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The use of CAR-T cells in the treatment of liver cancer
The use of CAR-T cells in the treatment of liver cancer. Liver cancer includes primary and metastatic cancers. Hepatocellular carcinoma (HCC) accounts for approximately 85% of primary liver cancers and represents the second most common cause of cancer-related deaths worldwide [1,2]. The main risk factors for liver cancer are chronic HBV and HCV infection, which cause chronic inflammation in the liver [3,4].
Since liver cancer is usually diagnosed at an advanced stage, few patients undergo surgery, and recurrence within 5 years complicates 70% of cases . Most HCC patients receive local or systemic treatments, such as chemotherapy, radiation therapy, percutaneous ethanol injection, radiofrequency ablation, various embolization procedures, and sorafenib. However, the efficacy of these therapies is insufficient .
Liver metastases are more common than primary liver cancer, because colon cancer, rectal cancer, pancreatic cancer, breast cancer, lung cancer and other organ cancers usually metastasize to the liver . Although survival rates vary, patients with untreated liver disease rarely metastasize and survive for more than 5 years.
Surgical resection is the only treatment. However, few patients are eligible for this treatment [9,10]. Therefore, there is an urgent need for a novel and effective treatment method for liver cancer.
In this review, we mainly discuss the immune characteristics of liver cancer, the obstacles faced by the application of CAR-T therapy in the treatment of liver cancer, and the recent preclinical and clinical trials of CAR-T therapy.
A typical CAR consists of three elements, namely an extracellular antigen recognition domain with or without hinges/spacers, a transmembrane domain and an intracellular signaling domain (Figure 1) [34,35].
Most CARs use a single-chain variable fragment composed of the variable heavy and light chains of a tumor-related antigen-specific monoclonal antibody as the extracellular antigen recognition domain. Ligands or receptors can also be used, such as CD27 receptor and vascular endothelial growth factor [36, 37].
The CAR hinge/spacer between the single-chain variable fragment and the transmembrane domain usually includes the CH2CH3 or Ig-like extracellular portion of CD4 or CD8 . The hinge/spacer is important because it ensures access to CAR-T cell epitopes and stabilizes CAR expression .
The intracellular signal transduction domain has two parts, namely the costimulatory signal and the activation signal. According to the number of costimulatory molecules, CAR proteins can be divided into three generations.
The basic process of CAR-T production (Figure 1) is as follows: (i) cut out from the patient’s peripheral blood and collect peripheral blood mononuclear cells; (ii) use immunoselective beads to separate T cells from peripheral blood mononuclear cells, And use anti-CD3 and IL-2 for activation; (iii) genetic modification of CAR-T; (iv) expansion of T cells in vitro; (v) evaluation to ensure CAR expression and the general condition of T cells; (vi) CAR-T is infused back into the patient.
In these steps, three key points should be noted. First, many studies have shown that, compared with typical methods, adoptive transfer of undifferentiated T cells, such as naive and central memory T cell subsets, can lead to better persistence and anti-tumor effects. The isolation of T cells with a low degree of differentiation and the application of culture conditions suitable for T cells with a low degree of differentiation have been reported [45,47]. Secondly, effective genetic modification methods are essential. In addition, the infusion route of CAR-T should also be considered. Compared with systemic intravenous injection, local injection, especially intratumor injection, may have a better therapeutic effect and lower toxicity, which is common in clinical practice [53,54].
Strategies to improve the efficacy and safety of CAR-T therapy in liver cancer have shown encouraging results in the treatment of hematological malignancies [55,56]. Therefore, many trials have been conducted to evaluate the efficacy of CAR-T therapy in solid tumors. However, the clinical results are not satisfactory, indicating a lack of treatment response and/or some serious side effects. Barriers to curative treatment include lack of specific tumor antigens, limited CAR-T cell transport and penetration to tumor sites, and immunosuppressive tumor microenvironment. Specific tumor antigens will be described later in this review.
Although CAR-T has an encouraging therapeutic effect, it still has some substantial side effects . According to the relationship between target and tumor, side effects can be divided into three types: target/tumor toxicity (tumor lysis syndrome and cytokine release syndrome), target/tumor toxicity and target/extra-tumor toxicity. In order to control these side effects, several strategies have been developed, including enhancing the selectivity of CAR and controlling the activity of CAR-T .
