CAR-T Cells in Cancer Therapy: From Structural Blueprint to Clinical Barriers

Abstract

Chimeric Antigen Receptor T-cell (CAR-T) therapy is a transformative immunotherapy, particularly effective against hematological malignancies. By engineering a patient’s T-cells to express synthetic receptors targeting tumor-associated antigens, CAR-T combines the specificity of monoclonal antibodies with T-cell cytotoxicity. Over successive generations, CAR designs have incorporated costimulatory domains, cytokine support, and advanced signaling to enhance persistence and efficacy. The therapeutic process involves autologous T-cell isolation, ex vivo genetic modification, and reinfusion to selectively destroy cancer cells. Despite remarkable success in B-cell leukemias, lymphomas, and multiple myeloma, challenges such as cytokine release syndrome, neurotoxicity, antigen escape, and poor efficacy in solid tumors persist. Strategies like dual-targeting CARs, armored CARs, suicide switches, and synthetic circuits are being developed to improve safety and control. Future directions include universal allogeneic CAR-T cells, CRISPR-based multiplex editing, off-the-shelf platforms, and integration with checkpoint inhibitors, targeted agents, nanotechnology, and synthetic biology to optimize delivery and tumor infiltration.

Keywords

Introduction

Figure 1: Structure of Car T Cell

Figure 1:Preparation of CAR -T cell.

Figure 2:Five generations of CAR molecules

Figure 3:mechanism of action of CAR-T cell

Combined Application of Multiple Treatment:

Clinical trials involving patients with malignant tumors have demonstrated that those receiving a combination of CAR-T cell immunotherapy with either chemotherapy or radiotherapy experienced significantly longer overall survival compared to patients treated with chemotherapy or radiotherapy alone. This highlights the important role of CAR-T cell therapy when used alongside these conventional treatments in improving patient outcomes. In a mouse model of ROR1-positive non-small cell lung cancer (NSCLC), pre-treatment with the chemotherapy agents oxaliplatin and cyclophosphamide (Ox/Cy) improved the ability of CAR-T cells to infiltrate tumor tissues. This enhanced penetration led to an increase in chemokine secretion by tumor-associated macrophages. Moreover, the surface expression of PD-L1 on these macrophages was significantly upregulated after combined treatment with CAR-T cells and Ox/Cy. Chemotherapy with Ox/Cy prior to CAR-T cell administration boosted CAR-T cell migration into tumors. When this strategy was paired with anti-PD-L1 checkpoint inhibition, it further improved patient survival [36]. Radiotherapy studies employing an IFN-gamma-dependent mechanism showed that levels of adhesion molecules within the tumor microenvironment (TME) increased, thereby enhancing the ability of T cells to adhere to tumor tissue [37,38]. Researchers have also engineered a novel CAR-T cell by integrating the targeting capacity and blood-brain barrier permeability of CAR-T cells with the therapeutic advantages of SN-38, a chemotherapeutic agent known for deep vascular penetration and effectiveness against tumor heterogeneity and TME barriers. Using a thin-film-probe supershot method, they developed SN-38-loaded liposomes and incorporated them into a CAR-T construct targeting EGFRvIII, resulting in SN-38-L-EGFRvIII-CAR-T cells. These engineered cells not only retain immune-mediated tumor-killing ability but also act as carriers to deliver chemotherapy drugs directly to tumors, offering a promising dual-function treatment approach [39]. Additionally, because dendritic cells (DCs) play a critical role in presenting antigens and promoting T-cell infiltration, recent studies have explored combining DC vaccines with MSLN-targeted CAR-T cells. Immunofluorescence analysis showed that the DC vaccines significantly improved the proliferation, infiltration, and persistence of the MSLN-CAR-T cells [40]. Beyond chemotherapy, combining CAR-T therapy with radioimmunotherapy presents a new method to eliminate tumors that show resistance to CAR-T treatment. In one study, researchers used a NSG mouse model implanted with tumor cells. They administered ICAM-1-targeting CAR-T cells intravenously and used 177Lu-DOTATATE for radioimmunotherapy. With the help of SPECT-CT imaging, they monitored the distribution and therapeutic impact of the CAR-T cells. Results indicated that this combination significantly enhanced treatment efficacy. The imaging confirmed that 177Lu-DOTATATE precisely targeted the tumor and measured the radiation dose absorbed. Although the findings suggest that this combination could be effective against tumors unresponsive to CAR-T therapy, the use of human xenograft tumor models limits broader applicability. Further research is needed to confirm the safety and effectiveness of this strategy [41]. Recent studies indicate that CAR-T cell therapy on its own shows limited success in treating solid tumors. To improve its effectiveness, various combination strategies are being explored.

