Recent advances in CAR-T cells therapy for colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer, with a high mortality rate and a serious impact on people's life and health. In recent years, adoptive chimeric antigen receptor T (CAR-T) cells therapy has shown well efficacy in the treatment of hematological malignancies, but there are still many problems and challenges in solid tumors such as CRC. For example, the tumor immunosuppressive microenvironment, the low targeting of CAR-T cells, the short time of CAR-T cells in vivo, and the limited proliferation capacity of CAR-T cells, CAR-T cells can not effectively infiltrate into the tumor and so on. New approaches have been proposed to address these challenges in CRC, and this review provides a comprehensive overview of the current state of CAR-T cells therapy in CRC.

Introduction

CRC occurs worldwide, has a high mortality rate and is the third most common cancer (1, 2), which seriously affects human life and health. Due to the rarity of early diagnosis of CRC, existing treatment methods including surgery, chemotherapy and radiotherapy cannot completely inhibit the progression, metastasis and recurrence of CRC when cancer cells infiltrate or metastasize to surrounding tissues (3).

CAR-T cells have shown significant efficacy in immunotargeted therapy of hematologic tumors (4). The United States Food and Drug Administration has approved CAR-T cells for the treatment of hematologic tumors (5). In recent years, basic and clinical studies on CAR-T cells therapy for CRC have been published, and some studies have made encouraging progress (6). However, CAR-T cells face many challenges in the treatment of CRC, limiting their clinical application (7). This article reviews the progress of CAR-T cells therapy for CRC.

Extracellular region, hinge region, transmembrane region and intracellular signal region are the four components of CAR, and each plays an important role (8). The extracellular domain is usually Fab or single chain variable fragment (scFv) of monoclonal antibody, which has flexible splicing function and determines antigen specificity (9). The hinge domain consists of (Cluster of differentiation4)CD4、CD8、CD28 or IgG4, which connects the extracellular domain to the transmembrane domain (10). The transmembrane domain consists of CD8α, CD4, CD3 ζ, CD28 or ICOS, linking the extracellular domain to the intracellular domain and acting as an anchor of the cell membrane (11, 12). Intracellular signaling domains transmit stimuli into the cell (13). First-generation CAR, which consist of scFv and intracellular CD3ζ molecular signaling domain (14–16), have limited antitumor activity due to the lack of co-stimulation and interleukin signaling (17). The costimulatory domain of the second generation CAR consists of 4-1BB (CD137) or CD28, which mimics costimulatory signals during activation (18). The third generation of CAR has two costimulatory domains, further enhancing the function of CAR (19). The fourth generation CAR is based on the second generation CAR and secretes cytokines such as interleukin2(IL-2) and IL-12 (20, 21). A schematic diagram of the different generations of CARs is shown in . Recently, researchers designed a combination of focused ultrasound (FUS) and CAR-T cells expressing heat-inducible genes (22). FUS activates heat-inducible genes by controlling local temperature in vivo ( 22). In animal experiments, CAR-T cells was injected into tumors in mice, and a small ultrasonic transducer was placed on the top of the skin of the tumor area (22). The tumor area was heated through the ultrasonic transducer in the FUS guided by magnetic resonance imaging. Only tumors exposed to ultrasound will be attacked by CAR-T, improving CAR-T targeting (22). This design is expected to be a promising CAR-T.

Figure 1

The chimeric antigen receptor (CAR) structure of four generations.

Basic experimental of CAR-T cells therapy for CRC

The genetic modification of peripherally derived T lymphocytes with CARs has achieved a remarkable effect in the treatment of hematologic malignancies (23, 24). CAR-T cells therapy for solid tumors still faces many challenges. Recently, there are some advances in CAR-T cells therapy for CRC. The targets of CAR-T cells therapy for CRC include carcino-embryonic antigen(CEA), Mesothelin (MSLN),Guanylyl cyclase C (GUCY2C), epithelial cell adhesion molecule(EpCAM), Human epidermal growth factor receptor-2 (HER2)、Doublecortin-like kinase 1 (DCLK1).