The most straightforward strategy is to choose a safer antigen to enhance selectivity (Figure 2Ci). So far, the epidermal growth factor receptor (EGFR) vIII is the only tumor-specific antigen strictly limited to tumor cells , and some clinical trials (NCT01454596, NCT02209376) are evaluating this target in nervous system tumors. Mucin-1 (MUC-1) may be an effective alternative target due to its abnormal glycosylation and loss of polarity in many tumors [77,78].
Chinese researchers are currently trying to use MUC-1 CAR-T therapy to treat HCC (NCT02587689). Since there are few highly specific antigens, a more practical strategy using a combination of antigens instead of an antigen target has been developed (Figure 2C ii-v).
Duong and colleagues produced a synergistic dual-specific CAR-T, which has complete anti-tumor effects only in the presence of two antigens, and is less active when stimulated by one antigen  (Figure 2C ii). The complementary bispecific CAR-T allows the two parts of the intracellular domain to be connected to two different CARs to achieve similar anti-cancer effects  (Figure 2Ciii).
In addition, a new method involving the synthesis of Notch (synNotch) receptors has been developed, which includes an antigen recognition domain, Notch core, and transcription factors. After synNotch receptor recognizes the first antigen, transcription factors are released to induce CAR expression of the second antigen. In this way, synNotch CAR-T can selectively eliminate tumor cells expressing two antigens without destroying cells expressing only one of the two antigens [81,82] (Figure 2C iv).
In addition to these methods, inhibitory CARs specific to antigens on normal cells can also be added to CAR-T, thereby protecting normal cells from CAR-T-mediated attacks through negative inhibitory CAR signaling [83 ] (Figure 2C v).
Since side effects are inevitable, CAR-T activity must be controlled in some way. This can be achieved by selective ablation of CAR-T in CAR-T cells that express suicide genes [84,85] (Figure 2Di). Transient expression of CARs by electroporation of in vitro transcribed mRNA can also overcome this problem, because T cells can be injected into patients as needed  (Figure 2D ii). Moreover, the activity of the newly designed switchable CAR-T cell can be controlled by an exogenous molecule, which can act as a bridge to connect two adjacent domains of the CAR structure, such as a hinge or an intracellular domain. These CAR-T cells can eliminate tumor cells only in the presence of these molecules [87,88] (Figure 2D iii, iv).
Preclinical and clinical trials for liver cancer
CAR-T therapy has been preclinically and clinically tested in liver cancer. Since the selection of tumor-associated antigens directly reflects the efficacy and safety of CAR-T cells, we will describe preclinical and clinical studies based on antigens (Table 1).
GPC-3 is a 70-kDa heparan sulfate proteoglycan, which is attached to the cell membrane through glycosylphosphatidylinositol anchors . GPC-3 is usually expressed in human embryos and certain adult tissues (ovary, breast, mesothelium, lung and kidney), but cannot be detected in healthy adult liver [89,90]. However, overexpression of GPC-3 has been detected in liver cancer and has been associated with diagnosis and poor prognosis [91,92].
The earliest GPC-3 CAR-T cells were constructed by Gao et al. . In their study, 1G and 3G (CD28 and 4-1BB) CAR-T cells were tested. They demonstrated that GPC-3 CAR-T cells can eliminate GPC-3 positive HCC cells in vitro, while 3G CAR-T cells can inhibit the growth of established orthotopic xenografts in vivo. Another group compared the contribution of CD28 and 4-1BB by evaluating the anti-tumor properties of T cells expressing GPC-3-CAR encoding CD28 and 4-1BB with or without internal domains . In vitro, T cells transduced by CD28 signal have higher cytotoxicity than T cells transduced by 4-1BB signal. However, 4-1BB induced excellent expansion of CAR-T cells both in vitro and in vivo.