• Combination with Chemotherapy

Research has shown that lymphodepletion enhances CAR-T therapy outcomes [42]. Several mechanisms have been proposed to explain the therapeutic benefits of lymphodepletion before administering adoptive T-cell immunotherapy [43]:

1. Nonmyeloablative chemotherapy creates temporary space for the incoming T cells.
2. The elimination of existing lymphocytes allows infused T-cells to better access vital cytokines like IL-2, IL-7, and IL-15 that support their survival and expansion.
3. Lymphodepletion can remove immunosuppressive cells, such as Tregs and MDSCs, while improving the function of antigen-presenting cells.
4. Some lymphodepleting drugs reduce immune suppression by targeting enzymes such as indoleamine 2,3-dioxygenase (IDO), which is involved in tryptophan metabolism.
5. Post-lymphodepletion, CAR-T cells show improved trafficking to tumor sites.

Common chemotherapy agents used for lymphodepletion in CAR-T therapy include cyclophosphamide and fludarabine [44]. Studies back the effectiveness of this combination. For instance, in patients with relapsed or treatment-resistant chronic lymphocytic leukemia (CLL) and other B-cell cancers, chemotherapy with cyclophosphamide before CD19 CAR-T therapy extended the persistence of CAR-T cells and improved efficacy [45]. Additionally, Srivastava et al. found that chemotherapy-induced lymphodepletion can trigger immunogenic cell death (ICD), releasing chemokines that attract CAR-T cells and increasing their infiltration into tumors [46]. Since the tumor microenvironment (TME) poses a major obstacle to CAR-T efficacy, chemotherapy can also help modify the TME to enhance therapy outcomes. Drugs like docetaxel [47] and gemcitabine [48,49] have been used as neoadjuvant therapies and shown to reduce MDSCs, thereby boosting the effects of GD2 CAR-T cells. Doxorubicin has demonstrated its ability to improve CAR-T cell activity by downregulating PD-L1 expression on osteosarcoma cells [50]. Oxaliplatin has been reported to alter the chemokine landscape in the TME, promoting recruitment of ROR1-CAR T cells. A Phase I clinical trial combining CAR-T therapy with paclitaxel and cyclophosphamide showed clinical benefits in 21 out of 28 patients who previously failed paclitaxel treatment [51].

• Combination with Radiotherapy

Solid tumors often develop an immunosuppressive and tolerant TME, transforming “hot” tumors into “cold” ones [52]. This suppresses CAR-T infiltration and promotes the recruitment of immune-suppressing cells, reducing the effectiveness of effector immune responses. However, recent evidence shows that radiotherapy (RT) can convert “cold” tumors into “hot” tumors by enhancing immune cell access to tumors and altering the suppressive TME [53]. As a result, combining RT with CAR-T therapy may yield stronger anti-tumor effects. In glioblastoma models, real-time imaging demonstrated that RT helped CAR-T cells rapidly infiltrate the tumor from blood vessels and expanded their presence in the TME, resulting in more durable immune responses [54]. Preclinical studies in mice with glioma also revealed that local tumor irradiation combined with NKG2D CAR-T cell therapy produced a synergistic effect—improving CAR-T cell tumor infiltration and increasing their cytotoxic function [55].

• Combination with Oncolytic Viruses

Oncolytic viruses (OVs) are gaining attention as cancer immunotherapy agents because of their ability to kill tumor cells and activate the immune system [56]. Despite this promise, OVs such as oncolytic adenoviruses (OAVs) face several hurdles like poor tumor penetration, off-target effects, resistance from the host immune system, and a suppressive TME [57]. As monotherapy, their efficacy remains modest, but when combined with CAR-T therapy, OVs may help overcome these limitations.

Mechanisms by which OVs support CAR-T therapy include:

1. Direct lysis of tumor cells, releasing tumor-specific antigens.
2. Activation of innate immune responses and recruitment of immune cells.
3. Delivery of immune-modulating genes to shift the TME to a pro-inflammatory state [58].