CEA is a glycoprotein formed by cells in the large intestine and a glycoprotein carcinoembryonic antigen, which has been considered as a sensitive marker of CRC (25). At present, there are many basic studies on CAR-T for CEA (26). CAR-T cells have excellent anti-tumor ability when dual targeting CEA and other targets such as CD30 antibody (27).The combination of CEA-CAR-T cells and recombinant human IL‐12(rhIL-12) significantly inhibited the growth of tumor xenografts (28).

MSLN is a cells surface glycoprotein, which is physiologically expressed in peritoneal, pleural and pericardial mesenchymal cells (29). Overexpression of MSLN can be detected in CRC (30). MSLN is an important CAR-T cells target in solid tumors (31, 32). In a recent study, the efficacy of MSLN-CAR-T cells on colon cancer xenografts was investigated. Compared with the control group, the mice in the MSLN-CAR-T cells group had more T lymphocytes in the peripheral blood and more granzyme B infiltrates in the tumor tissue (33). The experimental results showed that the MSLN-CAR-T cells group had a more significant anti-tumor effect (33).

GUCY2C is a binding receptor present in the enterocytes membranes that sustains balance by activating its hormone ligand guanosine or uridine to produce the second messenger cGMP (34). When GUCY2C signaling is blocked, it may lead to the pathogenesis of CRC. However, GUCY2C is expressed in both human primary and metastatic CRC, and GUCY2C is considered to be a tumor marker (35). GUCY2C is highly expressed in 95% of CRC metastasis (36). CAR-T cells targeting hGUCY2C mediated killing of CRC cells expressing hGUCY2C, and were nontoxic to intestinal epithelial cells expressing normal GUCY2C. Such CAR-T cells induce antigen-dependent T-cells activation and cytokine production, thereby enhancing antitumor efficacy (37).

EpCAM is one of the main surface tumor-associated antigens of CRC (38), which can promote the migration, proliferation and tumor growth of colon cancer cells (39). In the experimental treatment of CRC with EpCAM-CAR-T cells, compared with control T cells, EpCAM-CAR-T cells have greater lethality and specificity against cancer cells which express EpCAM (40).

HER2 is overexpressed in CRC (41), and is an important target for CAR-T cells therapy. HER2-CAR-T cells showed strong and particular cytotoxic capacity against colon cancer cells. In mouse models, HER2-CAR-T cells-treated mice showed significant tumor control, significantly improved overall survival, and suppressed distant metastasis of CRC to liver and lung (42).

DCLK1 is an enzyme that regulates epithelial mesenchymal transition (43). Mesenchymal DCLK1 labeling of tumor stem cells in a genetic mouse model of CRC (44). DCLK1-targeted CAR-T cells therapy inhibited xenograft tumor growth in mice without apparent toxicity (45).

Cbl-b is an E3 ubiquitin ligase that mediates ubiquitination, and removal of Cbl-b from CAR-T cells enhances the antitumor activity of CAR-T cells (46). Compared with the control group, Cbl-b -/-CAR-T cells significantly enhanced the killing ability of CAR-T cells against CRC cells, which was manifested by increased secretion of IFN-γ, TNF-α and granzyme B (47).

Challenges

Adoptive T cells therapy is a new option for tumor patients, but its efficacy is affected by various factors, it is imperative to find relevant strategies to solve the problem.

Immunosuppression in the tumor microenvironment

Hypoxia, acidic microenvironment and lack of substances necessary for the survival, proliferation and activation of T lymphocytes in tumor tissues will lead to immunosuppressive microenvironment, thereby weakening the killing effect of CAR-T cells on tumor cells (7). Tumor immunosuppressive microenvironment includes suppressive immune cells such as regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), immunosuppressive cytokines such as IL-10 and TGF-β, immunosuppressive molecules such as PD-1, and other molecules such as indoleamine dioxygenase 2-3 (IDO1) (7, 48). The immunosuppressive microenvironment promotes tumor immune escape (49). The occurrence and development of tumor are correlated strongly with immune escape (50), in which immune checkpoints play an important role (51). Programmed cell death protein 1 (PD-1) and its ligand PD-L1 are significant immune checkpoint proteins (52). PD-1 is an immune checkpoint receptor expressed in T lymphocytes, and PD-L1 is expressed mainly in the tumor microenvironment (53). When PD-1 encounters antigens, its expression is increased and binds to its ligand PD-L1, thereby inhibiting the immune response function of T cells and mediating immune suppression (54).

