Challenges and difficulties encountered in the application of CAR-T Cell Therapy

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Chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) immunotherapy is to genetically engineer T cells isolated from patients or allogeneic donors to express chimeric antigen receptor (chimeric antigen receptor). receptor, CAR), an adoptive cell therapy that specifically recognizes and kills tumor cells. CAR-T is one of the major breakthroughs in the field of cancer immunotherapy in recent years. It has great advantages in the treatment of hematological malignancies and has broad development prospects. At present, CAR-T cell therapy also faces huge challenges. The following content will take you to understand the challenges faced by CAR-T cell therapy, identify the mechanisms that lead to limitations and overcome these obstacles, so that CAR-T cells can better exert their potential, Optimize treatment strategies and improve patient outcomes. Several key factors that have been found to affect the efficacy of CAR-T cell therapy include the manufacture of CAR-T cells, the management of toxic side effects, and the recurrence of drug resistance.

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1 Problem in CAR-T Cell Manufacturing

The challenges faced by CAR-T cell manufacturing involve multiple links such as T cell acquisition, isolation and screening, transduction, culture expansion, and initial T cell phenotype selection. Through the optimization of methods in each link, CAR-T cell products can be realized with Higher clinical efficacy and less toxic side effects. At present, the CAR-T cells approved by the FDA are all autologous, and there is no risk of allogeneic rejection and graft-versus-host disease (GVHD), but it is difficult to obtain, and the cell quality is often not available. Assure. Using cells from healthy donors to produce CAR-T products is one solution to the problem of low-quality CAR-T cell sources. Early clinical studies have demonstrated the feasibility of using donor-derived CAR-T cells in patients with disease relapse after allogeneic transplantation with a lower risk of GVHD. In addition, donor-derived T cells facilitate the development of universal CAR-T products, which is of great significance to overcome the existing problems of insufficient CAR-T cell sources, poor quality, and long production cycles, but additional genetic modifications are required to reduce the risk of immune rejection and GVHD. In addition, CAR structures often contain exogenous sequences. Due to the difficulty of preparation, most scFvs of CAR-T cells are of murine origin and are immunogenic. Human anti-mouse antibodies against scFv have been detected in treated patients.

Studies have shown that the initial T cell phenotype of CAR-T cell products plays an important role in subsequent clinical responses. Specific T cell phenotypes, such as central memory T cells, stem-like memory T cells, and precursor T cells, may improve the expansion capacity and persistence of CAR-T cells. A study of CD19-targeted CAR-T cell therapy in CLL patients found that the CAR-T cell population of responders had abundant memory T cell-related gene expression compared with non-responders. Another research team induced the CAR protein to enter a quiescent state by forcing down-regulation of the CAR protein through a drug regulatory system or dasatinib, thereby obtaining a memory-like phenotype, successfully reversing the phenotype and transcriptional characteristics of exhausted CAR-T cells, and then restoring Antitumor function of CAR-T cells.

In addition, the timing of infusion of CAR-T cells also has an important impact on treatment response. By shortening the production cycle of CAR-T cells through technological optimization, it is expected to reduce the delay of patients' illness and benefit more patients. In addition, the genes encoding CAR structures are usually transduced into T cells by retrovirus or lentivirus, but with the development of transposon systems, it is more economical to use transposons instead of viral vectors for CAR-T cell production. At present, the Sleeping Beauty transposon system has been applied to the manufacture of CD19-targeted CAR-T cells.

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2 Toxic and side effects of CAR-T cell therapy

Almost all patients treated with CD19-targeted CAR-T cells developed toxic side effects of varying degrees, including cytokine release syndrome (CRS) and immune effector associated neurotoxicity syndrome, ICANS), etc., the latter also known as neurotoxic side effects. The American Society for Transplantation and Cellular Therapy (ASTCT) has developed and published standardized grading recommendations for CRS and ICANS, which have guiding significance for the management and treatment of CAR-T toxicity.