These mechanisms have been confirmed by various in vitro and in vivo experiments. One study used a combination of Ad5-ZD55-CCL5-IL12, an OAV armed with CCL5 and IL-12, along with CAR-T cells targeting carbonic anhydrase 9 (CA9) in renal cancer. Results showed enhanced CAR-T cell infiltration, expansion, and activation [59]. In addition, single-cell sequencing and spatial transcriptomics in animal tumor models confirmed that OncoViron, a chimeric OAV, significantly altered gene expression in tumor cells and recruited large numbers of lymphocytes, NK cells, and macrophages to the TME. It also promoted macrophage polarization from M2 to M1 and increased the release of immune-stimulating cytokines, thereby enhancing the anti-tumor activity of CAR-T therapy [60]. Moreover, extensive research supports the promising results of combining CAR-T therapy with immune checkpoint inhibitors (ICIs), as discussed further in Sections “Challenges and coping strategies of immunosuppressive microenvironment” and “Challenges and coping strategies of limited persistence”.

Major Challenges to Overcome for CAR T Cell Therapy:

Although CAR T cell therapy shows significant potential in the treatment of blood-related cancers, its broader application is hindered by the risk of severe, potentially life-threatening side effects. Two of the most commonly observed complications are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). CRS is caused by the excessive release of inflammatory cytokines such as IL-1 and IL-6, leading to symptoms like fever and hypotension. To manage these effects, the FDA approved tocilizumab in 2017—an antibody targeting the IL-6 receptor—for use during CAR T therapy. Other drugs under investigation include anakinra, an IL-1 receptor antagonist, and siltuximab, a chimeric antibody against IL-6 [61]. The pathophysiology of CRS and ICANS is complex. Animal studies have shown that monocytes and macrophages are the primary producers of IL-1 and IL-6 during CRS. Depleting these cells or inhibiting IL-6 signaling pathways has proven effective in preventing CRS, and anakinra has demonstrated efficacy in controlling both CRS and ICANS. Moreover, CD19—a common CAR T cell target in B cell malignancies—has also been detected in brain mural cells. This finding suggests a potential on-target, off-tumor mechanism for the neurotoxicity associated with CD19-CAR T cell therapy [62]. Several factors influence the severity of these adverse effects, including the patient’s initial tumor burden, the intensity of the lymphodepletion regimen, and the administered CAR T cell dose. Studies have linked elevated cytokine levels during therapy with higher tumor burdens at the start of treatment. Although tumor burden does not appear to affect the peak expansion of CAR T cells, it may reduce the chances of complete remission and negatively impact overall survival [63]. Evidence supporting the impact of tumor burden on adverse effects also comes from CAR T cell trials in autoimmune diseases. In small-scale clinical studies involving patients with systemic lupus erythematosus (SLE), CAR T cell therapy resulted in substantial expansion of CAR T cells, rapid symptom relief, and minimal side effects. A similar response was observed in a patient with refractory antisynthetase syndrome (ASyS), who showed significant CAR T cell expansion with only mild adverse reactions [64]. In contrast, patients with hematological malignancies such as chronic lymphocytic leukemia (CLL) and B-cell acute lymphoblastic leukemia (B-ALL) often present with high circulating B cell levels, whereas patients with autoimmune conditions like SLE or ASyS usually have normal or decreased B cell counts [65]. The relatively low toxicity observed in autoimmune patients suggests that severe side effects in cancer patients are more likely a result of extensive tumor cell destruction rather than the direct effects of CAR T cells. This massive cell death may release intracellular contents into the bloodstream, triggering tumor lysis syndrome (TLS), a potentially fatal complication [66]. These findings highlight the potential benefit of administering CAR T cell therapy during earlier stages of disease progression, when tumor burden is still low, to reduce the risk of severe toxicities.

