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Cytokine release syndrome and related neurotoxicity of CAR-T cell therapy
Cytokine release syndrome and related neurotoxicity of CAR-T cell therapy. Adoptively transferred tumor antigen-specific T cells have been genetically engineered to express chimeric antigen receptors (CAR) , and have achieved long-lasting clinical responses in certain types of cancer patients, especially those that express CD19 and recurrent B-cell malignancies.
CAR is a synthetic antigen recognition receptor, including antibody-derived single-chain variable fragments (scFv), hinge and transmembrane domains, and intracellular signaling domains.
The intracellular signal domain, usually the CD3ζ signal domain plus the costimulatory signal domain, such as the signal domain from CD28  and 4-1BB (also known as TNFRSF9) and the cell phenotype affected by engineered T cells.
Once the antigen is expressed Target cell activation , CAR T cell specific cytokine secretion characteristics and in vivo proliferation ability.
Despite their clinical success, the use of CAR T cells can lead to significant toxicities, which are directly related to the induction of strong immune response. The two main toxicities produced by CAR T cells were not found in early mouse models, but cytokine release syndrome (CRS) and immune effector cell-related neurotoxicity syndrome (ICANS; commonly referred to as Neurotoxicity). CRS usually starts with fever and systemic symptoms, such as chills, malaise, and anorexia .
Fever can be high and last for several days. In severe cases, CRS manifests as other features of systemic inflammation, including hypotension, hypoxia, and/or organ dysfunction. Organ dysfunction may be secondary to hypotension or hypoxia, but it may also be caused by the direct influence of cytokine release.
Dysfunctions of all major organ systems are observed in CRS patients, including the heart, lung, liver, kidney, and gastrointestinal system. However, if the symptoms and signs of CRS are recognized and treated in time, this organ dysfunction can be prevented or reversed in most patients [9-15].
ICANS usually manifests as toxic encephalopathy, and begins with difficulty in finding words, confusion, language impairment, aphasia, impaired fine motor skills, and lethargy [14,16,17]. In more severe cases, seizures, motor weakness, cerebral edema, and coma have been noted. Most patients with clinical features of ICANS have had CRS. Therefore, CRS can be regarded as the “starting event” or auxiliary factor of ICANS. ICANS usually occurs after the symptoms of CRS subsided (Figure 1), but occasionally concurrent manifestations of CRS and ICANS occur [1,18]. Similarly for CRS, ICANS is reversible in most patients without permanent neurological deficits [10,11,14-17].
Figure 1 | Schematic diagram showing the relative time scale of the occurrence and duration of CRS and ICAnS.
Pathophysiology of CRS
The pathophysiology of CRS can be divided into five main stages.
The first stage involves the infusion of CAR T cells into the patient’s body and after the CAR-mediated antigen expression target cells are recognized, they are transported to the tumor site.
In the second stage, CAR T cells proliferate at the tumor site, and the activated CAR T cells and cellular components of the tumor microenvironment produce cytokines in situ, activate “bystander” endogenous immune cells, and kill tumor cells directly and indirectly , And the onset of CRS.
In the third stage, the level of cytokines in the peripheral blood increases and the CAR T cell population expands, which is related to the systemic inflammatory response. This can lead to endothelial damage and vascular leakage in multiple tissues and organs, and its related effects, including hypoxia, hypotension, and/or organ damage.
The proliferation of cytokines and CAR T cells, endogenous T cells and peripherally activated monocytes enter the cerebrospinal fluid (CSF) and central nervous system (CNS) in the fourth stage, including the destruction of the blood-brain barrier (BBB), and ICANS s inception.
In the fifth stage, the activation of T cells after tumor eradication induces cell death, resulting in a decrease in serum cytokine levels and a decrease in systemic inflammation, the end of CRS and/or ICANS symptoms, and the persistence of CAR T cells for long-term memory. The relative time of occurrence and duration of CRS and ICANS is shown in Figure 1 .
Unexpected CRS before clinical
The study of CD19 CAR T cells was only discovered after the start of phase I clinical trials of CAR T cells [4,7]. Although the resulting fever and hypotension in patients are directly related to elevated serum cytokine levels, the underlying mechanism for this sporadic outcome (more common in patients with high tumor burden [4,7,21]) is not yet at first clear. It is now understood that almost all patients receiving CD19 CAR T cell therapy will have a certain degree of CRS.
