CAR T-cells can amplify or attenuate the immune response due to well-thought out the intracellular domains design, by generating signals of varying strengths, durations, and intensities. An expected complication of this therapy is that excessive CAR T-cell activation drives a cytokine storm, leading to “on-target toxicity.” Another potential issue is “off-target toxicity” to normal cells when the current targets do not allow distinction between normal and cancer cells (37). On-target and off-target toxicities of CAR T-cells need to be monitored and measures taken quickly to abrogate them. In contrast, CAR T-cell exhaustion induced by sustained signaling of chimeric receptors contributes to poor persistence and limits the durability of CAR T-cell immunotherapy.

T-cell exhaustion is a dysfunctional state of differentiated T-cells that differs from anergy and senescence. The exhausted state is a complex phenotype associated with progressive loss of effector functions and poor memory T-cell response (41). This exhausted state is associated with increased expression of multiple inhibitory receptors, including PD1 (42). Another concern about conventional CAR T-cells is fixed antigen specificity, which means that only one TAA can be targeted at a time (37). This limitation harbors the risk of tumor-variant appearance and limits the eficacy of CAR T-cell therapy.

CAR T-cells do not follow the classical T-cell–target cell immunological synapse topology. The epitope location, structural dimensions of the target molecule, and extracellular spacer length of CAR allows for the specificity of CAR T-cell dimensional interactions (43, 44). Steric constraints due to the location of the target epitope may require structural modeling of the CAR for adapting/modulating the extracellular spacer length and promoting synapse formation between effector T-cells and tumor cells (45, 46). Other aspects, including the density and accessibility of target molecules to tumor cells, need to be considered (46). In the context of immunological synapse dynamics, tumor cells and their microenvironment underpin specificity and complexity of immune responses through secretion of immunosuppressive soluble factors, such as indoleamine-2,3-dioxygenase-1 (IDO1), PD-L1, and IL-10 (47–50), and recruitment of immunosuppressive cells, such as tumor-associated macrophages and myeloid-derived suppressor cells (51). This immunosuppressive microenvironment limits immune cell homing and infiltration and inhibits effector T-cell activity (52).

The metabolic landscape created by cancer cells in the TME dampens the ability of T-cells to respond (53). The energetic state of the TME and catabolites generated by tumor metabolism play a critical role in T-cell biology, affecting T-cell infiltration, survival, and function (54). The production of immunosuppressive enzymes, such as IDO, tryptophan-2,3-dioxygenase, nitric oxide synthase, and arginase-1, impairs the activation and effector function of T-cells by causing tryptophan and L-arginine deficiency. These aspects of the TME impact CAR T-cell functions and lead to significant clinical implications (55).

According to data from clinical trials on CAR T-cells in solid tumors, immunotherapy outcomes are associated with multiple parameters that reflect cross-talk between the TME and the immune system, including tumor mutational burden (TMB), immunologically hot tumors with inflammatory microenvironments containing high levels of infiltrating cytotoxic T-cells, immune escape mechanisms, timing of administration, and delivery systems of immunotherapeutic agents (56).

These considerations indicate that the following strategies are key for the generation of potent CAR T-cells with limited side effects: improving CAR T-cell chimeric receptor flexibility, engineering an appropriate extracellular domain for antigen recognition and binding ability, and improving intracellular signaling capacities. Further understanding of TME variables will help in the design of therapeutic strategies that would enhance the clinical effectiveness of CAR T-cells, especially in solid tumors ( Figure 2 ).

One of the first preclinical studies, published by Ang et al., focused on the cytotoxic effects of epithelial cell adhesion molecule (EpCAM or CD326)-directed CAR T-cells. EpCAM is a transmembrane glycoprotein expressed on the surface of normal epithelial cells (88). EpCAM is one of the first cancer stem cell antigens to be reported. Its overexpression is associated with increased cell proliferation, migration, invasion, and tumor metastasis (88, 89). In CRC, EpCAM is overexpressed in more than 90% of all cancer cells and is associated with poor prognosis (89, 90). In the study conducted by Ang et al., EpCAM-targeting CAR T-cells were developed using a humanized monoclonal antibody, and their ability to treat CRC-xenografted mice was assessed (76). This in vivo immunodeficient mouse model displayed a phenotype similar to late-stage metastatic cancer in humans, including extensive peritoneal metastasis and ascites production (76). Interestingly, repeated injections of EpCAM-CAR T-cells suppressed peritoneal disease progression in tumor-bearing xenografted mice.

