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ChallengeWhat are challenges and toxicity control of CAR-T for solid tumors?
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What are challenges and toxicity control of CAR-T for solid tumors?
CD19 targeting chimeric antigen receptors opens the way for new immunotherapies in oncology and other medical fields. This therapeutic innovation is based on advances in genetic engineering, tumor immunology, and cell manufacturing science. The basic principle is to use genes to instruct T cells to recognize any selected antigen, thereby bypassing library limitations and immune tolerance limitations, and accelerating the establishment of immunity by providing patients with effective artificial immune cells, rather than inducing uncertain The endogenous response is through active immunization.
CD19 is a cell surface molecule found in most B cell malignancies. We initially chose it as a CAR target rather than other B cell surface molecules, such as CD20 and CD22, because of its relatively high expression. Based on the evidence that CD19-specific CAR can eliminate leukemia and lymphoma in mice. The National Cancer Institute and the University of Pennsylvania conducted clinical trials on patients with refractory/relapsed CD19+ malignancies. Early anecdotal results obtained in NHL, CLL, and ALL were quickly confirmed in larger single-center and multi-center studies.
The first batch of CARs approved by the U.S. Food and Drug Administration in 2017 and the European Medicines Agency in 2018 were the second-generation CARs for CD19. These CAR molecules contain CD28 or 4-1BB costimulatory domains (Figure 1A). So far, a variety of CAR products have been approved (Tisagenlecleucel, Axicabtagene ciloleucel, Brexucabtagene autoleucel, Lisocabtagene maraleucel), etc., for relapsed or refractory (r/r) children and young adults B-cell acute lymphoblastic leukemia, diffuse Large B-cell lymphoma, high B-cell lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, and certain follicular lymphomas. Some excellent clinical reviews deal with the efficacy and toxicity of CD19 CAR T cells in different disease settings, including B cell hypoplasia, cytokine release syndrome (CRS), and immune effector cell-related neurotoxicity syndrome (ICANS).
Figure 1 CAR structure, function and design.
(A) Prototype CD19 CAR.
(B) Novel 28z CAR design. Left: 1928z CAR T cells have a single functional ITAM at the membrane proximal position (1XX CAR), showing increased persistence while retaining powerful effector functions.
Right: CARs containing truncated cytoplasmic IL2Rb and YXXQ STAT3 binding motifs (28-ΔIL2RB-z(YXXQ) CAR) can achieve antigen-induced JAK-STAT activation.
(C) Novel BBz CAR design. Left: Increasing ITAM from 3 to 6 in 4-1BB-based CAR (BBzz CAR) can enhance antigen sensitivity and IL2 secretion.
Right: Changing the length of extracellular and intracellular domains in 4-1BB-based CAR (BBz(86) CAR) reduces the secretion of cytokines and the severity of CRS.
The success of CD19 CAR therapy has sparked widespread interest in this new type of immunotherapy. Currently, there are more than 700 clinical trials listed on clinicaltrials.gov, of which 41% target CD19, and the rest are either hematological malignancies (32%) or solid tumors (27%) (Figure 2A). CD22 and CD20 have been proven to be effective CAR targets for B-cell malignancies, albeit to a lower degree than CD19, while BCMA shows great promise for the treatment of multiple myeloma. BCMA CAR T cells have been approved by the FDA. This review discusses the next frontiers and challenges in implementing CAR therapy in oncology and other fields.
Figure 2 Summary of clinical trials for CAR T cells. (A) Clinical trials for CD19, other hematology and solid malignancies. According to clinicaltrial.gov (March 2021): 41% of global clinical trials use CD19 CAR T cells.
This includes the dual targeting of CD19 and CD22 CAR T cell therapy.
(A) total of 32% of clinical trials target blood cancers, but not CD19. A total of 27% of clinical trials target solid tumors.
(B) CAR targets in clinical studies of hematology and solid tumors.Based on clinicaltrial.gov (March 2021): Analysis of the distribution of antigens of interest used to guide CAR T cells in clinical trials, showing that anti-CD19 scFv is the main CAR T used (37%), followed by anti-BCMA scFv (9 %). Other CAR T cells targeting other hematological or non-hematological antigens lag behind all CAR T clinical trials by less than 4%.