The clinical symptoms of CRS often start with fever, and severe cases can lead to systemic inflammatory response, hypotension, hypoxia, and organ failure. ICANS is mainly manifested as toxic encephalopathy, severe cases can lead to seizures, cerebral edema and coma. Most of the patients with ICANS had a history of CRS, suggesting that CRS may act as an initiating factor or promoting factor for ICANS. If the early symptoms of CRS and ICANS can be detected and effectively intervened, the clinical course of both is reversible, but severe CRS and ICANS can be fatal. Understanding the pathophysiological mechanisms of CRS and ICANS is helpful for the development of targeted drugs to suppress the toxicity of CAR-T cells on the basis of retaining the anti-tumor activity of CAR-T cells as much as possible. CRS is associated with elevated levels of various cytokines, among which IL-6 is an important immune molecule mediating CRS. Tocilizumab, which blocks the IL-6 receptor, is currently the main treatment for CRS. Preclinical trials have shown that CRS is triggered by a multicellular network of CAR-T cells and host cells, with the monocyte-macrophage system playing a central role in the activation process. IL-1 is one of the main cytokine products of the monocyte-macrophage system, and may be involved in the driving link of CRS, and blocking this target is effective in relieving CRS. TNF, interferon-γ (IFN-γ), granulocyte/macrophage colony-stimulating factor (GM-CSF) and other pro-inflammatory cytokines are also involved in the process of CRS, which may be the potential's target. Currently, low-grade CRS is mainly treated with antipyretic and supportive treatment, and other complications that may lead to fever, such as infection, are actively prevented. For moderate to severe CRS, tocilizumab is generally used, and steroids are selectively used as adjuvant therapy according to the patient's condition, and the effect is more significant. For severe CRS patients, steroids are generally used to inhibit the proliferation and cytokine secretion of CAR-T cells and other "bystander" cells. It should be noted that steroids cannot be used in large doses, and their inhibitory effect on the immune system will lead to a decrease in the efficacy of CAR-T. Some small molecule inhibitors such as ruxolitinib and ibrutinib can extensively inhibit the production and signal transduction of various cytokines and can bind to multiple targets, thereby regulating the immune function of CAR-T cells and reducing side effects.

The mechanism of ICANS may be related to the accumulation of CAR-T cells and pro-inflammatory cytokines in the central nervous system. Preclinical studies have observed a correlation between the number of CAR-T cells in the cerebrospinal fluid and the level of cytokines and the severity of ICANS. The incidence of ICANS in CD19-targeted CAR-T therapy is higher than that in CD22-targeted CAR-T cell therapy, which may be because CD19 is expressed in human brain medial parietal cells. The clinical treatment method for ICANS patients is to give steroids, and the dosage should be the lowest to avoid the impact on the efficacy of CAR-T and serious immunosuppression. Tocilizumab has achieved good results in the treatment of CRS, but its effect on ICANS is very limited, which may be related to its difficulty in passing through the blood-brain barrier.

Toxic side effects are currently an important factor limiting the efficacy of CAR-T, hindering the enhancement of the anti-tumor effect of CAR-T cells by increasing the dose of CAR-T cells or enhancing effector activity. High tumor burden, advanced age, and high-intensity lymphodepleting preconditioning are thought to be associated with the occurrence of immunotoxic side effects. With the increase of treatment cases and the extension of follow-up time, more toxic and side effects appeared, such as hemophagocytic lymphohistiocytosis/macrophage activation syndrome-like toxicity, B cell aplastic anemia-related immune dysfunction Damaged state complicated by fatal infection, fatal cerebral edema, etc. Existing studies have found that adding suicide genes, such as inducible caspase-9 or herpes simplex virus thymidine kinase, to CAR is a possible way to reduce the cytotoxic side effects of CAR-T, but it will cause irreversible clearance of CAR-T cells and reduce resistance. Tumor efficacy.

