Chimeric antigen receptor (CAR) T-cell therapy is a successful adoptive cell therapy strategy that has significantly improved the treatment and prognosis of hematological malignancies. So far, the Food and Drug Administration (FDA) has approved seven CAR-T cell products to treat hematological malignancies (1–3). CAR-T cells can have a single chain variable fragment (scFv) on their surface that recognizes tumor-related surface antigens after genetic engineering modification and pass through CD3ξ, 4-1BB (a costimulatory protein, a component of CAR intracellular modules), CD28 and other intracellular signal domains to initiate an anti-tumor immune response and release INF-γ, TNF-α, perforin and granzyme can kill tumor cells (4–7). Compared with traditional T-cell receptors (TCRs), CARs can independently recognize tumor antigens and do not rely on antigen-derived peptides presented by class I molecules of the major histocompatibility complex (MHC). Moreover, when CARs are added to T-cells, they can be designed to specifically recognize and bind to these proteins or gangliosides on the surface of cancer cells (8). This recognition triggers the activation of the T-cells and leads to the destruction of the cancer cells; to date, four generations of CARs have been created (9). The first-generation CARs only possess an intracellular signaling domain of CD3ξ to activate T cells (10). In the second-generation CARs, the intracellular signaling domain added a co-stimulatory domain to CD3ξ, usually 4-1BB (CD137) or CD28, to enhance T cell activation and proliferation (11). CD3ζ is a T-cell-activated switch in CARs, and the co-stimulatory domain increases the effectiveness and durability of the CAR (12). The third-generation CARs have three intracellular signaling domains, with two co-stimulatory signaling domains [4-1BB (CD137) and CD28] used in conjunction with the CD3ξ domain. The fourth-generation CARs are referred to as T cells redirected for universal cytokine-mediated killing. Here, the addition of cytokine expression cassettes to the second-generation CARs enables the secretion of proteins, including interleukin (IL)-2, IL-12, IL-10, IL-17 and IL-15, and promotes enhanced T cell activation, while recruiting a patient's immune cells (4,13).

CAR-T cell therapy is an up-and-coming adoptive immunotherapy technique with remarkable efficacy in the treatment of hematological tumors. However, despite its potential, several obstacles still limit its effectiveness in hematological and solid tumors (14,15). There is a need for continued research to address these challenges and optimize the use of CAR-T cell therapy in the treatment of hematological and solid tumors. This includes developing strategies to overcome antigen escape, reducing the toxicity associated with CAR-T cell therapy, improving the penetration of CAR-T cells into solid tumors, and enhancing the long-term efficacy of CAR-T cell therapy. By addressing these challenges, CAR-T cell therapy has the potential to become an even more effective and widely used therapy for cancer treatment.

CAR-T cell therapy is a promising cancer treatment that genetically modifies a patient's T cells to recognize and attack cancer cells. While CAR-T cell therapy has been reported to show great success in treating certain types of cancer, such as lymphoma, this approach has certain limitations and challenges (16). Reportedly, the main limitations of CAR-T cell therapy for hematological malignancies are antigen escape, CAR-T cell associated toxicities (17). There are also challenges such as insufficient CAR-T cell infiltration and immunosuppression when it is used to treat solid tumors (18).

Antigen escape and toxicity are significant challenges associated with CAR-T cell therapy when used alone or in combination with radiotherapy (19). One approach to reducing antigen escape is to develop dual-targeting CAR-T cells that target multiple antigens on tumor cells, which increase the breadth of the CAR-T cell response and reduce the likelihood of antigen escape. CAR-T cell therapy can also target antigens which are essential for cancer cells, which reduces the risk of antigen escape, and controlled expression of the target antigen, such as through inducible promoters or RNA interference, helps to reduce off-target effects and toxicity (20). Optimizing toe dose of CAR-T cells also helps to reduce toxicity and the risk of antigen escape. Previous studies have reported that lower doses of CAR-T cells were more effective than higher doses, as higher doses caused more severe toxicity and increased the risk of off-target effects (21). Reducing the risk of antigen escape and toxicity associated with CAR-T cell therapy when used alone or in combination with radiotherapy is a critical area of research. These strategies can help improve CAR-T cell therapy's safety and efficacy and reduce the risk of treatment-related side effects (22).