CAR-T cells does not effectively chemotaxis to tumor tissue

One of the challenges of CAR-T cells therapy for solid malignancies is the specific recognition of targeted antigens (55). Currently, the majority of tumor target antigens recognized by CAR-T cells are also expressed in normal cells, so when CAR-T cells are used to treat tumors, the therapeutic effect is ineffective (7). Meanwhile, CAR-T cells can also injury normal tissues and cause toxicity in vitro ( 7).

CAR-T cells can not proliferate and persist in the blood or tumor area

The persistence and proliferation of CAR-T cells in blood or tumor are important factors for the efficacy of CAR-T cells in cancer treatment (56). Firstly, different costimulatory molecules of CAR affect the survival and proliferation of CAR-T cells (57). Secondly, in the tumor microenvironment, there are a series of factors that affect the survival, proliferation and induce the failure of CAR-T cells. For example, when CAR-T cells are in chronic T helper 2 cells(Th2) inflammation state, their expansion ability is weakened and the number of apoptotic cells is increased (58). Thirdly, TGF-β and adenosine significantly inhibit the tumor cytotoxicity of CD8 + T cells by inhibiting the expression of granzyme (59, 60). In addition, the hypoxic acid microenvironment in the local tumor can cause damage to CAR-T cells, in which lactic acid accumulation can inhibit the production of IL-2, thereby affecting the proliferation and function of CAR-T cells (61). Further, the PD-1/PD-L1 axis affects the survival and function of CAR-T cells (62). Transcription factors T-bet and B lymphocyte-induced maturation protein 1 (Blimp1) regulate early CD8+T lymphocytes (63, 64). Forkhead box protein O1 (FoxO1) can regulate memory CD8+ T cell differentiation (65).

The level of CAR-T cells invasion in tumor tissue was low

When a CAR-T cells is used to treat a tumor, the CAR-T cells must reach the site of the tumor to perform their tumor-killing function (7). In solid tumors, CAR-T cells must overcome multiple obstacles to reach the tumor site, such as blood vessels and the tumor’s stroma (66, 67). Primarily, when intravenous infusion of CAR-T cells in the treatment of CRC, CAR-T cells must cross the vascular barrier and interstitial barrier to enter the tumor site to exert its efficacy (66). Intratumoral vascular beds and interstitial abnormalities are the key factors affecting the efficacy (66). Then, the inability of many T cells to reach the cancer cells may depend on the lack of surface-expressed chemokine receptor that match chemokine expressed in the tumor or tumor stroma (68). When the chemokines/chemokine receptors axis is mismatched, tumor cells secrete trace amounts of chemokines, resulting in the inability of T cells to reach the tumor tissues (68). For example, CXCL10 can make a variety of antitumor lymphocytes chemotactic to tumor tissues, such as CD8+ T cells, and is associated with T-lymphocytes infiltration in solid tumors (69).

Strategies

Develop drugs and measures that can improve the tumor microenvironment

In order to improve the tumor microenvironment to improve the anti-tumor efficacy and durability of CAR-T cells, there are currently the following methods.

It is essential that CAR-T cells secrete pro-inflammatory cytokines to protect them from the inhibitory tumor microenvironment. Studies have shown that secreted cytokines such as IL-7 and IL-12 CAR T cells can improve the immunosuppressive microenvironment (70, 71). Mesenchymal Stem Cells (MSCs) are the main components of tumor stroma and have the ability to actively migrate into tumor tissues (72, 73).By making MSCs capable of releasing IL-7 and IL-12 and combining CAR-T cells, researchers found that CAR-T cells could prolong the time of T cells attack on tumors and improve the tumor immunosuppressive microenvironment (74). IDO1 degrades tryptophan, an essential amino acid for T cells, which is required for T cells survival and immune responses (75). The expression of IDO1 is inhibited by miR-153 (76). When miR-153 was overexpressed in tumor cells, the tumor immunosuppressive environment was improved, CAR-T cells targeting epidermal growth factor receptor variant III(EGFRIII) were more effective in killing colon cancer cells overexpressing miR-153 (77). CD30 signaling can promote the differentiation of T cells to Th2, which has immunosuppressive function (78). In CRC, CAR-T cells dual targeting CD30 and CEA can produce a more significant proinflammatory response, manifested by higher granzyme B and perforin levels In T cells, which improves the ability of CAR-T cells to attack the tumor (27). IL-10 binds to its cognate receptor IL-10R to cause a wide range of immunosuppressive functions (79, 80). Recent studies have shown that CAR-T cells combined with IL-10 monoclonal antibody (mAb) can partially alleviate bone marrow cell-mediated immunosuppression by blocking IL-10 signaling, while promoting CAR-T cells expansion and enhancing killing effect, thereby increasing anti-tumor function (81).