In registered trials, up to one-third of patients with B-cell acute lymphoblastic leukemia developed severe CRS and up to one-half of patients developed ICANS , although the true incidence of CRS and ICANS may be low Here. Empirical testing of different blocking antibodies quickly determined that IL-6 is the key mediator of CRS[4,7], and tocilizumab (a monoclonal that blocks IL-6 receptor (IL-6R) signaling) Antibody) has become the main component of CRS management [3,23]. Since activated T cells can produce IL-6, it was initially assumed that CAR T cells themselves are the main source, although some observations indicate that there may be other contributors [3,24]. However, follow-up studies discussed below have identified macrophages and monocyte lineage cells as sources of IL-6.
It is worth noting that although there seem to be significant differences in some studies between the activation kinetics and cytokine production capacity of CAR T cells including CD28 or 4-1BB signal domains [4,7,10,21, 25,26], but the CRS associated with any type of CAR is highly similar [12,27,28]. Although CRS induced by CD28-based CAR T cells tends to develop earlier than CRS induced by 4-1BB-based CAR T cells, the cytokine profile observed in patient serum is at the peak levels of cytokines and chemokines There is almost no difference in this aspect, which is consistent with a common pathophysiological mechanism. The levels of IL-6, IL-8, IL-1 receptor antagonist (IL-1Ra), CC-chemokine ligand 2 (CCL2) and CCL3 (mainly not T cell-derived products) usually increase [7 ,17,25,29], which indicates that there may be a common mechanism of CRS, which is beyond the CAR T cell itself and involves the host cell.
The schematic diagram of CRS pathophysiology is shown in Figure 2 .
Figure 2 | Working model of the pathophysiological mechanism of CRS.
Cell interactions and molecular mediators. The two mouse models finally showed that CRS is caused by a multicellular network involving CAR T cells and host cells, in which macrophages and monocyte lineage cells are involved. A study showed that in a CD19+ lymphoma xenograft model, several endogenous cell populations at the tumor site, including dendritic cells, monocytes, and macrophages, produce IL-6, and the number of macrophages is far More than other cell types . In this heterogeneous environment, the level of mouse IL-6 greatly exceeds the level of human IL-6 produced by CAR T cells. The production of IL-6 is not induced at the remote tumor-free sites, which supports the view that CRS originates locally but produces systemic pathology.
Another study showed in humanized NSG mice using patient-derived leukemia cell lines [33,34] that clodronate or CAR T cell-mediated targeting was used prior to the administration of therapeutic CAR T cells Depleting phagocytes will eliminate IL-6 production and apparent CRS. Single-cell RNA sequencing data of white blood cells isolated during CRS confirmed that cells of the monocyte lineage are the origin of IL-6. In these two models, based on clinical experience, IL-6 blockade greatly reduced the toxicity associated with CRS [29,33]. These two studies further show that IL-1 is a monocyte and macrophage-derived cytokine and an effective driver of CRS-related toxicity.
Potential triggers for the recruitment or activation of macrophages during CRS are emerging. CAR T cells themselves must be activated to induce bone marrow cells to produce cytokines , which is consistent with clinical observations that CRS is mild or absent in patients who do not respond to CAR T cell therapy [4,10,11,28, 35]. It is unclear whether a contact-dependent interaction between CAR T cells and host bone marrow cells is required. Although the CD40-CD40L interaction is not necessary to trigger CRS, it will aggravate the activation of macrophages, thereby increasing the production of IL-6 and aggravating CRS . It remains to be determined which other contact-dependent mechanisms (if any) may be critical to the development or amplification of CRS. T cell surface molecules, such as the integrin LFA1 and the costimulatory molecule CD28, interact with cognate receptors strongly expressed by bone marrow cells (ICAM1 and CD80 or CD86, respectively) [36, 37]. The CD28-CD80/CD86 axis may be of particular interest in the context of CRS because it reports the potential role of bidirectional signaling and induction of IL-6 production by bone marrow cells . Blocking these interactions may reduce the severity of CRS [38,39].