Radiotherapy (RT) is a traditional first-line anti-tumor therapy. In addition to directly killing the tumor cells, radiotherapy also activates immune responses in the TME (56) (Fig. 1D). Dying immunogenic cells secrete radiation-associated antigen proteins, tumor-associated antigens, and cell death-associated molecular patterns that increase target antigen expression, which leads to the activation of immune surveillance (6,57). Radiotherapy also increases the number of MHC molecules on antigen-presenting cells (APCs), which leads to the translocation of calreticulin (CRT), which releases high mobility group box 1 protein as well as ATP and heat shock protein, which activated dendritic cells, APCs, etc., and enhanced endogenous target antigen-specific immunity (13,58).

In addition, radiation converts immunosuppressive cells in the TME into immune-promoting cells, which strengthens the anti-tumor behavior of CAR-T cells (59) (Fig. 1B). Radiotherapy increases the number of M1-like macrophages and decreases M2-like macrophages and MDSCs in the TME (6). Although radiotherapy offers strong advantages in tumor treatment, it also promotes radio resistance in tumor cells, which results in the failure of local cancer treatment (60). However, radioresistant tumor cells are sensitive to T-cell retargeting, therefore, combining radiation with CAR-T cell immunotherapy can achieve the greatest efficacy. According to previous reports, radiotherapy boosts the efficacy of CAR-T cells through multiple mechanisms (61,62).

In the TME, radiotherapy triggers M1-type macrophages to secrete more chemokines, such as CXCR3, CXCL9-11 or CXCL16, in response to IFN-γ, which promotes CAR-T cell recruitment (63). Irradiation also induces an increase in the production of the integrins ICAM-1 and VCAM-1 in vascular endothelial cells, which facilitates the movement of CAR-T cells across the vascular endothelium into the tumor tissue (64,65) (Fig. 1C). In addition, radiotherapy normalizes tumor blood vessels and improves tumor reoxygenation, which improves hypoxia-induced therapy resistance (60). Radiotherapy also promotes the local expansion of CAR-T cells in tumors (Fig. 1A). In conclusion, radiotherapy may improve the effectiveness of CAR-T cell therapy by activating and enhancing endogenous target antigen-specific immune responses, which promotes CAR-T cell recruitment and expansion, and modulates the inflammatory TME (66,67).

Although there is ample evidence that the combination of radiotherapy and CAR-T cell therapy exerts synergistic antitumor effects, radiation therapy can cause immunosuppression and CAR-T cell death, which reduces the efficacy of the combination therapy. Radiotherapy induces the transformation of immune cells to immunosuppressive cells, such as Tregs, and enhances the effect of immunosuppressive molecules, such as IL-10, TGF-β and adenosine in the TME (68–70). Tregs are key cells that promote immunosuppression in the tumor immune microenvironment. Radiotherapy causes increased Tregs infiltration in irradiated tumor areas, and Tregs show increased Akt expression, which makes them more radioresistant than other T cell subsets (71). A preclinical study reported that targeted depletion of Tregs after radiotherapy promoted long-lasting antitumor effects (72). Notably, it has been reported that the infiltration efficiency of Tregs depends on the dose at each administration rather than the total dose and that this is positively correlated with each radiation dose (73). Therefore, when combining radiotherapy with CAR-T cell therapy, the optimal fractionation and radiation dose should be evaluated to maximize the efficacy and minimize the toxic side effects (74).

In addition, radiotherapy also directly damages CAR-T cells. Although there are few reports on the direct damaging effects of radiotherapy on CAR-T cells, high-dose radiotherapy administration immediately after the infusion of CAR T cells needs to be carefully evaluated for its effects on the health of the CAR-T cells (75). Notably, in a clinical study, it was shown that administration of 2 Gy of radiotherapy per dose induced in vivo expansion of CAR-T cells (76).