Guo and his team demonstrated that intravenous injection of live attenuated Brucella in mice can promote the tumor microenvironment to a proinflammatory state, enhance the anti-tumor immunity of T cells, and reduce the resistance of tumors to CAR-T cells (82). Dopamine treatment can promote the differentiation of CD8+ T lymphocytes into CD103+ tissue-resident memory CD8+ T lymphocytes (TRM), and TRM can trigger stronger anti-tumor immunity. Moreover, dopamine treatment enhanced the anti-tumor function of CAR-T cells (83).

In addition, blockade of immune checkpoints can improve immunosuppression. Adding genes expressing PD-1 negative receptors to CAR-T cells can block intracellular immune checkpoints and enhance the lethality to target cells (84, 85). Investigators also used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) systems to knock down the expression of PD-1 in CAR-T cells and achieved excellent preclinical efficacy by blocking PD-1/PD-L1 induced suppression of T-cell immune function (86–89).

Improve CAR-T cells targeting of tumors

Targeting multiple antigens and application of novel CAR can improve the targeting of CAR-T cells. Jiang and colleagues constructed a dual CAR system containing two extracellular domains, NKG2D and PD-1, and showed that such CAR-T cells effectively eliminated target cancer cells (90). CAR-T cells that dual target CD30 and CEA have a more specific ability to kill tumor cells, which is manifested by blocking the inhibition of cytotoxic T lymphocyte immune function induced by CD30 (27). In addition, when using the novel inhibitory CAR (iCAR) construct, the iCAR can trigger inhibitory signals when CAR-T cells are present in normal tissues, thereby inhibiting T cell function, avoiding the attack of normal tissues, and enhancing the targeting of tumor tissues (7). Additionally, switchable CAR T cells can increase their targeting, with the “switch” acting as a bridge between tumor cells and T cells, allowing T cells to specifically kill tumor cells (91). Besides, the combination of focused ultrasound (FUS) and CAR-T cells, in which only tumors exposed to ultrasound are attacked by CAR-T cells, also improves CAR-T cells targeting (22).

Amplification and long-term presence of CAR-T cells

How to maintain sustained expansion of CAR-T cells in vivo is a common consideration in the treatment of solid tumors with CAR-T cells. Cytokines such as IL-2, IL-7, IL-12 and IL-15 play an important role in T cells activation, proliferation and immune response (92–94). However, the content of immune stimulatory cytokines in the tumor microenvironment is very low. There are now several therapies for combining cytokines with CAR-T cells to treat tumors. CEA-CAR-T cells combined with rhIL-12 can increase the multiplication, persistence and cytokines release of CEA-CAR-T cells in vivo ( 28). When MSCs that can release IL-7 and IL-12 are used in combination with CAR-T cells, CAR-T cells survive longer and have better expansion ability in vivo, thereby improving the anti-tumor response (74). Li and his team demonstrated that inhibition of Wnt significantly inhibited TGF-β expression in tumor tissues and improved T cells infiltration (95). Moreover, after the inhibition of Wnt, the contents of T-bet and FoxO1 in the nucleus of CAR-T cells increased, and the expression of BLIMP1 increased, indicating that the inhibition of Wnt can make CAR-T cells early kill tumor function and differentiate into memory T lymphocytes (95). CD133 is expressed in cancer cells of various epithelial cell origins (96). A phase I trial of CAR-T cells targeting CD133 (CAR-T-133) in the treatment of advanced metastatic malignancies has found that CAR-T-133 cells can persist in vivo through multiple infusions and increase the content of immunostimulatory cytokines, which makes valid disease clearance and prevention of relapse possible (97). Previous studies have shown that increasing telomerase activity in CAR-T cells can enhance their proliferation ability and delay senescence (98). Other studies have shown that the costimulatory domain 4-1BB of CAR-T cells can improve the exhaustion of T cells and enhance their persistence in vivo (99).