Cytokine mediators: IL-6 and IL-1. IL-6 is a pleiotropic cytokine with pro-inflammatory and anti-inflammatory effects. IL-6 is mainly composed of macrophages and other cells of the myeloid lineage, which can act in an autocrine manner and combine with other inflammatory signals to promote the maturation and activation of macrophages. IL-6R is mainly expressed by immune cells (including microglia) and liver cells [40,41]. IL-6 can signal and promote inflammation through cis and trans signals. When soluble IL-6–IL-6R forms a complex with the ubiquitously expressed membrane-bound gp1 , trans-signaling has extensive effects outside the immune system. For example, in addition to controlling the acute phase response, IL-6 is also involved in the regulation of hyperthermia, glucose metabolism, neuroendocrine system and appetite [41,42]. However, the exact role of IL-6 in CRS is still elusive. In many patients, IL-6 blockade can lead to the reversal of most symptoms and overall cytokine downregulation [4,7,10,11]. In preclinical models, IL-6 has also been found to mediate the mortality of CRS [29,33], and promote macrophage activation by inducing nitric oxide synthase (iNOS) and producing nitric oxide (NO)  .
IL-1 is also a pleiotropic cytokine with multiple functions. It is mainly produced by monocytes and macrophages. The IL-1 receptor (IL-1R1) is ubiquitously expressed and is responsible for transducing pro-inflammatory signals. IL-1 can induce tissues to produce downstream pro-inflammatory cytokines, such as IL-6, and a series of chemokines for maturation and recruitment of tissue immune cells. In addition, IL-1 can activate the production of pro-inflammatory lipid mediators (such as prostaglandin E 2, which can promote edema 44), induce acute-phase proteins and send signals to the hypothalamus to induce fever, as well as the pituitary and adrenal glands, which affect the circulation The system has direct and indirect effects 43,45,46.
In two independent studies, IL-1 was found to be a key mediator of CRS (Figure 2). In a humanized xenograft model, blocking IL-1 induced signaling with the IL-1R antagonist anakinra protects mice from weight loss and fever, and prevents CRS-related death33. In the SCiD-beige xenograft model, anakinra protects mice from CRS-related mortality and reduces iNOS macrophage expression. CAR T cells designed to overexpress IL-1Ra also provide protection from the lethal effects of CRS29. Importantly, in both mouse models, when IL-1 inhibition was initiated prophylactically, the anti-tumor efficacy of CAR T cells remained unchanged while inhibiting CRS. The timing of anti-IL-1 therapy may be critical to its effectiveness. Interestingly, in the humanized NSG mouse model, IL-6 blockade did not improve the infiltration of macrophages into the brain compared with IL-1R blockade.
Based on these animal studies, several clinical trials have been initiated to evaluate the administration of anakinra in the context of CRS and neurotoxicity prevention (ClinicalTrials.gov NCT04148430, NCT04205838, NCT03430011, NCT04432506, and NCT04359784). In view of the clinical availability of IL-1β blocking drugs (such as the monoclonal antibody canakinumab), future preclinical studies should clarify the specific role of IL-1α and IL-1β in the CRS cascade, because these cytokines are in pro-Inflammatory function, but it also differs due to differences in expression and disease background45.
Damage-related molecular patterns and other soluble media. CAR T cells tend to induce cell death through the inflammatory process of pyrolysis rather than apoptosis, leading to the release of damage-related molecular patterns, such as ATP and HMGB1 (Ref. 47). The release of damage-related molecular patterns by tumor cells can lead to the activation of macrophages in vitro to produce IL-6 and IL-1, and in vivo inhibition of pyrolysis in the SCID-beige xenograft model can reduce CRS-related mortality (but may also affect CAR The cytolytic activity of T cells) .
Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced by a variety of cell types, including activated CAR T cells, and its blocking effect can eliminate the production of IL-6 and other cytokines by monocytes in vitro. However, in the xenograft model , IL-6 production is robust, and CRS is ultimately fatal, in which human T cell-derived GM-CSF does not cross-react with homologous mouse recipients . Nevertheless, GM-CSF may still be a contributing factor to CRS, rather than a necessary condition for it. In the human PBMC xenograft model, blockade of mouse and human GM-CSF reduced IL-6 production by 50 (Figure 2, 3).
Figure 3 | Schematic diagram of CRS current and potential therapeutic interventions.