Along with EpCAM, carcinoembryonic antigen (CEA) is another studied target of anti-CRC CAR T-cells. CEA is a glycoprotein belonging to the immunoglobulin family and is overexpressed in many types of human cancers, including colon, pancreatic, gastric, lung, and ovarian cancers. CEA is overexpressed in 98.8% of CRC tissue samples and is one of the most important diagnostic and prognostic tumor markers (91, 92). Thus, CEA-targeted therapies hold promise for generating novel therapeutic strategies for CRC treatment. CAR T-cells targeting CEA have demonstrated excellent antitumor activity both in vitro and in vivo, which was significantly increased when combined with interleukins, such as IL-12 (68). Schlimper et al. generated cytokine-induced killer cells from blood lymphocytes of patients with CRC and transformed them into CEA-targeting cells by engineering a new CAR. Such cytokine-induced killer cells showed enhanced selectivity towards autologous CEA⁺ colon carcinoma cells and increased secretion of proinflammatory cytokines, such as IFN-γ and cytolysis (57, 93). Owing to the potent effects of IL-12 on T-cell functions, Chi et al. designed CEA-specific CAR T-cells and used them in combination with recombinant human IL‐12 to improve CRC tumor responses (68). The authors showed that CAR T-cells used simultaneously with recombinant human IL‐12 significantly enhanced antitumor activity in a xenografted tumor model. These data highlight the importance of adding cytokines to increase CAR T-cell activity in solid tumors.

To improve efficacy against peritoneal metastases, intraperitoneal (IP) delivery of CAR T-cells have been assessed. Compared with systemic intravenous delivery, regional IP infusion of anti-CEA CAR T-cells effectively protects against CEA+ peritoneal tumors and metastasis, enhances the effector memory response and is associated with increased IFNγ production (70). Therefore, it has been suggested that regional delivery improves CAR T-cell penetration deep inside solid tumors without increasing toxicity or overcoming barriers associated with systemic delivery. Furthermore, intratumoral accumulation of myeloid-derived suppressor cells and regulatory T-cells (Tregs) was detected in IP tumors, and the removal of these cells enhanced intra-tumoral T-cell cytotoxicity (94). Combination of IP CAR T-cell delivery and suppressor cell targeting appears to be a viable strategy for circumventing the immunosuppressive microenvironment in solid tumors.

In a xenograft model induced by co-inoculation of CAR T-cells and tumor cells, Huang et al. reported complete eradication of the tumor using EGFRvIII-CAR T-cells in combination with miR-153. These results indicate that miR-153 inhibits IDO1 expression in CRC cells and enhances CAR T-cell therapy. Thus, combination of IDO1 inhibitors and CAR T-cells can prospectively serve as an attractive therapeutic strategy for treating CRC and solid tumors (73).

To generate an adaptable gene-based vector that confers immune-cell specificity to various cancer types, Tamada et al. genetically engineered cells to express a CAR that binds a fluorescein isothiocyanate (FITC) molecule (anti-FITC CAR T-cells). The results indicated an improvement in the ability of the CAR T-cells to treat EGFR-positive CRC tumor-bearing mice (95). This versatile model confers T-cell specificity to a tag, which is easily conjugated with various clinically used tumor-reactive antibodies such as cetuximab, rendering the process economical. The results presented by the authors showed that specific interactions between FITC-labeled cetuximab and anti-FITC CAR T-cells in an immunocompromised mouse model, delayed colon cancer growth (57, 95).

A pivotal challenge in solid tumor CAR T-cell therapy is the potential toxicity of on-target/off-tumor effects owing to shared antigens in normal tissues. Placental alkaline phosphatase (PLAP) is a TSA that is not expressed in normal tissues but is overexpressed in CRC, rendering it an attractive target for CAR T-cell therapy (96). PLAP has been detected in more than 20% of colorectal adenocarcinomas. To assess the therapeutic potential of PLAP, a second-generation humanized PLAP-CAR T-cell construct was used in a xenograft model of CRC, which showed significantly decreased tumor growth in vivo (83). Humanized PLAP-CAR T-cell activity was further amplified by the addition of ICB therapy (anti-PD-1 or LAG-3 antibodies). This suggests that combining CAR T-cell therapy with ICB may be an effective therapeutic approach for CRC. Hence, the synergistic effect of the combination can be achieved through two steps: infiltration of immunogenically silent tumors by CAR T-cells followed by ICB therapy, which reverses CAR T-cell inhibition and restores functional persistence.

