults, adequate lymphatic clearance is critical to the success of treatment and may prevent CAR rejection.
1.3 T cell exhaustion
In TME, the presence of immunosuppressive cytokines produced by myeloid-derived suppressor cells ( MDSC ), cancer-associated fibroblasts ( CAF ) and tumor cells can cause T cell depletion.
Chronic exposure to antigens can also lead to T cell failure. If antigen stimulation persists, T cells will undergo a series of epigenetic, metabolic and transcriptional changes, showing signs of exhaustion.
This process occurs in a progressive manner, IL-2 and TNF-α are lost in the early stage, and the secretion of IFN-γ and chemokines decreases in the later stage of exhaustion.
Although the ability of high proliferation is also lost in the early stage, depleted T cells can still proliferate to a limited extent when stimulated in the body.
Depleted cells also showed high expression of inhibitory receptors, such as PD-1, TIM-3, LAG-3, CD160, BTLA, CTLA-4 and TIGIT.
2. Strategies to improve the persistence and effectiveness of CAR-T cells
2.1 Expression of cytokines and their receptors
The fourth-generation CAR T cells have recently been developed to resist the immunosuppressive environment in TME and at the same time overcome immune exhaustion.
TRUMKS is designed to combine the cytotoxic function of CAR-T cells with the in situ delivery of immunomodulatory cytokines.
Under the action of the induction system, after the CAR binds to the antigen, cytokines are synthesized and act in an autocrine manner to increase the survival and expansion of T cells.
Cytokines can also play a role in a paracrine manner, regulate the surrounding environment, and interfere with the immunosuppressive cytokines present in TME.
A series of cytokines including IL-12, IL-7, IL-15, IL-18, IL-21 and IL-23 are currently being studied and have entered the early stage of clinical trials.
The I L-12 is a pro-inflammatory cytokine, can induce Th1CD4 + T cell responses, promote clonal expansion of CD8 + and persistence.
It is also responsible for regulating the cytotoxic activity of CTL and natural killer ( NK ) cells, reactivating incompetent tumor-infiltrating lymphocytes, recruiting NK cells, and inhibiting Treg.
Preclinical studies on CD19-CAR-T cells that structurally express IL-12 have shown that it enhances tumor-killing effects and immune memory against cancer antigens.
However, the potentially lethal toxicity associated with IL-12 makes it necessary to develop an induction system that restricts IL-12 secretion only when CAR is activated. Some clinical studies ( NCT02498912, NCT03932565 and NCT03542799 ) are ongoing and recruiting.
IL-15 is an stimuli CD8 + T cell and NK cell activation, cytokine proliferation and cytotoxic activity of the cells.
IL-18 increases Th1 cell cytokine production, while inhibiting IL-10 synthesis. Both IL-15 and IL-18 are immune response enhancers and have been tested on CAR-T cells. Compared with traditional CAR-T cells, CAR-T cells that secrete IL-18 and IL-15 show enhanced expandability and persistence in tumor-bearing mice, as well as enhanced tumor cells in vitro and in vivo toxicity.
A number of clinical trials are currently being recruited to use engineered T cells and NK cells to test the effects of CARs secreted by IL-15 and IL-18 in solid tumors and hematomas.
Recent studies have shown that it is possible to co-express multiple cytokines in the same CAR-T cell.
NCT04833504 is a recently completed clinical trial in which CD19+CART cells expressing IL-7 and CCL19 were detected in patients with relapsed or refractory B-cell lymphoma, but the results have not been reported.
Two other clinical trials are currently being recruited to detect the combination of CAR-T cells expressing IL-7 with the secretion of PD-1 blockers or other cytokines.
2.2 Combined checkpoint blocking therapy
Strategies to reduce depletion by suppressing checkpoint signals include knocking out co-inhibitory molecules through shRNA expression vectors or CRISPR/Cas9.
Many studies have reported that blocking checkpoint inhibition can restore cytokine production and promote CAR-T cell survival.
In addition, blocking multiple immune checkpoints at the same time, such as PD-1, TIM-3 and LAG-3, can synergistically increase the effector function of CAR-T cells.
Combining CAR-T cells with immune checkpoint blocking therapy may be an effective strategy to enhance anti-tumor activity, persistence and memory cell formation.
In glioblastoma and breast cancer cell lines, anti-PD-1 antibodies enhanced the anti-tumor activity of anti-HER2 CAR-T cells.
However, some clinical trials have reported negative results after using PD-1 inhibitors and anti-GD2 CAR-T cells to treat patients with neuroblastoma.
In addition, some studies have adopted other strategies to block PD-1, such as gene editing to make CAR-T cells secrete PD-1 blocking antibodies or down-regulate PD-1.
2.3 Use stem T cells
Effector T cells were initially considered to be the best product of ACT because of their ability to kill tumor cells.
However, their durability is limited, and their expansion capacity is poor, and they are prone to exhaustion.
T SCM is an ideal candidate for ACT due to its long lifespan, strong self-renewal ability and differentiation ability in different T cell populations.
Clinical studies have shown that the infusion of phenotypic and functional T SCM -like CAR-T cells ( CD62L+, CD28+ and CD27+ ) can produce good results.
For example, patients with CLL and multiple myeloma treated with anti-CD-19 CAR-T cells showed good responses related to the CD27 + CD45RO − CD8 cell population.
In addition, studies in mice have shown that infusion of CD62L+-rich T cell populations can increase expansion and persistence, leading to durable tumor regression.
Although CAR-T cell therapy has made significant progress in the past decade, the limited persistence of CAR-T cells in patients remains a challenge, mainly due to T cell depletion.
By designing CAR structure, changing production conditions, or introducing new treatment methods, we may create engineered T cells that are resistant to exhaustion, thereby further expanding the clinical application of CAR-T cells.
Although the results so far are still limited, the possibilities are endless, and a breakthrough may be imminent.
1.Improving CAR T-Cell Persistence. Int J MolSci. 2021 Oct; 22(19): 10828.
Why is the durability of CAR-T cell therapy limited?
(source:internet, reference only)