Interferon-γ (IFNγ) is produced in large quantities by activated T cells, which can be induced by iNOS to promote the maturation of macrophages and enhance NO production [53,54]. In addition, IFNγ can promote the permeability of other tissues, including BBB, by loosening tight junctions [49-51,55-58].
The adrenal system is also involved in the development and maintenance of CRS, because catecholamine epinephrine and norepinephrine directly affect the activation of CAR T cells and subsequent cytokine release. Administration of atrial natriuretic peptide (ANP), electrolytes and extracellular fluid volume regulators, and cytokine secretion inhibitors  or inhibition of catecholamine synthesis by methotrexate can reduce the cytokines produced by CAR T cells in vitro and in vivo, and reduce Mortality xenograft mouse model  (Figure 2, 3). In a syngeneic mouse model of B-cell acute lymphoblastic leukemia, lower doses of methotrexate will not compromise the anti-tumor efficacy. Interestingly, according to reports, immune cells are not only activated by catecholamines, but also naturally produce catecholamines after activation, thus creating a positive feedback loop . However, it is known that the imbalance of ANP levels promotes edema, lower blood pressure, and electrolyte imbalance, all of which are common in CRS pathology. Therefore, the safety and feasibility of blocking catecholamine receptors, especially in the onset of CRS, will cause some concerns.
Pathophysiology of ICANS
Although the clinical features of ICANS are easy to identify, their pathophysiology is still poorly understood. In addition to the effects of various pro-inflammatory cytokines, recent animal models also involve endothelial cell activation and BBB destruction, leading to direct neuronal cell damage. However, these CAR T cell-mediated neurotoxicity models are limited by the lack of human cytokines and hematopoietic cells, and the accompanying xenograft versus host disease [33,62-64]. Nonetheless, recent studies on mice and non-human primates have produced important insights. These insights, together with clinical trial data from patients who developed ICANS after CAR T cell infusion, have improved our understanding of the pathophysiology of ICANS. Understand (Figure 4).
Figure 4 | Pathophysiology of ICANS.
Vascular permeability, endothelial destruction and glial cell damage. The levels of protein, CD4+ T cells, CD8+ T cells, and CAR T cells in the cerebrospinal fluid of ICANS patients are elevated, which indicates that the integrity of the BBB is lost. Clinical studies have shown that there is a correlation between the number of CAR T cells and the level of cytokines in the cerebrospinal fluid and the severity of ICANS [17,21,65].
Some of the biochemical characteristics of severe CRS and ICANS observed in patients, such as hypofibrinogenemia and increased fibrin degradation products, are the same as the characteristics of diffuse intravascular coagulation and endothelial cell destruction, which are usually seen in sepsis And critical illnesses related to the increase. Vascular permeability . In fact, there is evidence that patients with severe ICANS have vascular leakage. In a single-center study of 133 patients receiving CD19 CAR T cell therapy, changes in the angiogenin (ANG)-TIE2 axis have been described, which regulates endothelial cell activation in health .
ANG1 is constitutively produced by platelets and perivascular cells. When it binds to its endothelial receptor TIE2, it can stabilize endothelial cells. After inflammatory cytokines activate endothelial cells, ANG2 is released from the endothelial Weibel-Palade bodies and replaces ANG1, thereby further increasing endothelial cell activation and microvascular permeability. Consistent with this mechanism, compared with patients with less neurotoxicity, the ratio of serum ANG2 to ANG1 in patients with severe ICANS was statistically significantly increased, and the combination of von Willebrand factor (vWF) and CXC chemokine Body 8 (CXCL8) has a higher concentration, all of which are produced by platelets and perivascular cells.
In addition, patients with an increased serum ANG2 to ANG1 ratio before lymphocyte clearance and CAR T cell administration have a higher risk of ICANS. Further data indicate that high molecular weight vWF multimers are isolated in patients with severe ICANS, leading to coagulation dysfunction. However, the factors that control the baseline and disturbance levels of ANG2 and ANG1 are still elusive.
In addition to the destruction of the BBB and the increase in vascular permeability, glial cell damage has also been reported in children and young adults with ICANS after CD19CAR T cell therapy . In a cohort of 43 patients, GFAP and S100b levels in the cerebrospinal fluid of patients with acute neurotoxicity were found to increase significantly. GFAP is a well-validated marker of astrocyte damage, regardless of the cause , while S100b in cerebrospinal fluid is a marker of astrocyte activation . In the same study, it was also found that increased levels of IL-6, IL-10, IFNγ and granzyme B in cerebrospinal fluid were related to neurotoxicity.