• High Cost and Challenges in Autologous CAR T Cell Production

One of the primary obstacles in applying CAR T cell therapy broadly is the high expense involved in producing autologous CAR T cells. Treatment costs can soar up to $500,000, especially for patients suffering from severe cytokine release syndrome (CRS). Additionally, producing autologous CAR T cells takes around 21 to 35 days. This delay can be critical, as patients might need interim (bridging) therapies during this period, and in rapidly progressing cases, some may deteriorate before receiving the CAR T cells. Furthermore, T cells extracted from patients with advanced disease often exhibit functional exhaustion, reducing their therapeutic effectiveness compared to T cells from healthy individuals. To make CAR T therapy more accessible and cost-effective, alternative strategies are under investigation. These include the use of “off-the-shelf” allogeneic CAR T cells (allo-CAR T) and direct in vivo CAR T cell generation. Using allogeneic CAR T cells comes with immune-related risks such as graft-versus-host disease (GvHD) and potential rejection by the patient’s immune system. To address this, gene-editing tools like CRISPR/Cas9 are employed to eliminate TCR genes from donor T cells, which helps reduce GvHD. Simultaneously, deleting the beta-2-microglobulin (B2M) gene prevents the formation of the HLA class I complex, thereby lowering the risk of immune rejection [67]. Despite these innovations, a significant limitation of allo-CAR T therapy is the short lifespan of these cells inside the patient’s body, which may reduce their ability to fight cancer effectively. This reduced persistence might be due to natural killer (NK) cells targeting the HLA class I-deficient CAR T cells through a “missing-self” recognition process. Moreover, HLA class I molecules may be essential for T cell survival, as studies in B2M-deficient mice show a complete absence of CD8+ T cells. Another complication is the potential for CRISPR/Cas9 to introduce unintended genetic disruptions, such as large chromosomal deletions or karyotype abnormalities. To avoid these risks, base editing—which does not produce double-stranded DNA breaks—is being explored. This technique has been applied in creating base-edited allo-CAR T cells for patients with refractory T-cell acute lymphoblastic leukemia (T-ALL). Notably, in that study, the B2M gene remained intact, meaning the HLA class I structure was preserved. In addition, another genome-editing method, TALEN, has been utilized to disrupt both the TRAC and B2M genes, aiming to engineer “immune-evasive” universal CAR T cells that can be used across different patients without triggering rejection [68].

LIMITATIONS OF CAR-T CELL THERAPY:

Antigen escape

A significant challenge of CAR-T cell therapy is the emergence of tumor resistance to CAR constructs that target a single antigen. While CAR-T cells aimed at a specific antigen initially yield high response rates, many cancer cells in a considerable number of patients treated with these cells exhibit either a partial or complete loss of the targeted antigen expression. This occurrence is referred to as antigen escape. For instance, although 70–90% of patients with relapsed and/or refractory acute lymphoblastic leukemia (ALL) respond positively to CD19-targeted CAR-T cell therapy, recent follow-up findings indicate the development of a common resistance mechanism. This includes the downregulation or loss of CD19 antigen in 30–70% of patients who experience a recurrence of the disease after treatment. [69] Likewise, a decrease or absence of BCMA expression has been noted in multiple myeloma patients who are receiving treatment with BCMA-targeted CAR-T cells. [70,71] Comparable patterns of antigen escape resistance have been noted in solid tumors. For instance, a case report on CAR-T cell therapy aimed at IL13Ra2 in glioblastoma indicated that tumor recurrences showed reduced expression of IL13Ra2 In solid tumors, various tandem CARs have undergone testing in preclinical models, such as HER2 and IL13Ra2 in glioblastoma, as well as HER2 and MUC1 in breast cancer. In both instances, targeting two pathways led to better anti-tumor responses than therapies focused on a single target. In the glioblastoma research, CARs that target HER2 and IL13Ra2 demonstrated enhanced anti-tumor effectiveness and reduced antigen escape in comparison to two alternative dual-targeting treatments. [72] This research highlights the significance of enhancing the choice of target antigens that not only boost antitumor responses but also reduce the mechanisms of antigen escape to avert relapse.

On-target effects that occur outside of the tumor.      

Identifying solid tumor antigens is difficult since they frequently appear in normal tissues as well, which raises the risk of “on-target off-tumor” toxicity. Consequently, meticulous antigen selection is vital for the design of CARs. An effective approach to enhance specificity involves targeting tumor-specific post-translational modifications, such as the overexpressed truncated O-glycans like Tn and sialyl-Tn.While initial generation CAR-T cells aimed at TAG72 in colorectal cancer did not generate an anti-tumor effect, newer models of second generation TAG72-CAR-T cells along with various tumor-specific post-translational modifications are now under exploration.[73]Continued advancement of creative approaches to diminish antigen escape and identify antigens that can trigger adequate antitumor effectiveness, while reducing toxicity issues, will be essential to broaden the clinical application of CAR-T cell therapies for both hematological cancers and solid tumors.