Increased CAR-T cells invasion in tumors

Targeting tumor blood vessels and stroma and increasing the expression of chemokines are important methods to improve CAR-T cells infiltration into tumor tissues (100). Vascular blocker combretastatin A4 phosphate (CA4P) is a vascular interfering agent with high selectivity for tumor vascular system (101). Targeting CA4P can block the VE-cadherin signaling pathway, affect the stability of microtubule polymerization of tumor cell-related vascular endothelial cells, induce cell apoptosis, destroy the vascular system, reduce the blood supply in the tumor, and lead to tumor cell necrosis in the tumor tissue (101). CA4P combined with HER2-CAR-T cells therapy has a better antitumor effect than CA4P or HER2-CAR-T cell therapy alone, which can destroy tumor blood vessels, thereby promoting the infiltration of T cells into tumor tissues and enhancing the proliferation of CAR T cells (102). Vascular endothelial growth factor (VEGF)/VEGFR axis can promote the generation of vascular endothelial cells, which is a key signaling pathway of angiogenesis (103). VEGFR -targeting CAR T cells can disrupt vascular structures and obviously inhibit xenograft tumor growth, invasion, and metastasis (104). Cancer-associated fibroblasts (CAFs) are important components of tumor stroma (105). Fibroblast activating protein (FAP) is over expression in CAFs and suppresses tumor immune response by promoting the recruitment of immunosuppressive cells (106). At present, FAP-targeted CAR-T cells have achieved certain preclinical and clinical efficacy in solid tumors (107, 108). When the Wnt signaling pathway is blocked, it can up-regulate the expression of chemokine CXCL10, improve T cells tumor infiltration in cancer models, and improve the efficacy of CAR-T cells in CRC treatment (95, 109).

Clinical trials

In the past few years, immune cell therapy has been increasingly used in multicentre clinical trials. Multiple clinical trials targeting tumor antigens have been approved, including CEA, MSLN, EpCAM, HER2 and antigens, as well as NK group 2 member D ligands (NKG2DL), Mucin-1 (MUC1), B7-H3 (CD276), CD133, mesenchymal epithelial transfer factor(c-Met), which is overexpressed in colorectal cancer, can be used as a target for CAR-T cells. In , we summarized the clinical information available on ClinicalTrials.gov regarding CAR-T-cells therapy for CRC.

Table 1

Clinical trial of CAR-T cells in CRC(https://clinicaltrials.gov/).