Cytokines and their cell sources. In the humanized NSG mouse model, infusion of human CD19CAR T cells with CD28 or 4-1BB costimulatory domains resulted in B cell hypoplasia, CRS, and neurotoxicity. These mice developed delayed lethal ICANS after initial CRS, and this pattern is also observed in some patients (usually <1%) [14,72]. However, in this model, blocking signal transduction via IL-6R has no effect on neurotoxicity. In contrast, IL-1R blockade eliminates CRS and neurotoxicity without affecting the efficacy of CAR T cells. Monocyte ablation has a negative impact on CAR T cell proliferation and population expansion. These differences may be due to the known IL-1R antagonist Anakinra can cross the BBB [73,74], but there is no clear evidence that the IL-6R specific monoclonal antibody tocilizumab can cross the CNS.
A non-human primate ICANS model using immunocompetent rhesus monkeys has recently been reported . These primates developed characteristics consistent with ICANS after infusion of CD20 CAR T cells with a 4-1BB costimulatory domain. Although an increase in the number of CAR T cells and non-CAR T cells in CSF and brain parenchyma was observed during peak neurotoxicity, the number of CAR T cells was proportionally higher than the number of non-CAR T cells. This is related to high concentrations of IL-6, CXCL8, IL-1Rα, CXCL9, CXCL11, GM-CSF and vascular endothelial growth factor. The content of these substances in CSF is higher than the corresponding serum samples. Various degrees of histological panencephalitis were observed 8 days after CAR T cell infusion, including multifocal meningitis and perivascular T cell infiltration, which coincided with the peak level of CAR T cell proliferation in peripheral blood. Detailed phenotypic analysis found that compared with non-CAR T cells, CAR T cells have a significant increase in the cell surface expression of integrin VLA4, which may promote the increased transport of CAR T cells to the CNS. Taken together, these findings suggest that the mechanisms driving ICANS may include the accumulation of pro-inflammatory cytokines and CAR T cells in the CNS, although their relative contribution is currently unclear.
Many clinical trials have shown that there is an association between increased serum levels of various cytokines and the risk of ICANS [10, 13, 16, 17] [10, 13, 16, 17]. In multiple studies (using various CAR constructs and different target malignancies), the cytokines that continue to increase in patients include IL-2, IL-6, IL-10, and IL-15. However, although mouse models have shown that immune cells derived from recipient monocytes have a clear role in the cytokine secretion and pathogenesis of CRS and ICANS, in clinical trials, it is impossible to determine the specific cell source of cytokines in ICANS patients. . Nevertheless, a significant increase in the number of bone marrow cells has been observed in the cerebrospinal fluid of patients with severe ICANS .
Target antigen expression in the central nervous system. Data from clinical trials indicate that the presence of antigen-positive tumor cells in CNS is not necessary for the development of ICANS . In addition, it is worth noting that when patients with glioblastoma multiforme are infused with CAR T cells intrathecal or intratumorally, these patients do not develop ICANS. However, in a recent study, single-cell RNA sequencing analysis showed that CD19 is expressed in human brain wall cells, including pericytes and vascular smooth muscle cells. This increased targeting extra-tumor effects may contribute to the correlation with CD19CAR T cells The neurotoxicity . This observation may explain the higher incidence of ICANS observed with CD19-directed therapy compared with therapies that target CD20, CD22, and BCMA (also known as TNFRSF17), although alternative or additional mechanisms may exist. CD22 is expressed by microglia in the human brain [76,77] and is not associated with the higher incidence or severity of ICANS in CD22 CAR T cell therapy.