Movement of CAR-T cells and their penetration into tumors

One approach to address these limitations is by employing delivery methods beyond systemic administration, as localized treatment (1) reduces the requirement for CAR-T cells to travel to the sites of disease and (2) decreases on-target off-tumor toxicities because the CAR-T cells’ activity is specifically directed at tumor cells, minimizing engagement with healthy tissues. Preclinical studies have shown enhanced therapeutic effectiveness of intraventricular administration of CAR-T cells targeting HER2[ref.14] and IL13Ra2 in brain metastases of breast cancer and glioblastoma. These findings have prompted three current clinical trials examining the intraventricular delivery of CAR-T cells for glioblastoma (NCT02208362, NCT03389230) and for recurrent brain or leptomeningeal metastases (NCT03696030). In a similar vein, preclinical research indicated that intrapleural injection of CAR-T cells leads to improved treatment outcomes for malignant pleural mesothelioma, resulting in an ongoing phase 1 clinical trial (NCT02414269). [74] While localized injection seems to demonstrate greater effectiveness, this method is theoretically constrained to individual tumor lesions or oligometastatic conditions. A newly developd approach that seems to greatly enhance CAR-T cell movement entails the expression of chemokine receptors on CAR-T cells that align with and react to chemokines produced by tumors. Physical obstacles like the tumor stroma hinder CAR-T cell therapy since these barriers prevent the effective penetration of tumor cells. The stroma mainly consists of an extracellular matrix, with heparan sulfate proteoglycan (HSPG) being the key element that CAR-T cells need to break down to infiltrate the tumor. CAR-T cells modified to produce heparanase, an enzyme that breaks down heparan sulfate proteoglycans (HSPG), exhibit improved infiltration into tumors and increased antitumor effectiveness. Likewise, CAR-T cells targeting fibroblast activation protein (FAP) show enhanced cytotoxic abilities by diminishing tumor-associated fibroblasts in animal studies. [75]

Future Developments and Latest Prospects In CAR-T Cell Therapy:

1. Structural and Functional Modification of CAR-T Cells

There are two approaches for designing CAR-T cells that target multiple antigens. The first involves introducing CAR molecules with different scFvs into the same T cell to create a parallel configuration of dual/multi-specific CAR-T cells, while the second involves incorporating two or more scFvs into a single CAR molecule that is then transferred into one T cell to establish a tandem configuration of dual/multi-specific CAR-T cells.[76] Multi-targeted CAR-T cells can effectively mitigate the issue of antigen loss that can occur during therapy and also decrease the likelihood of CRS. The innovative cell therapy product STAR-T incorporates a TCRab-based dual-chain receptor that links immunoglobulin heavy and light chain variable regions (VH and VL) with TCR-Ca and TCR-Cb, respectively, differing from AbTCR, which utilizes TCRgd, and the single-chain scFv-derived TAC and TRuC receptors. The structure of STAR closely resembles that of a natural TCR. This receptor has both TCR and antigen recognition capabilities, and its sensitivity to antigens surpasses that of CAR-T cells, which significantly lowers the chances of tumor recurrence caused by antigen loss in various solid tumor models. [77] Allo-715 is designed to eliminate GvHD and reduce the rejection of CAR-T cells. The excessive expression of RUNX3, a key modulator of T-cell immune response, was discovered to effectively decrease the depletion of CAR-T cells when activated by antigens. [78]

2. Boolean Logic Gate Designs

Employing Boolean logic gates has become a promising approach that can control the function of CAR-T cells under particular circumstances by programming them to function as switches, thus enhancing their specificity. (LINK) CAR is a Boolean logic and gated CAR-T cell platform that operates quickly and reversibly, demonstrating superior performance compared to other systems. AdCAR-T comprises a dual-component signaling system utilizing a split recognition/activation mechanism, where the labeled adapter molecules (AM) convey the antigen-recognition signal to stimulate T cells through the anti-labeled CAR, enhancing specificity and effector control. [79]

3. Armored CAR-T Cells

The idea of "armored" CAR-T cells holds significant promise in the field of immunotherapy. This approach has been proposed to address challenges related to tumor evasion and unintended toxicity, and it involves enhancing cells with additional protective features alongside second- or third-generation CAR-T cells. This includes the simultaneous expression of essential cytokines, chemokines, or co-stimulatory ligands to boost their immunomodulatory capabilities and anti-tumor effectiveness. For instance, mice given “armored” CAR-T cells demonstrated a much greater reduction in tumor size compared to those receiving standard CAR-T cell treatment. [80] The release of chemokines or cytokines by “armored” CAR-T cells can improve the cells’ ability to move towards the tumor location and mitigate immunosuppressive signals.