Similarly, Dezfouli et al. reported that the stress-inducible membrane form of heat shock protein 70 (mHsp70) was an ideal CAR T-cell target for CRC treatment, due to its sufficient affinity, specificity, density, and valence, while being undetectable in normal cells and tissues (97–99). The antitumor activity of anti-Hsp70 CAR T-cells was demonstrated by increased granzyme B degranulation and IFN‐g secretion, associated with enhanced cytotoxicity against CRC cells expressing mHsp70. Furthermore, the greater in vitro persistence of anti-Hsp70 CAR T-cells compared to that of normal T-cells promises long-term persistence in a clinical context (84). Despite the promising in vitro results, which support the applicability and specificity of the anti-Hsp70 CAR T-cell approach, and the reduced likelihood of off-target effects, further studies are needed for exploring the safety and efficacy of this approach in relevant in vivo models.

Daly et al. used genetically modified T-cells to target the CRC-associated antigen EGP40. The CAR construct contained two EGP40-specific monoclonal antibodies fused in line with the Fc receptor gamma-signaling chain (57, 100). Human redirected T-cells specifically recognize and lyse EGP40-expressing colon carcinoma cells (100).

A major drawback in the development of CAR T-cell therapy is the toxicity to vital organs (78). Guanylyl cyclase C (GUCY2C) is a membrane-bound receptor that is expressed in >95% of CRC metastases (101, 102). The GUCY2C receptor expressed in intestinal epithelial cells triggers signaling pathways that regulate epithelial cell proliferation, differentiation, and apoptosis (103). In a syngeneic murine CRC model with lung metastases, Magee et al. reported that GUCY2C-targeting CAR T-cells provided long-term protection in the absence of intestinal toxicity (104). CAR T-cells exhibited T-cell activation, cytokine production, and enhanced cytolytic activity in an antigen-dependent manner. In a human xenograft model, GUCY2C-targeting CAR T-cells eliminated human CRC metastatic cells, resulting in survival benefits (105).

Natural killer group 2 member D (NKG2D) is an activating immunoreceptor associated with tumor immunosurveillance owing its ability to recognize tumor cells and initiate an antitumor immune response. In humans, it is expressed on NK cells, invariant natural killer T-cells, CD8+ T-cells, γ δ T-cells, and certain autoreactive or immunosuppressive CD4+ T-cells (106). Ligands for NKG2D are expressed in response to external signals, such as stress or pathogens, and during neoplastic cell transformation; however, they are typically absent in healthy tissues, rendering it a promising CAR candidate (106, 107). Soluble NKG2D ligands impair NKG2D-dependent functions, resulting in an impaired antitumor response. Thus, they are associated with poor clinical prognosis and metastasis (108). High expression of NKG2DL in human colorectal carcinomas and its correlation with improved disease-free survival rationalized the use of NKG2D as a potential immunotherapy target (109). Deng et al. reported the role of third-generation NKG2D CAR T-cells and investigated their cytotoxicity against human CRC cells in a xenograft model (82). In this model, NKG2D CAR T-cells showed significantly high killing activity, inhibited tumor growth, reduced tumor size, and produced longer overall survival with no severe toxicity to vital organs. Moreover, tumor sections from treated mice showed lymphocyte infiltration (82). However, regarding the genetic modification strategy, the authors showed that electroporation of T-cells in the presence of a NKG2D-CAR minicircle DNA vector reduced cell viability and CAR expression in a time-dependent manner (82). This strategy, based on CAR transient expression, produces less toxic effects but requires re-inoculation for achieving stable antitumor activity (76).

There is growing evidence that CRC is derived from cancer stem cells, which are slow-cycling, self-renewing, and pluripotent (110–112). This small fraction of the total cell population can undergo epithelial–mesenchymal transition, a key process for metastasis formation and therapeutic drug resistance (113, 114). Double cortin-like kinase 1 (DCLK1), a microtubule-associated protein, maintains stemness in CRC cells, and it is a putative tumor stem cell (TSC) marker in CRC mouse models (115–118). Sureban et al. described DCLK1-targeting CAR T-cells as an efficient therapeutic approach for eradicating CRC stem cells (110). DCLK1-targeting CAR T-cells have been shown to display higher cytotoxicity in vitro and exhibit antitumor activity in in vivo xenograft mouse models in vivo. Intravenous injections of DCLK1-directed CAR T-cells have been shown to reduce tumor size in tumor-bearing mice, strongly suggesting that DCLK1-based CAR T-cells inhibit CRC tumor growth in vivo without obvious cytotoxicity to normal tissues (110).