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3 Relapse of drug resistance after CAR-T cell therapy

Although CAR-T cells have made great breakthroughs in the treatment of hematological malignancies, among patients receiving CD19-targeted CAR-T cell therapy, the rate of drug resistance relapse is as high as 30% to 50%, and most of the relapses occur 12 years after treatment. within a month. However, this kind of relapse is not just for the CD19 target, and related studies on other targets such as CD22 and BCMA have also proved that drug-resistant relapse is a major challenge commonly faced by CAR-T cell therapy. At present, relapse events are usually divided into antigen-negative relapse and antigen-positive relapse.

The primary mechanism of antigen-negative relapse is antigen loss. Currently recognized mechanisms of antigen loss include splicing mutations, epitope cryptic, and cell lineage changes due to loss of target epitopes. However, even if the antigen is not completely lost, reduced antigen expression or density through immunomodulation is sufficient to allow tumor cells to escape. In a clinical trial using CD22-targeted CAR-T cells to treat leukemia patients, it was found that leukemia patients with positive antigens relapsed, suggesting that the maintenance of CAR-T cell activity needs to reach the minimum antigen expression threshold. Combinatorial multi-molecular-targeted CAR-T cells are expected to overcome tumor cell escape from antigen loss or downregulation mechanisms. For patients who relapse after CD19-targeted CRA-T cell therapy, CD22 is an ideal target because most CD19-negative patients remain positive for CD22 expression. In clinical trials, CAR-T cells targeting CD22 were effective in the treatment of patients with CD19-negative B-cell lymphoma and relapsed leukemia, but in the process of sequential immunotherapy, drug resistance relapse caused by down-regulation of CD22 expression by tumor cells was also found. Therefore, the development of CAR-T cells that simultaneously target CD19 and CD22 may have greater potential in overcoming drug resistance. In addition to the selection of target antigens, it is also necessary to pay attention to the precise mechanism of CAR-T in the formation of immune synapses and the killing of target cells. Natural TCRs can recognize antigens at low-density levels, and it is speculated that structural differences between CARs and natural TCRs may lead to differences in the requirements for antigen recognition density.

It is worth noting that not all relapsed patients are CD19 negative, which also shows that in addition to antigen loss and tumor cell escape, there are other factors that lead to CAR-T resistance. The main cause of antigen-positive relapse is CAR-T cell exhaustion, which leads to self-function decline due to long-term exposure to high levels of antigen. It is generally believed that the antigen-independent signal transduction of CAR-T cells is closely related to cell exhaustion, and high tumor burden is also an important factor leading to exhaustion. Immune checkpoint blockade technology combined with CAR-T cells holds promise in overcoming exhaustion and enhancing the effector and persistence of CAR-T cells. Co-expression of IL-7 receptor with CAR can avoid stimulation of "bystander" cells and improve the proliferative capacity, antitumor activity and persistence of CAR-T cells. The CAR structure of CAR-T cells contains non-self components, which are immunogenic and may induce humoral and cellular anti-CAR immunity, thereby limiting efficacy and affecting the proliferative capacity and persistence of CAR-T cells. Studies have shown that 5% of DLBCL patients and 36.7% of B-ALL patients have increased levels of anti-CAR antibodies after infusion of CAR-T cells. Cyclophosphamide or fludarabine pretreatment is considered to be an important factor in reducing the degree of anti-CAR cellular immunity. The development of humanized CAR-T products is an effective means to solve this problem, which has shown durable efficacy in relapsed/refractory B-ALL in clinical trials. The co-stimulatory domain in the CAR structure also affects the persistence of CAR-T cells. It is generally believed that compared with the 4-1BB costimulatory domain, CD28-derived CARs are less durable and more prone to exhaustion; whereas CAR-T cells containing the 4-1BB costimulatory domain have higher levels of anti-apoptotic proteins BCL-2 and BCL-XL, and there may be a mechanism to promote the formation of memory phenotype T cells. Improvements currently in clinical trials include the use of artificial antigen-presenting cells to activate CAR-T cells, regulation of CAR-T phenotypes, and combined inhibition of immune checkpoint molecules. The results are worth looking forward to.

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