Microglia are a special kind of phagocytic cells that can persist in the central nervous system for decades. They engulf myelin fragments and protein aggregates, thereby preventing damage to neuronal cells and maintaining brain homeostasis and function [78-80]. Using CRISPR-Cas9 knockout screening combined with RNA sequencing, a previously undescribed role of CD22 as a negative regulator of microglia phagocytosis has been established. In elderly mice with senescent microglia, CD22 is up-regulated, which impairs the elimination of myelin fragments, β-amyloid oligomers, and α-synuclein fibrils in the body. The administration of CD22 blocking antibody reversed microglia dysfunction and improved phagocytosis and cognitive function. The role of microglia phagocytosis in the pathogenesis of ICANS and the impact of CD22 expression by microglia on CD22CAR T cell therapy remains to be elucidated, but cytokine-mediated microglia have been described in children with cerebral malaria Activation . These children will develop diffuse encephalopathy with interrupted BBB and cerebral edema. These clinical features are not different from those of ICANS.
Brain edema. Cerebral edema is a rare but potentially fatal neurological complication that has been observed after CAR T cell therapy [16,65,81]. Existing evidence suggests that the pathophysiology of cerebral edema may be different from the more common encephalopathy manifestations in ICANS. In a clinical trial evaluating CD19 CAR T cells in adult B-cell acute lymphoblastic leukemia patients, five patients developed fatal brain edema, which led to the termination of the trial . The root cause analysis of evaluating patient characteristics, conditioning therapy and product attributes shows that patients with cerebral edema are younger than 30 years old, the percentage of CD8+ T cells in CAR T cell products is higher, and the level of serum IL-15 before CAR T cell infusion The platelet level is low, and the CAR T cell population rapidly expands to a peak within the first week, which is related to the sharp increase in serum IL-2 and TNF81 levels. Importantly, the autopsy of two patients showed that the BBB was completely broken down, but there were no activated T cells in the central nervous system. Although they are not definitive, these results suggest that the destruction of the BBB and subsequent brain edema may be due to a surge of inflammatory cytokines rather than the infiltration of CAR T cells into the central nervous system. In addition, analysis shows that brain edema may be caused by multiple factors including patient characteristics and product attributes.
Clinical management of CRS and ICANS
Low-grade CRS is managed with supportive treatment and antipyretic drugs, while ensuring that there are no concurrent causes of fever, such as infection. Moderate to severe CRS is treated with IL-6R blocking antibody tocilizumab, with or without corticosteroid immunosuppression, and intensive supportive treatment, including fluid resuscitation and vasopressors for hypotension and hypoxia requiring supplemental oxygen [9,14,82-84]. Low-grade ICANS are also usually managed through diagnostic tests and supportive care, while severe ICANS are usually treated with corticosteroids in most centers [14,84]. The use of tocilizumab significantly reduced the incidence of severe CRS, which may be because IL-6 levels reached a peak in the early stage of CRS, and it is a key mediator of the downstream inflammatory cascade . However, in very severe cases of CRS, corticosteroids are also needed for treatment; a variety of cytokines may play a role in the later stage of CRS , corticosteroids can induce overall immunosuppression, including suppression of CAR T cells and other bystander immunity Proliferation and cytokine secretion of cells, especially bone marrow cells.
Although tocilizumab is very effective in the management of CRS, it is not effective in most cases of ICANS [14, 16, 17, 84]. This may be due to the pathophysiological difference between CRS and ICANS and/or the poor permeability of tocilizumab in the BBB. In fact, the preventive use of tocilizumab reduced the incidence of severe CRS, but increased the incidence of severe ICANS, which may be due to the increased serum IL-6 levels observed after tocilizumab administration , This is because receptor block prevents it from being absorbed by peripheral tissues [65,85]. However, these observations need to be interpreted with caution because the study was not randomized and the sample size was small65. Although corticosteroids can cross the BBB and are commonly used to treat ICANS10,86, there is no definitive evidence of their clinical benefit on the severity or duration of ICANS. Most studies have shown that the use of tocilizumab does not seem to affect the efficacy of CAR T cells [10,15,86]. However, data on the effects of corticosteroids on CAR T cell efficacy are conflicting. Some studies have shown no effect, but other studies have shown that adverse clinical outcomes increase the risk of early progression and death [10,87,88]. This further emphasizes the need to better understand the pathophysiological mechanisms of CRS and ICANS and to develop new management strategies.