(C) Cell types designed for CAR therapy. Based on clinicaltrial.gov (March 2021): Most CAR T cells are transduced with αβ T cells (>96%). Other cell types are being studied and account for 4% of listed clinical trials. These cells include: γδ T cells, NK and NK/T cells, immune cells derived from pluripotent stem cells, and monocytes.
The evolution of CAR design
The current CAR contains a single-chain Fv (scFv) for antigen recognition and a dual signal tail, usually containing CD3-ζ and CD28 or 4-1BB cytoplasmic domains. Different scaffolds adapted to the epitope position on the target molecule can further improve the overall CAR function. In some cases, the binding domain is derived from a receptor-ligand pair, or may contain VHH elements instead of scFv. This architecture is modular and can be adapted to a wide range of goals (Figure 1A).
CAR design continues to evolve, using affinity-optimized human binding domains and recruiting different signaling pathways. One example of this evolution is the reduction in activation intensity in the effective 28z CAR design, and the other is the enhancement in the less effective BBz (Figure 1B and C). Other examples are the additional recruitment of JAK/STAT signals in 28z CAR (Figure 1B) or the addition of spacer elements to reduce cytokine secretion in BBz CAR (Figure 1C).
Contrary to the single specificity of natural T cells, CAR T cells can bind multiple antigens by designing multi-specific CARs or CAR co-expression. Targeting two independent antigens may reduce tumor escape and increase selective pressure on tumor cells. The first dual-targeting CAR T cell has entered the clinic, combining two BBz CARs targeting CD19 and CD22. Combining 28z CAR with BBz CAR provides superior antigen sensitivity and efficacy, which is further enhanced by matching signal characteristics to antigen density.
A variant approach requires two different CARs to successfully recognize two up-regulated tumor antigens in order to initiate complete immune cell function, as Eshhar’s team demonstrated using dual-targeting CAR T cells against myeloma cells. The results not only show an effective and effective CAR T, but also a safety strategy to protect normal healthy tissues.
CAR T cell therapy to treat solid tumors
At present, CAR T cells, which have been approved by the US FDA and EMA, target CD19 and are used to treat some refractory leukemias and lymphomas. Adapting CAR therapy to the treatment of solid tumors is a major goal and challenge. The first result obtained in the solid tumor trial, based on the successful strategy implemented for B-cell malignant tumors, has been lacklustre, indicating that further improvements and special adjustments to CAR treatment for solid tumors are needed. Overcoming the resistance mechanisms of CAR therapy that have been encountered in B-cell malignancies may help achieve this goal.
One mechanism of resistance is the absence or insufficient expression of CAR targets in tumor cells. Reports from multiple trials have shown that up to 25% of patients receiving CD19-targeted CAR T-cell therapy have relapsed with CD19-negative or low-CD19 disease. This phenomenon is called antigen escape.
Another drug resistance mechanism can be traced to the limited ability of CAR T cells to enter and infiltrate tumors due to physical barriers and/or immunosuppressive tumor microenvironment (TME). In the field of B-cell malignancies, it is often noted that the response may be complete in BM, but not in other disease sites such as LN or retroperitoneal space. Even with infiltration, it can be expected that solid tumors will use a series of mechanisms to suppress T cells and CAR T cells, including myeloid-derived suppressor cells, tumor-associated macrophages (TAM) and Treg.
Therefore, CAR T cells successfully treat solid tumors at least to determine the appropriate target antigen, so that CAR T cells can enter all tumor sites and overcome the immunosuppressive effect of TME.
The first attempt to solve solid tumors replicated the methods established in B-cell malignancies, using the same CAR design (28z or BBz) and targeting a single antigen. Although occasionally good responses have been obtained in glioblastoma multiforme (GBM), head and neck squamous cell carcinoma, and prostate cancer, the overall response cannot be compared to CD19 CAR therapy.
One limitation is due to the lack of potential targets, such as CD19, which is expressed on the surface of almost all tumor cells and only in dispensable normal cells. Although few differentiation antigens meet these two criteria, some proteins that are overexpressed in tumors are not tumor-specific, but show promise, such as mesothelin, PSMA, GPC3, and so on.
However, if the CAR function is to be calibrated within a specific treatment window to limit the response to normal cells, carefully selecting scFv, signal components, and transcriptional regulation, it may be possible to target tumor antigens found in normal tissues. Antigen-specific inhibitor CAR can also be used to protect normal tissues. Figure 2B illustrates the current distribution of CAR target antigens in hematology and solid tumor clinical studies based on cilinicaltrial.gov.