Jiang et al.  found similar results in xenografts derived from patients. It was found that the co-expression of GPC-3 characterized by CD28 and 4-1BB and dual-target CAR-T cells targeting asialoglycoprotein receptor 1 can reduce the risk of in vivo and in vitro target/extra-tumor toxicity . Based on these findings, some clinical trials have been conducted to evaluate the safety and effectiveness of GPC-3 CAR-T cells. Researchers at Chongqing Xinqiao Hospital are trying to combine CAR-T therapy with transcatheter arterial chemoembolization (NCT03084380). Other researchers have used intratumoral injections to enhance the efficacy of CAR-T therapy (NCT02715362, NCT03130712).
In summary, these findings indicate that GPC-3 may be a popular target for the treatment of HCC.
MUC-1 is a transmembrane glycoprotein that is usually expressed in gland or luminal epithelial cells of the breast, esophagus, stomach, duodenum, pancreas, uterus, prostate and lung [97,98]. Due to transformation and loss of polarity, glycosylated MUC-1 is abnormally overexpressed in many tumor tissues, such as breast cancer, gastric cancer, colon cancer, and liver cancer. Therefore, MUC-1 can be used as a cancer biomarker and therapeutic target. 1G and 3G CAR-T cells targeting glycosylated MUC-1 on HCC were tested in HCC cells (QGY-7701) . The results of the study show that MUC-1 CAR-T treatment can specifically kill HCC cells overexpressing MUC-1. The clinical trial of MUC-1CAR-T cells is currently underway (NCT02587689).
Epithelial cell adhesion molecule (EpCAM)
EpCAM is another transmembrane glycoprotein involved in cell signal transduction, migration, proliferation and differentiation . Identified as a marker of liver stem/progenitor cells, EpCAM has also been found to be up-regulated in liver cancer and is associated with poorly differentiated liver cancer and poor prognosis . Therefore, EpCAM may be a potential therapeutic target. In some solid tumors, such as prostate cancer, breast cancer and peritoneal cancer, EpCAM CAR-T cells have been preclinically tested [102-104]. However, no preclinical studies have examined EpCAM CAR-T cells in liver cancer.
There are currently two medical centers testing EpCAM CAR-T cells (NCT03013712, NCT02729493) in a clinical setting. Alpha-fetoprotein (AFP) AFP is a glycoprotein that is usually overexpressed in HCC and is a potential therapeutic target. However, AFP is expressed and secreted in cells, so it is considered unsuitable for CAR.
In order to overcome this problem, Liu et al.  designed a highly specific antibody against the AFP-MHC complex and engineered the antibody to be expressed as a CAR. They found that these “T cell receptor-like” CAR-T cells caused significant inhibition of tumor growth in an in vivo liver cancer xenograft model. “T cell receptor-like” CAR-T provides more options for antigen selection, and may have more potential applications in the treatment of liver cancer.
Viral infection, especially infection by HBV and HCV, is one of the main risk factors for HCC. Both HBV and HCV are known cancer viruses. Therefore, as an etiological treatment, targeting viral antigens may be effective for the treatment of liver cancer.
Bohne et al.  first designed a 2G CAR for HBV-S and HBV-L proteins, which enables T cells to recognize HBsAg-positive HepG2 cells, release interferon-γ and IL-2, and lyse HBV replicating cells. In particular, HBV-S CAR-T cells are more effective than HBV-L CAR-T cells, and soluble HBV will not block the function of CAR-T.
Krebs et al.  tested HBV-S CAR-T cells in HBV transgenic mice with immune function, and the results showed that CAR-T cells were located in the liver of mice to block HBV replication and only caused Transient liver injury. Alternatively, a 2G CAR specific to HCV E2 glycoprotein was constructed to test the CAR’s ability to control HCV infection . HCV E2 CAR-T cells can secrete antiviral and pro-inflammatory cytokines, and lyse liver cancer cells infected by HCV. Unfortunately, researchers have not tested the anti-tumor effects of these CAR-T cells in vivo and clinically.
In any case, CAR-T cells specific for viral antigens may have a good therapeutic effect, although further research is still needed.
Carcinoembryonic Antigen (CEA)
As a 180 kDa glycoprotein immobilized by glycosylphosphatidylinositol, CEA is widely expressed on the surface of many cells including colon, stomach, pancreas, ovarian and lung tumors . The expression of CEA in normal adult tissues is limited to the top surface of the epithelial cell membrane facing the lumen of the lung and gastrointestinal tract, making it invisible to immune cells .