The influence of other clinical factors. The occurrence, severity and duration of CRS and ICANS after CAR T cell therapy may be affected by factors related to the host, tumor and/or treatment. Patients with a high baseline inflammatory status (defined by C-reactive protein, ferritin, D-dimer, and pro-inflammatory cytokine levels) have an increased risk of CRS and ICANS89,90. These patients may be more prone to inflammation after infusion of CAR T cells. More severe CRS and/or ICANS cases are also associated with a variety of malignancies (including leukemia, lymphoma, and multiple myeloma).
Tumor burden is related, which may be related to the greater expansion and simultaneous activation of CAR T cell populations [13,31,86,91-93]. The intensity of conditioning therapy before CAR T cell infusion also seems to affect the severity of these toxicities. More intense regimens increase the risk of severe CRS and/or ICANS, which may be by inducing greater lymphopenia, thereby eliminating cytokines Sinking and making higher levels of steady-state cytokines, such as IL-2 and IL-15, can be used for CAR T cell proliferation [5,94-96]. Interestingly, old age is not associated with an increased risk of severe CRS or ICANS86,90,97.
The impact of CAR design. Recent studies have shown that the design of the CAR molecule itself can significantly affect the proliferation and cytokine profile of CAR T cells, thereby affecting the incidence and severity of CRS and/or ICANS. As mentioned above, CAR T cell products with CD28 costimulatory signal domain appear to proliferate faster after infusion, and their numbers seem to peak earlier than products designed with 4-1BB costimulatory signal domain. In clinical studies, this is related to the earlier onset and higher incidence of more severe CRS and ICANS [10,15]. However, so far no direct comparison of CAR T cell products with CD28 and 4-1BB costimulatory domains has been made. The differences in patient characteristics such as changes in tumor burden and toxicity monitoring and grading between studies may explain some of the reported differences in pharmacokinetics and adverse events between the CD28 and 4-1BB domains.
Evidence in the literature also suggests that changing the non-signaling domains of CAR molecules, including hinges and transmembrane regions or antigen binding domains (scFv), may affect the toxicity associated with CAR T cell therapy. For example, changing the length of the CD8α hinge and transmembrane domain will reduce the cytokine production of CD19CAR T cells with 4-1BB and CD3ζ signal domains and reduce their proliferation, but it is retained in preclinical models and phase I trials. Clinical trials of their cytolytic activity . Importantly, after testing this product in patients with B-cell lymphoma, the rate of complete remission is very high, with only low-grade CRS and no ICANS . Compared with published studies using tisagenlecleucel (a CD19CAR T cell therapy using high-affinity scFv [11,99]), CD19CAR T cells with low-affinity scFv have higher anti-tumor efficacy, but will not Acute lymphocytic leukemia causes severe CRS in pediatric patients. Patients with B-cell lymphoma receive CD19CAR T cell therapy. CD19CAR T cells contain the same CD28 and CD3ζ signal domains as the approved CD19CAR T cell therapy axiabtagene ciloleucel[95,100], but have different scFv and hinge and transmembrane domains, and have similar Anti-lymphoma activity but more ICANS101 has a lower incidence and severity.
In a small study testing CD19CAR natural killer cells, a high complete response rate was observed in patients with B-cell malignancies that did not induce CRS or ICANS, indicating that different immune effector cells may have different toxicity characteristics . Finally, preclinical studies have shown that CARs encoding a single immunoreceptor tyrosine-based activation motif (instead of three of them) in CD3ζ can enhance therapeutic effects and memory T cell differentiation without increasing inflammatory activity [ 103].
In general, these reports indicate that reducing tumor burden and baseline inflammatory status, adjusting pretreatment regimens, and optimizing the design of CAR molecules and/or CAR T cell products may reduce the incidence and/or severity of CRS and ICANS.