The availability of clinically relevant CRC models is essential for CAR T-cell testing and molecular analysis. In this regard, patient-derived xenograft mouse models of CRC have been generated and used in preclinical studies of anti-CRC CAR T-cells. These models reflect the clinical and molecular heterogeneity of patients, and they have been used in two preclinical studies on anti-CRC CAR T-cells (79, 81, 119). One study selected highly expressed mesothelin (MSLN/MESO) as a target for CAR T-cells, and MSLN-CAR T-cells showed significant antitumor efficacy (81). MSLN expressed at low levels in normal tissues acts as a tumor differentiation antigen.MSLN is a glycosylphosphatidylinositol-anchored protein that was first described as a membrane protein expressed in normal and neoplastic mesothelial cells. In subsequent studies, a broader expression pattern has been demonstrated mainly in cancer, with overexpression observed in multiple solid tumor types, including CRC (120). A significant expression of MSLN is significantly expressed in CRC, with up to 60% of cases showing positivity (120–122). In in vitro and iIn vivo models, MSLN overexpression activates the downstream PI3K/AkT, NF-κB, and MAPK/ERK pathways, which promote cancer cell proliferation, migration, and metastasis and hinder apoptosis (120–128). Although the biological significance of MSLN expression in CRC remains poorly understood, it is certain that MSLN plays a positive role in CRC cell proliferation. Furthermore, MSLN has shown potential as a prognostic biomarker and potential target for CRC treatment.

In another study, a patient-derived xenograft mouse model implanted with CRC xenograft has been used for assessing the antitumor capacity of human epidermal growth factor receptor 2 (HER2)-CAR T-cells (79). HER2 is an oncoprotein that is overexpressed in a small but relevant proportion of patients with CRC, especially in anti-EGFR-resistant and RAS wild-type tumors (79, 129). HER2-targeted CAR T-cells have been engineered and their in vivo efficacy and safety was tested in a patient-derived xenograft model in vivo (79, 80). Significant regression of CRC xenografts has been observed, with protection from relapses upon tumor rechallenge in this mouse model. This enhanced efficacy results in a significant survival benefit (79). Consequently, HER2 has become a promising target for CAR T-cell therapy in CRC. Nevertheless, as clinical trials are full of uncertainty, one of the first clinical trials testing third-generation HER2 CAR T-cellsin patients with metastatic cancers (NCT00924287) was terminated after the first patient died of severe acute respiratory failure as a result of the treatment.

Recently, Feng and his coworkers used CDH17 as a target for CAR T-cells. In humans, CDH17 is a CA²⁺-dependent adhesion switch mainly expressed in the intestinal system (130, 131). CDH17 is upregulated in gastrointestinal cancers and is used as a marker for adenocarcinomas of the gastrointestinal system (132, 133). The results demonstrated that CDH17-CAR T-cells eradicated tumors in a xenografted NSG mouse model without damaging healthy normal tissues expressing CDH17 as it is sequestered and hidden between normal cells (87). This study is the first preclinical investigation of CDH17 as a target for CAR T-cell development, and it uncovers a new class of TAAs that are accessible to CAR T-cells in tumors but are masked in normal cells, protecting the latter from CAR T-cell attack. Hence, in solid tumors, re-investigation of previously identified TAAs is of great interest.

As the clinical safety and effectiveness of CAR T-cell therapy are closely associated with the intrinsic quality and fitness of CAR T-cells, comprehensive preclinical investigations of CAR T-cell biological products are crucial. Such investigations are usually performed in different in vitro and in vivo models that can prove to be powerful and versatile tools for disease modeling and therapeutic experimentation and generate reliable data that indicate the clinical therapeutic potency of CAR T-cell therapies.

Owing to the promising results obtained in preclinical studies, many CAR T-cell therapies developed against CRC are being evaluated for antitumor efficacy and safety in clinical trials.

The efforts of CAR T-cell research teams have yielded outstanding results for the treatment of leukemia (168–174). However, clinical evaluation of this new immunological weapon for fighting solid cancers is surrounded by disappointment. Past experiences, notably with ICIs, have highlighted significant contribution of multiple factors that significantly contribute to the strengths and weaknesses of immunotherapies and forecast their clinical outcome in solid cancer. These aspects must be considered when identifying the therapeutic strategies for solid tumor treatment.