The first clinical trial of CEA CAR-T cells was conducted in the UK (NCT01212887). Researchers used 1G CAR-T cells to treat CEACAM5-positive cancers and collected 14 patients before premature closure due to acute respiratory toxicity . Ten of these patients had liver metastases. However, it is worth noting that the clinical efficacy of 1G CAR-T cells is limited by poor persistence and transient respiratory toxicity that depends on pretreatment.
Subsequently, Roger Williams Medical Center conducted a trial to test the safety and potential efficacy of 2G CAR-T hepatic artery infusion for unresectable CEA-positive liver metastases (NCT01373047). The trial revealed the safety of CEA CAR-T hepatic arterial infusion in a large number of pretreated populations with a large tumor burden, and has encouraging clinical activity, and showed that the increase in neutrophil/lymphocyte ratio is comparable to CAR- T is related to adverse reactions after hepatic artery infusion [112,113]. Based on these results, two follow-up phase Ib trials are currently underway (NCT02850536, NCT02416466).
In addition, due to the unique immunobiological characteristics of the liver, the same researcher studied CEA CAR-T cells in a mouse CEA-positive liver metastasis model, and determined the mechanism of liver myeloid suppressor cell expansion and inhibition of CAR-T function . Their findings suggest that combining CEA CAR-T cells with drugs targeting liver myeloid suppressor cells may be a reasonable strategy for future clinical trials.
China Southwest Hospital is currently trying to inject CEA CAR-T cells intravenously to treat liver metastases from CEA-positive cancers (NCT02349724). Recently, they recruited 10 patients with colorectal cancer and proved that even at high doses, CEA CAR-T treatment is well tolerated for these patients. In addition, some therapeutic effects have been observed in most patients receiving treatment .
A recent study reported two phase 1 clinical trials of 1G CAR-T cells targeting TAG-72. These are the first human trials of CAR-T cells in the treatment of solid tumors . In the second trial, 9 patients with colorectal liver metastases were treated with CAR-T cells via hepatic artery infusion. The results reveal the relative safety of TAG-72 CAR-T cells and the limited persistence of 1G CAR-T cells, indirectly supporting the incorporation of costimulatory domains in CAR design and the use of complete human CAR constructs to reduce immunogen Sex.
Abnormal expression of EGFR family proteins is often observed in liver cancer. Morgan et al.  reported a case of serious adverse events after the administration of 2GCAR-T cells targeting ERBB2 to treat colorectal liver metastasis. By recognizing low levels of ERBB2 in lung epithelial cells, the massively administered cells trigger the release of cytokines and cause acute respiratory distress syndrome. In addition, researchers from the Chinese People’s Liberation Army General Hospital reported another case in which a patient with cholangiocarcinoma who progressed after EGFR CAR-T treatment (NCT01869166) obtained another partial switch to severe but controllable epidermal/endothelial toxicity The response after CD133-specific CAR-T immunotherapy (NCT02541370) . These two trials are still in progress.
In this review, we summarize the current preclinical and clinical trials of CAR-T treatments for primary and metastatic liver cancer, especially for the treatment of GPC-3, MUC-1 and CEA. To date, clinical trials of HCC have not been completed. However, preclinical studies in vitro and in vivo have shown effective anti-tumor efficacy. Although the safety of CAR-T in liver metastasis has been confirmed, some serious side effects have been observed. In addition, unlike CAR-T in hematological malignancies, the successful application of CAR-T in liver cancer still has to overcome several obstacles.
Therefore, based on the specific immunobiology of the liver, some specific strategies should be further studied, including local injection (hepatic artery infusion and intratumor infusion) and drugs targeting liver myeloid suppressor cells.
This review focuses on CAR-T treatment of primary and metastatic liver tumors. The research on CAR-T treatment of liver cancer is still in the preliminary stage, and the safety and effectiveness of CAR-T clinical application are still unclear. In addition, other methods of combination therapy should also be explored. Nevertheless, we are optimistic that CAR-T treatment will provide new methods for liver cancer treatment in the near future.
(source:internet, reference only)