New strategy for managing CRS and ICANS
Excessive secretion and imbalance of cytokines are the core of CRS pathology. In many cases, non-specific immunosuppressive agents (such as corticosteroids) can relieve symptoms in patients [4,7,12,27,104]. In addition, other broad-spectrum cytokine inhibitors, such as Ruxotinib, which blocks JAK1 and JAK2, or itatinib, which blocks JAK1 (kinase required for cytokine receptor signaling), are expected to attenuate pro-inflammatory cytokines For example, IFNγ and IL-6 [37,105]. In fact, in preclinical models of CAR T cell-related toxicity, ruxotinib and itaatinib reduced toxicity and cytokine secretion . Similarly, Ibrutinib is a Bruton’s tyrosine kinase (BTK) inhibitor and also inhibits IL-2 tyrosine kinase (ITK; a kinase involved in proximal T cell receptor signaling) [106 ], resulting in decreased secretion of cytokines in the mouse model, including decreased secretion of IL-6, but also decreased levels of CAR T cell-derived cytokines, such as IFNγ, which may indicate decreased CAR T cell activation. In this study, the anti-tumor efficacy was monitored only in the short term (day 4 after CAR T cell transfer), and no difference was observed . Interestingly, when co-cultured with the mantle cell lymphoma cell line, Ibrutinib reduced the cytokine secretion of tumor cells, demonstrating the wide range of effects of this treatment. Small molecule kinase inhibitors can often bind to multiple targets. These results suggest the possibility that ruxotinib and ibrutinib can directly affect the level of CAR T cell activation, thereby affecting the clinical outcome.
Another study used the incomplete specificity of kinase inhibitors to create an on/off switch for CD3ζ chain-based CAR T cells . Dasatinib is a BCR-ABL targeted kinase inhibitor, which has been approved for the treatment of various hematological malignancies and has been shown to effectively inhibit CAR T cell-mediated cytotoxicity and cytokines in a rapid and reversible manner. produce. In addition, short-term administration of dasatinib in preclinical models can reduce CRS-related mortality without impairing the anti-tumor efficacy in vivo after drug removal and CAR T cell activity is restored  (Figure 3). To further support the indispensable role of activated CAR T cells in initiating CRS, another recent preclinical study showed that by recruiting the phosphatase SHP1 engineering to the immune synapse to attenuate LCK signaling can reduce the production of CAR T cells. Effector cytokines and the severity of CRS . In addition, BBz CAR (with 4-1BB and CD3ζ costimulatory domains) has an improved hinge structure, transmembrane domain, and intracellular domain near the membrane, and has the appearance of inactivating activation and reducing effector cytokine production. Type, leading to reduced preclinical toxicity  in the SCID-beige xenograft model 30 and compared with clinical and historical data 98.
Cumulatively, these studies have shown that the inhibition of broad-spectrum cytokines through different mechanisms can reduce the pathology of CRS. Given our understanding of the pathophysiological mechanisms of CRS, this is the expected result. Nevertheless, long-term inhibition of cytokines may be detrimental to the anti-tumor efficacy of CAR T cells. Therefore, targeted interventions aimed at selectively destroying specific cytokine signaling pathways may be favored. In a small series of 8 patients with severe ICANS or hemophagocytic lymphohistiocytosis after receiving CD19 CAR T cell therapy, blocking IL-1R by anakinra seemed to benefit half of the patients . The judicious use of new drugs that target other cytokines, such as IFNγ (e.g., emapalumab), may help clarify their role in the management of severe CRS111. Future clinical studies should be able to prove the benefits of these methods when used in a preventive and/or therapeutic setting.
Currently, the widespread adoption of CAR T cell therapy is limited, partly because of the need for treatment by centers experienced in managing common toxicities of CRS and ICANS, as well as the financial and health burdens that result from it. A deeper understanding of the molecular and cellular pathophysiology of CRS and ICANS will help develop effective targeted therapies to reduce toxicity without affecting anti-tumor activity. New CAR constructs are already being designed to minimize the risk of triggering CRS and ICANS, while optimizing the recognition of tumor antigens and effective T cell signaling.
Another current limitation is the need for a deeper understanding of CAR T cell biology and mechanism of action. This includes the biophysical properties of CAR and the impact of its costimulatory domain on gene expression profiles, which may change T cell subsets, function, memory potential and exhaustion. In order to improve the clinical efficacy of CAR T cell therapy, there is an urgent need to generate T cells with the best in vivo adaptability, durability and efficacy.
In addition to oncology, in the field of autoimmunity and solid organ transplantation, people are very interested in using CAR-based technology to generate antigen-specific regulatory T cells, which may provide targeted immunosuppression [112-115]. The in vivo biology and function of these regulatory CAR T cells will be different from those of the effector CAR T cells observed so far, and some new toxicities may also appear. The safe and widespread use of engineered T cell therapy in oncology and non-malignant indications will depend on effective and reliable prevention of these complications.
(source:internet, reference only)