Nevertheless, it must be considered that even for hematological malignancies with complete remission rates as high as 90%, patients remain at risk of relapse because of the poor persistence of CAR T-cells in vivo. Limited CAR T-cell persistence is closely related to the immunological properties of the cellular infusion product.

In the last decade, cancer treatment has undergone a major revolution owing to therapies that target the TME. Deep characterization of the immune response and TME, a continuously evolving entity, along with the molecular and phenotypic analysis of tumor cells, is an attractive strategy for cancer treatment. Using accumulated data from recent immunotherapy studies, notably ICI, to design more effective CAR T cells, can be of a great interest.

The complex spatiotemporal changes in the TME complicate cancer treatment by driving various responses to immunotherapies. The landscape of tumor-infiltrating immune cells defines prognosis and treatment efficacy (175, 176), supporting the idea that treatment strategy designs need to be guided by mapping the composition and functional state of immune cell infiltrates (177). Schematically, the TME can be broadly classified into cold (non-T-cell inflamed) or hot (T-cell inflamed), on the basis of accumulation of proinflammatory cytokines and T-cell infiltration (178). Commonly, hot tumors, characterized by T-cell infiltration and immune activation signatures, exhibit better response to ICB treatment (179). Meanwhile, cold tumors lack such infiltrates because of several factors: poor tumor antigenicity, impaired antigen presentation, T-cell activation failure, and/or lack of T-cell homing into the tumor bed. A limited number of T-cells surround cold tumors and localize to the tumor periphery with poor T-cell activation and infiltration (180, 181). Thus, the high-inflammatory state of the TME is associated with high TMB and increased tumor-related antigenicity, resulting in T-cell tumor-infiltration, amplification, and tumor necrosis induction (178, 182). Remarkably, CRC tumors with MSI and a higher mutational burden respond well to ICI-based immunotherapy (183–186). Primary lesions, such as metastatic colon cancer, are highly enriched in immune infiltrating cells with increased mutational load, and a high immunoscore is associated with a significantly lower number of metastases (187).

Several research teams are focused on converting cold tumors into hot tumors to render them more receptive to immunotherapies, leading to an objective antitumor response. The second goal was to prevent hot tumors from cooling. In this way, creating a beneficial TME goes through vascular bed remodeling to enhance the circulation of CAR T-cells and activate the immune response at the tumor side. Innate immune sensing and signaling, dysregulated oncologic pathways, altered cellular metabolism, and epigenetic changes in the TME must be integrated as tightly as possible into the global antitumor strategy. Immunogenic cell death induced by chemo-, radio-, and targeted therapies plays a key role in stimulating altered antitumor immune responses, potentially converting non-inflamed cold tumors into hot tumors (188–192). Hence, the future of antitumor combination therapies lies in well-designed sequential strategies that successfully prepare patients for immunotherapeutic regimens, including CAR T-cell adoptive cell transfer (193, 194). Tumor immune microenvironment characterization can help identify good responders to immunotherapies, such as CAR T-cell therapy.

Currently, 28 ongoing clinical trials are evaluating TMB as a stratification biomarker for predicting immunotherapy responses (195–197). The frequency of tumor mutations varies widely across, and within cancer subtypes (198). Greater mutational load, notably nonsynonymous mutations, increases the possibility of neoantigen generation and expression by tumor cells, allowing the conversion of immune tolerance to antitumor immune responses. Consistent with this hypothesis, TMB has been shown to predict the clinical benefit of anti-PD-1/PD-L1 immunotherapy (199, 200). Patients in the immune desert cluster with a low T-cell infiltration microenvironment respond less favorably to ICI therapies (201–203). Moreover, several recent preclinical and clinical reports have clearly highlighted the major role of neoantigen-specific T-cell activity in recognizing and eliminating cancer cells (204–213). Several studies have reported a highly significant association between neoantigen burden and clinical benefits (183, 214–216). Therefore, TMB has emerged as a powerful determinant in response to immunotherapies.

Based on current data and trends, TMB appears to be a robust predictive biomarker of the response to immune checkpoint immunotherapy. Generally, two types of cancer tend to harbor high TMB: (i) tumors induced by carcinogenic and mutagenic substances and (ii) tumors caused by germline mutations in DNA repair and replication factors. Thus, TMB promises to be useful for MSI-H CRCs, marked by frequent mutations in dMMR and DNA replication genes (195, 196). The model combining TMB with other immunotherapy biomarkers provides a promising method with greater predictive power to identify immunotherapy responders and predict outcomes.