Cell surface antigens that have been specifically modified in tumor cells are an attractive class of CAR targets. Glycans, including glycolipids, N- and O-linked glycoproteins, and glycosaminoglycans, can be specifically glycosylated in cancer cells. They include LewisY in advanced epithelial cancer, sialyl-LewisA in pancreatic adenocarcinoma, GD2 in neuroblastoma and some gliomas, and Tn-MUC1 in various adenocarcinomas. The involvement of various enzymes in glycan biosynthesis and their redundancy are the reasons for their stable expression and may reduce the risk of antigen escape. Tumor cells must indeed lose a few of the >20 GalNAc polypeptide transferases that contribute to O-glycosylation in order to stop producing Tn antigen. However, if the protein backbone is mutated (for example, the MUC1 of Tn-MUC1 is lost), glycoprotein expression may still be lost.
Like any T cell, CAR T cells need to extravasate to the tumor site and overcome the immunosuppression in TME to be effective (Figure 3). Immune cells transported to peripheral tissues are regulated by complex signal transduction and physical processes, which may be significantly disrupted in some tumors. The challenge of immunological “cold tumor” is a common obstacle to all immunotherapy.
However, T cell engineering allows unique solutions. For example, in some cases, the targeted migration of chemokines, such as the overexpression of the IL8 receptor CXCR2R in CAR T cells (Figure 3A), can alleviate the rate limitation of tumor entry .
Another method is to direct CAR T cells around the tumor. For example, CAR T cells can target fibroblast-associated protein (FAP), which is present in the stromal fibroblasts of TME. CAR T cells targeting FAP can effectively infiltrate solid tumors, but they may also have bone marrow toxicity (Figure 3B). Another method is to use CAR T cells that secrete enzymes to form a pathway, such as heparanase, to degrade heparan sulfate proteoglycans (Figure 3C).
Figure 3 Selected new strategies to enhance the efficacy of CAR-T cell therapy on solid tumors:
(A) CAR-modified T cells express chemokine receptors to increase their ability to transport/return to the tumor site (such as CXCR2).
(B) FAP-specific CAR-T cells can guide CAR-T cells around the tumor.
(C) Heparanase expression enhances the pathway of CAR-T cells in TME.
(D) Intratumoral administration of CAR T cells in solid tumors.
(E) Combination therapy with monoclonal antibodies targeting immune checkpoint suppression receptors to relieve immunosuppression.
(F) siRNA that reduces receptor function or depletion markers.
(G) Engineered CAR T cells can secrete pro-inflammatory or CAR-T supporting cytokines, such as IL-2, IL-7, IL-12 or IL-15.
(H) It is proposed to integrate multiple metabolic strategies (such as l-arginine) to optimize the manufacturing process and maximize the therapeutic efficacy of CAR T cells.
Local or regional CAR T cell delivery in solid tumors can not only maximize the accumulation of CAR T cells at the tumor site, but also improve their safety by limiting their systemic distribution (Figure 3D). Regional CAR therapy targeting mesothelin in mesothelioma has shown effective and long-lasting CD4-dependent tumor immunity, and can provide greater anti-tumor efficacy compared with intravenous administration.
Tumors and their stroma attract and support a variety of immunosuppressive cell types, including a heterogeneous population of myeloid-derived suppressor cells, macrophages, Foxp3+ Treg, fibroblasts, and platelets. A variety of inhibitors that act on TME-related immunosuppression are being studied.
Overcoming TME by combining such drugs with CAR T cells may prove beneficial (Figure 3). Blockade of suppressive immune checkpoints for mandatory immune suppression is a promising option.
Checkpoint inhibition can be achieved by combining CAR T cells with the systemic administration of immune checkpoint inhibitory antibodies or by ablation, for example, PD-1 or reducing its function and dominant negative receptors in a cellular manner.
Interfering RNA provides another way to reduce but not completely eliminate receptor function, for example, silencing adenosine receptors and other depletion markers such as TIM-3, LAG-3, TIGIT, and KLGR-1 (Figure 3E).
Adenosine-mediated T cell suppression can be counteracted by other means, such as using shRNA (Figure 3F) or by fusing the extracellular domain of the adenosine receptor to the intracellular costimulatory domain of adenosine in response to CAR’s A2AR gene target In this way, the usual suppression signal is switched to an activation signal.