Antigen	phase	Clinicaltrials. gov identifier	CAR-T Cells Treatment		Recruitment Status
NKG2DL	Early Phase 1	NCT05248048	NA		Recruiting
	Phase 1	NCT04550663	NA		Not yet recruiting
	Phase 1	NCT03370198	3 DL: 3 × 108–3 × 109 cells/d(3ds)		Active, not recruiting
	Phase 1	NCT04107142	3DL:3 x 108- 3 x 109 CAR-γδ T cells/d(4ds)		Unknown
	Phase 1	NCT03310008	3 DL: 108–109cells/d(3 ds) and FOLFOX		Active, not recruiting
	Phase 1	NCT03692429	3 DL:1-100x108cells/d (3 ds) andFOLFOX		Recruiting
CEA	Phase 1	NCT02850536	1 × 1010 cells/d(3 ds) with IL2		Completed
	Phase 1	NCT02416466	1 × 1010 cells/d(3ds) with IL-2		Completed
	Early Phase 1	NCT04513431	NA		Not yet recruiting
	Phase 1	NCT05240950	3DL:1- 6×106/kg anti-CEA CAR-T (+) cells(1d)		Recruiting
	Phase 1Phase 2	NCT04348643	NA		Recruiting
	Phase 1	NCT02349724	5 DL: 105–108 CAR+ cells/kg (split: 10%,30% and 60% per day)		Completed
	Phase 1	NCT03682744	NA		Active, not recruiting
	Phase 1Phase 2	NCT02959151	1.25~4×107 CAR+T cells/cm3 tumor bulk(1d)		Unknown
MSLN	Phase 1	NCT05089266	NA		Not yet recruiting
	Early Phase 1	NCT04503980	4DL:1×105-3×106αPD1 MSLN-CAR+ T cells/kg(1d)		Recruiting
EpCAM	Phase 1	NCT05028933	3DL:3-10×105EPCAM CAR-T/kg(1d)		Recruiting
MUC1	Phase 1	NCT05239143	NA		Recruiting
	Phase 1Phase 2	NCT02617134	NA		Unknown
HER2	Phase 1	NCT03740256	7 DL: 1–100 × 106Cells(1d) and oncolytic adenovirus CAdVEC intra-tumor injection		Recruiting
B7-H3	Phase 1	NCT05190185	3DL:1-100×106 CAR-T/kg(1d)		Recruiting
EGFR	Phase 1	NCT03542799	NA		Unknown
	Phase 1Phase 2	NCT03152435	NA		Unknown
CD133	Phase 1Phase 2	NCT02541370	0.5–2 × 106 cells/kg(2ds)		Completed
c-Met	Phase 1Phase 2	NCT03638206	NA		Recruiting

NA, not available; d(s), dose(s); DL, dose levels.

In a phase I trial of CEA + CRC patients treated with CEA-CAR-T cells (NCT02349724), five dose-escalation CAR-T cells were administered to 10 patients with relapsed and refractory CRC metastases. No serious adverse events related to CAR-T cells therapy were observed in the trial (6). Among the 10 patients, 7 were stable after CAR-T cells therapy, of which 2 were stable for more than 30 weeks and 2 showed tumor shrinkage (6).

A phase 1B hepatic Immunotherapy for Metastases-selective internal irradiation therapy (HITM-SIR) trial was conducted in patients with liver metastases from CRC (NCT01373047). Six of them received anti-CEA CAR-T hepatic artery infusions (HAIs) and SIRT. Significant reductions in Granulocyte macrophage colony stimulating factor (GM-CSF), GM-CSF-R, IDO, and Programmed death ligand-1(PD-L1) were observed after HITM CAR-T HAI treatment, suggesting a reversal of immunosuppressed hepatic tumor microenvironment (TME). Subsequent increases in IL-2 and IL-6 in tumor biopsies after infusion further demonstrated pro-inflammatory liver TME. The median survival of patients in the trial was 8 months (110).

Conclusions

There are many approaches to CRC adoptive cell therapy, of which CAR-T cells are one of the most researched and promising, although clinical studies are still in the early stages of clinical trials. Many studies have demonstrated the efficacy and safety of CAR-T cells in the treatment of CRC. However, the therapy faces many challenges that limit its clinical application. In addition, CAR-T cells therapy can cause a number of toxic effects, the most common of which is cytokine release syndrome (CRS), which is a cytokine secretion response after CAR-T cells infusion (111). CRS has a series of non-characteristic manifestations, such as fever, nausea, decreased cardiac function, and hypotension (112). It can also cause other systemic toxicity, such as dyspnea, respiratory failure, arrhythmia, elevated myocardial enzymes, cardiac insufficiency, liver insufficiency, gastrointestinal reaction, coagulation dysfunction, muscle injury, neurotoxic allergy, etc (112). Only when these problems are effectively addressed can the efficacy of CAR-T cells therapy for CRC be improved and more patients receive effective treatment. In conclusion, CAR-T cells are a promising treatment for CRC and further research is needed.

Author contributions

CC and QL designed this manuscript. XQ wrote the main manuscript text. FW prepared figure and table. CC and QL revised the article. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the National Natural Science Foundation of China [grant number 81800242 and 81970382]; The Science foundation of Haiyan [grant number JJMS2022-16].

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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