In dMMR and MSI-H CRC tumors, neoplastic foci present lymphoid tertiary structures deeply infiltrated with activated CD8⁺ T-cells (217–219). Consistent with this observation, dMMR cancers are sensitive to checkpoint inhibitors, leading to pembrolizumab approval for MSI-H or dMMR cancers (22, 220, 221). In colon cancer, the potent local immune response associated with high mutational load allows neoantigens to be targeted by T and B lymphocytes with an appreciable clinical antitumor reactivity under ICI treatment (183, 222–224).

The complex TME plays an essential role in the clinical response of solid tumors to CAR T-cell therapy. Thus, the inhibition of proliferation and/or lack of persistence can limit effective antitumor immune response and lead to the poor treatment response in CRC. The immunosuppressive microenvironment plays a decisive role in inhibiting CAR T-cell persistence in the tumor milieu. On the one hand, immunosuppressive cells and cytokines hamper the function of CAR T-cells by blocking them from tumor sites or inducing apoptosis (36). On the other hand, the immunosuppressive microenvironment lacking the cytokines necessary for T-cell persistence can foster the apoptotic pathway.

Once at the tumor site, CAR T-cells can undergo activation-induced cell death upon functional activation and proliferation/expansion. B-cell lymphoma-extra-large (Bcl-xL), a critical regulator for T-cell survival, has been reported to contribute to CAR T-cell persistence and antitumor activity (225–227). Interestingly, the inclusion of exogenous Bcl-xL gene into a second-generation anti-CEA CAR retroviral construct promoted CAR T-cell persistence both in vitro and in vivo, with enhanced antitumor potency and sustained survival in a CRC mouse model. The novel CAR T-cell platform which is based on the exogenous expression of persistent genes, can help overcome the lack of persistence of CAR T-cells in solid tumors.

Exploring better combination therapies is another approach to improve CAR T-cell efficacy and decrease their shortcomings for solid-tumor treatment. Emerging novel combined CAR T-cell therapies and immunotherapy or small-molecule drugs have shown promise in preclinical studies, and they can overcome the limitations of CAR T-cells targeting solid tumors. The combination of EpCAM CAR T-cells with the Wnt pathway inhibitor hsBCL9CT-24 has shown a synergistic effect against EpCAM-positive colon cells in vitro and in vivo. The combined strategies modulate the TME to induce the inflltration of T-cells, effector function and prevent the exhaustion of CAR T-cells (77). Induction of antibody-dependent cell cytotoxicity (ADCC) via the Fc-gamma receptor is another multi-target CAR T-cell approach that enables ADCC to CAR T-cells. Hence, by endowing CAR T-cells with ADCC activity via the Fc-gamma receptor IIIa (CD16), they exhibit sustained proliferation and cytotoxicity to antibody-targeted cancer cells (228). In CRC, CD16-CAR T-cells combined with cetuximab decreased the viability of KRAS-mutated HCT116 CRC cells in vitro, decreased tumor growth in a SCID mouse model, and increased disease-free survival (229). Recently, dendritic cell (DC) vaccines have been studied in combination with immunotherapy strategies, such as those targeting PD-1/PD-L1. Regarding cancer stem cell resistance to conventional therapies, the synergistic killing effects of PD-L1-CAR T-cells and CRC stem cell (CCSC)-dendritic cell vaccine-sensitized T-cells in CCSC have been investigated. A study has shown that PD-L1 is highly expressed in CCSCs and that PD-L1-CAR T-cells specifically recognize the PD-L1 molecule on CCSCs (85). In this study, combined therapy markedly relieved the tumor burden in mice compared to monotherapy with PD-L1-CAR T-cells or CCSC-DC vaccine. This showed that there was moderate tumor remission both in vitro and in vivo.

Finally, combination therapies are being developed for improving immune response, minimizing on-target off-tumor toxicities, and transforming immunologically “cold” to “hot” tumors (230). A combination of CAR NK and CAR T-cells can harness the advantages of both CAR NK and CAR T-cells, allowing for both rapid and persistent killing while potentially minimizing the toxicities associated with CAR-T cells (228, 231–233). The development of these therapies is poised to be the next frontier in immunotherapy as it will result in more robust immune attacks and effective clinical outcomes.