Radiotherapy is a double-edged sword, with differences in dose and fractionation times resulting in different immunomodulatory responses. On the one hand, it synergistically promotes anti-tumor effects when used in combination with CAR-T cell treatment (77). On the other hand, radiotherapy promotes an immunosuppressive microenvironment and reduces the anti-tumor effects of CAR-T cells (78). Therefore, the timing, total radiation dose and the number of fractions of radiation therapy during CAR-T cell treatment are critical for the success of the combination therapy (67). When CAR-T-cell therapy is used in union with radiotherapy, the standard total radiation dose and fractionations need to be optimized (56,79). Few previous studies have explored this, but the conclusions of studies on other immunotherapies such as immune checkpoint blockage and their combination with radiotherapy can be used for reference. Existing research indicates that the dose-fraction regimen of 8 Gy ×3 is the best standard fractionation regimen for immunotherapy combinations (80,81). However, certain studies have suggested that low-dose radiotherapy (<2-4 Gy) may be a better complement to CAR-T cell treatment based on the radioimmunological effects (82).

Substantial evidence suggests that hypo-fractionated RT (>2 Gy/fraction) or standard fractionated RT (1.8-2 Gy/fraction) enhances the ability of radiation to promote anti-tumor immune response by the mechanisms described above (18,83). Hypo-fractionated irradiation has been reported to be the best radiotherapy method for extending the benefits of immunotherapy (84). CAR-T cell treatment combined with photon radiation is the type of combination therapy that has reported to demonstrate the best anti-tumor effects and minimal side effects (85). However, other types of radiation therapy such as iodine 125 seed brachytherapy combined with Robo1 (a member of the axon guidance receptor family that has also been reported to play a role in modulating chemotaxis of T cells and tumor angiogenesis) dimeric antigen receptor-natural killer cells has also been reported to suppress human pancreatic tumor growth in mice (39,86). Minimizing immunosuppression and CAR-T cell death due to radiotherapy and optimizing the dose and fractionation of radiotherapy when combined with CAR-T cell therapy are essential considerations for enhancing treatment efficacy and reducing toxicity (87). These strategies are still being studied, and more research is needed to fully understand the optimal parameters for combining radiotherapy and CAR-T cell therapy (88).

The implementation and feasibility of combined radiotherapy and CAR-T cell therapy is an area of active research and clinical investigation. The combination of these two therapies has the potential to enhance treatment efficacy; however, certain challenges and considerations need to be addressed (89). One potential benefit of combining radiotherapy and CAR-T cell therapy is that radiotherapy can help to create a more favorable TME for CAR-T cells to function. Radiation can cause DNA damage in cancer cells, which leads to the release of tumor-associated antigens and the activation of immune cells (90). One concern is that radiation can cause damage to CAR-T cells, which may compromise their function and reduce treatment efficacy. Several clinical trials to investigate the feasibility and efficacy of combining radiotherapy and CAR-T cell therapy to treat various types of cancer are understood to be currently underway (60,91,92). These studies are reported to be evaluating dosing schedules, radiation techniques and CAR-T cell products. The combination of radiotherapy and CAR-T cell therapy has the potential to be a powerful treatment option for certain types of cancer such as prostate cancer, pancreatic cancer and glioblastoma (93). The below section enumerates and summarizes the existing preclinical and clinical studies which have reported on the combination of CAR-T cell therapy and radiotherapy, to understand its safety and efficacy and to assess the optimal timing, total radiation dose and the number of fractionations of radiotherapy, and CAR-T cell infusion.

CAR-T cell therapy is a revolutionary and promising immunotherapy technology. Mature CAR-T cell products have been approved for treating hematological tumors and have demonstrated promising results in the clinic. However, its application in hematological and solid tumors faces many unexpected difficulties and reduces the therapeutic effect. As a conventional anti-tumor therapy, radiotherapy can overcome certain obstacles by enhancing the specific immunity against endogenous tumor antigens, which promotes the amplification and expansion of CAR-T cells and improves the hypoxic TME (108). However, radiotherapy has advantages and disadvantages, and differences in dose and the number of divisions generates different immune regulatory responses. Therefore, the timing of radiotherapy, the total radiation dose and the number of fractionations, and the type of radiation are critical parameters that influence the success of the combination therapy (107).

In preclinical and clinical studies on the combination of CAR-T cells and radiotherapy, radiotherapy has been reported to promote the rapid exosmosis and migration of CAR-T cells from the vascular system into the tumor site, activate anti-tumor immunity, reduce tumor load, improve the therapeutic effect and reduce the occurrence of CAR-T cell related severe toxic reactions. Although the antitumor effects of this combined treatment strategy are definite, there is still a long way to go before a standard treatment plan is put forward, and more research is needed to fully understand the optimal parameters for combining therapies to address the associated challenges for clinical translation.