CAR T cells can be used to reprogram the TME. For example, CAR T cells can provide local cytokine secretion to enhance their infiltration, proliferation and persistence. CAR-expressing T cells can be designed to produce a variety of cytokines (such as IL-4, IL-2, IL-7, IL-21 and IL-15) (Figure 3G) or their homologous receptors, with the aim of Improve the anti-tumor activity and durability of these cells.
These CAR T cells are sometimes referred to as armored CARs, fourth-generation CARs, or TRUCK (redirected T cells are used for antigen-unrestricted cytokine-initiated killing). For example, TRUCK, which has been shown to induce or constitutively express IL-12, activates the innate immune anti-tumor response and changes tumor immunosuppression.
The enhancement of glycolytic metabolism of cancer cells may limit the nutrients available to CAR T cells in the tumor bed, hinder their function or survival, thereby affecting the microenvironment and suppressing immune cells. It has been shown that the use of pretreatment in vitro feeding or tumor-inhibiting specific metabolic pathways to provide nutrients for CAR T can improve CAR T survival, such as providing CAR T cells with l-arginine before adoptive transfer (Figure 3H).
Toxicity of CAR T cell therapy
CAR T cells are powerful immune effectors, and in some cases may cause severe toxicity that requires expert and emergency medical management. For any specific patient, the occurrence of these acute toxicity is still difficult to predict and hinders the widespread implementation of CAR therapy.
The first type of toxicity is expected, due to CAR T cell targeting/non-tumor, leading to CD19 CAR B cell aplasia. Different methods have been proposed to inhibit the toxicity to normal cells. A second-generation CAR with a split costimulatory structure, combined with traditional activation constructs and inhibitory CARs, is possible to achieve higher levels by ensuring that effector cells are fully activated only when there is a certain tumor-associated antigen array. Target accuracy. Another method combines inhibitory AR to retrain T cell function.
The unknown toxicity of new CAR T cells can also be limited by injecting T cells transiently expressing the CAR construct after RNA transfection, thereby minimizing the duration of unforeseen toxicity. In the context of solid tumors, local delivery of transgenic T cells can also reduce toxicity while improving efficacy.
Another way to reduce side effects is to find the smallest effective dose. Globerson Levin et al. modeled the number of CAR T cells required to achieve the therapeutic effect, while reducing the number of CAR T cells injected to reduce side effects. It also shows the possibility of using much less CAR-T lymphocytes than currently used CAR-T lymphocytes.
The other two toxicities, which were not expected at the beginning of the first CD19 CAR test, have posed greater challenges so far: CRS and ICANS. Both of these toxicities occur early after CAR T cell administration and may be significant in as many as one-third of CAR T cell recipients.
The mechanism of CRS is beginning to be well understood. The secretion of pathological cytokines is mainly attributed to the interaction between CAR T cells and bone marrow cells in TME. Although activated T cells produce chemokines and cytokines, including IL-2, soluble IL-2R-α, IFN-γ, IL-6, soluble IL-6R and granulocyte-macrophage colony stimulating factor, IL6 The main sources of IL1 and IL1 are macrophages and CAR T cells near the tumor. The cornerstones of CRS management are IL6R blockers and corticosteroids, but new interventions, including IL1Ra and dasatinib, are emerging. Little is known about neurotoxicity, and it is mainly treated with corticosteroids.
Through the action of suicide genes or other elimination switches, CAR T cell activity can be terminated by reversibly turning off CAR signal transduction or irreversibly eliminating CAR T cells. The suicide gene can be included in the transfection construct to trigger apoptosis after induction with a specific drug.
Therefore, although acute CAR T cytotoxicity currently limits the widespread use of CAR T cells, given the rapid progress in CAR design and our understanding of the pathophysiology of CRS, it can be expected that this limitation will eventually be eliminated.
CD19 CAR T therapy opens Pandora’s box for cell-based immunotherapy and synthetic immunology. These advancements will depend on advances in receptor and circuit design, tumor immunology, genetic engineering, and cell manufacturing science. The clinical results achieved by CD19 CAR T cells in the past 10 years have prompted the pharmaceutical industry to explore and invest in cell-based immunotherapies, which was not possible in the past with adoptive cell therapies. Developing a series of personalized therapies requires a lot of work, which will require identifying suitable targets, overcoming TME, entering cold tumors, and minimizing toxicity.
(source:internet, reference only)