4. Research on Novel Targets

CD317 is a novel target antigen for glioblastoma, which is among the most aggressive solid tumors. Normal neurons and microglial cells do not express CD317, but it promotes the proliferation of various malignant cells, such as those from liver and breast cancers, as well as glioblastoma. A recent investigation described the development of CAR-T cells through lentiviral transduction of CD317. In a mouse model of glioma, these CAR-T cells demonstrated significant anti-tumor efficacy. B7-H3 CAR-T cell therapy demonstrated enhanced targeted suppression of tumor cell proliferation and cytokine release without inducing significant toxic side effects, as evidenced by experiments conducted on mice. The modified MUC1-CAR-T cells exhibited notable anti-tumor activity against esophageal cancer cells. [81]

5. CAR-NK Cell Therapy

CAR-NK cells exhibit considerable potential. Similar to the structure and overall design of CAR-T cells, CAR-NK cells are composed of extracellular signaling domains, transmembrane segments, and intracellular domains. Clinical studies have shown that the safety profile of CAR-NK cells is better than that of CAR-T cells, as the cytokines they produce are primarily GM-CSF and IFN-g, which are not associated with the cytokines that trigger CRS, resulting in a significantly lower risk of CRS. By employing bifunctional lipid nanoparticles (DLNPs) to stimulate and effectively transport mRNA that encodes CAR. The research also investigated how DLNPs activate and regulate NK cells, in addition to optimizing the concentration of the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) within lipid nanoparticles to enhance gene delivery effectiveness and minimize cytotoxicity. CAR-NK cells that target Glypcan-3 showed notable therapeutic effectiveness. [82]

6. Combination Therapies

Individuals who underwent treatment with a combination of CAR-T cell immunotherapy and chemotherapy or radiotherapy experienced considerably improved overall survival compared to those who received chemotherapy or radiotherapy alone.SN-38-L-EGFRvIII-CAR-T cells possess the ability not only to produce their immunological effects but also to serve as a vehicle for the precise delivery of chemotherapy agents. DC vaccines greatly improved the growth, infiltration, and longevity of the MSLN-CAR-T cells. Combining radioimmunotherapy with CAR-T cell therapy may significantly enhance treatment efficacy. [83]

7. Advanced Manufacturing and Delivery Technologies

The study utilized an LNP (antigenic lipid-like nanoparticle), which incorporates CD3 and CD28 antibody fragments, to activate and expand T cells. Initially, the lipid-like nanoparticles were conjugated with CD3 and CD28 antibody fragments. Subsequently, CAR mRNA was encapsulated within aLNP to promote effective transcription in T cells. Lastly, the aLNP was co-cultured with T cells to facilitate the uptake of CAR mRNA by the T cells. The results of the experiment revealed that aLNP effectively delivered CAR mRNA to T cells, leading them to express CAR. [84]

8. Dual-Targeting CAR Platforms

AbTCR-CSR was effective in eliminating AML cells. Analysis through flow cytometry revealed that AbTCR-CSR demonstrated a notable capacity for recognizing and killing AML cells, while showing no cytotoxic effects on normal peripheral blood mononuclear cells (PBMCs). [85]

CONCLUSION:

CAR-T cell therapy is a paradigm shift in cancer treatment, providing long-lasting remissions in otherwise treatment-refractory hematological malignancies. The development of CARs from the first to the next generation has shown how important it is to integrate costimulatory domains, cytokine support, and safety switches to improve efficacy and control. However, there are still major obstacles to overcome, such as severe toxicities like cytokine release syndrome and neurotoxicity, antigen escape, limited trafficking to solid tumors, and the immunosuppressive tumor microenvironment. To overcome these obstacles, a multifaceted approach is needed, ranging from dual-targeting and armored CAR designs to synthetic circuits and gene-edited universal cell products. The field's future lies in combining CAR-T therapy with checkpoint inhibitors, targeted drugs, and advanced delivery systems, while utilizing nanotechnology and synthetic biology to improve precision, persistence, and monitoring.

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