Introduction

Among the basic methods of treating tumors, radiotherapy and chemotherapy still occupy an important position, but the accompanying toxic side effects cannot be ignored, which seriously affect the daily life of patients. To solve these problems, researchers have gradually turned to research on the role of immune cells and inflammatory factors in the tumor microenvironment, hoping to use the normal body immune system to monitor and kill cancer cells. So immune cell therapy has begun to be used in the treatment of tumors [1].

Among them, chimeric antigen receptor T-cell immunotherapy (CAR-T), which introduces Chimeric Antigen Receptors (CARs) into T cells, has a specific killing effect on cancer cells by using modified CAR-T to specifically recognize and bind to Tumor Associated Antigens (TAAs) on the surface of cancer cells, thus achieving specific treatment for cancer [24]. This method can be used for specific treatment of cancer cells. At the same time, this method can show better anti-tumor effects because it does not require the participation of antigenpresenting cells (APCs) and is therefore not limited by the major histocompatibility complex (MHC) [5]. In the clinical treatment of hematologic tumors, CAR-T cell immunotherapy has achieved better results, especially in CD19-positive B-cell leukemia and lymphoma, with an overall remission rate of more than 80%, which has again increased the interest of researchers in modifying T-cell immunotherapy and gradually extending it to clinical treatment of various tumors [6,7].

In this paper, we first describe the basic components and principles of CAR-T, then review the progress of CAR-T cell immunotherapy for various tumors in recent years, and finally summarize the future of CAR-T cell immunotherapy.

CAR-T cell immunotherapy

Composition of CAR

The key site in CAR-T cell immunotherapy is the chimeric antigen receptor (CAR), which consists of three main components, namely the extracellular antigen-binding domain, the hinge region and the intracellular structural domain, and is a fusion protein expressed on T cells [8,9]. The extracellular antigen binding domain can be composed of a monoclonal antibody heavy chain and a light chain with a single variable fragment, and this binding domain can determine the target of CAR-T cell attack through its antigen specificity [10]. The hinge region, as a transmembrane spatial domain with a certain degree of toughness, mainly functions as a link between the extracellular antigen binding domain and the intracellular structural domain, which determines the stability and signalling function of CAR, and thus facilitates the binding of CAR-T cells to target cells [10,11]. The intracellular structural domains are mainly divided into co-stimulatory structural domains and T cell activation structural domains, which serve to trigger and activate antigens to generate specific immune responses and transmit first and second signals to influence the activation, proliferation and killing of T cells [12]. Currently, researchers have conducted research on the design of CARs for their structure and have reached the fifth generation. The first-generation CARs consist of an extracellular single-chain variable fragment scFv, a transmembrane region and a single intracellular activation signal molecule [13]. These CARs lack the second signal molecule to induce T-cell activation, and can only transmit the activation signal to the cell, causing a short period of T-cell proliferation and a small amount of cytokine secretion, which is not conducive to the long-term anti-tumor activity of CAR-T cells in patients [14]. The second-generation CARs add co-stimulatory molecules to the original ones, which can enhance the killing and toxic function of T cells on tumor cells by activating the second signal. The thirdgeneration CARs add multiple costimulatory molecules, which interact with each other to enhance multiple signaling pathways in T cells, resulting in more significant enhancement of T cells in various aspects of anti-tumor cell functions, prolonging T cell proliferation activity, survival cycle and promoting the release of multiple cytokines. The fourth-generation CAR, on the other hand, adds selectable markers to multiple co-stimulatory molecular structures, allowing T-cell activity to be enhanced by the genes encoding CARs, inducing them to secrete cytokines and chemokines, recruiting and activating their own intrinsic immune cells, and improving T-cell survival in the tumor microenvironment. Fifth-generation CART cells are general-purpose CAR-T cells designed on the basis of gene editing technology to recognize more target proteins and improve the flexibility of CART cells to treat different cancers. Its genes are designed to prevent rejection by the body and to pre-prepare allogeneic T cells for patients for ready supply, but so far, fifth-generation CAR-T cells have not been widely used [15] (Figure 1).

Figure 1: The way of Car-T cell therapy in human.

Principles of CAR-T cell immunotherapy

Activation signalling of T cells and tumor-specific recognition are the basic principles of CAR-T cell immunotherapy [16]. A dual signalling pathway induces T-cell activation, consisting of antigen-specific signals, i.e., the binding of T-cell receptors (TCRs) to major histocompatibility complexes (MHCs) - antigenic peptides, as the first signalling pathway, and antigennonspecific signals, consisting of the binding of T cells to CMs on the surface of antigen-presenting cells (APCs), as the second signalling pathway [17]. The activation and cascade of T cells are informed by both signalling pathways, with the ultimate goal of converting T cells into cytotoxic T cells (CLT). This binding can induce the production of perforin, which can lyse tumor cells, or release a large amount of cytokines, which can change the living environment of tumor cells, thereby directly killing them or inhibiting their growth [18-20]. CAR-T cell immunotherapy, on the other hand, utilizes the combination of the effect of CLT on target cell toxicity and the specific recognition of antigens by antibodies to enable CAR to kill tumor cells by generating activated T cells through the specific recognition of antigens that target tumor cells [21]. In this treatment, most of the T cells are derived from the peripheral blood of the patient, and by introducing the designed exogenous gene into the T cells, a stably expressed CAR-T cell can be obtained, then the stably expressed CAR-T cells are cultured and proliferated in vitro, and finally the proliferated CAR-T cells are injected back into the patient to kill the tumor cells.

Preparation of CAR-T

Firstly, T cells are isolated from the patient’s peripheral blood cells, and then the T cells are cultured and proliferated in vitro. And secondly, the CAR gene is introduced into the patient’s isolated T cells by electroporation, lentivirus or retrovirus transduction, so that the T cells are modified to express the CAR gene to obtain stably expressed CAR-T cells. The retroviral transduction method has a wide range of application and high transduction efficiency, but has a high risk of tumorigenicity due to the loss of the ability to infect non-dividing cells due to the small DNA fragments carried by the transduction method. For safety reasons, electroporation is safe, but has the disadvantage of transient expression, which requires multiple injections [22-24].

Current status of research on CAR-T therapy for various types of cancer treatment

Chimeric antigen receptor (CAR) T-cell technology was originally proposed by Eshhar and Gross in the 1980s with the idea of directing T-cell responses through genetic editing of T-cell receptors (TCRs) to enable them to play a greater role in the immune response and fight against tumors. In recent years, various gene editing techniques have enabled CAR-T cells to be targeted to different sites, thus greatly improving the therapeutic effect on various types of cancers [12].

CAR-T cell immunotherapy for head and neck squamous cell carcinoma

Head and neck squamous cell carcinomas (HNSCCs), as malignant lesions of the mucosal epithelium of the oral cavity, pharynx and larynx, account for 90% of all head and neck cancers and are among the sixth most common malignancies worldwide(25-27). Surgery with conventional radiotherapy, chemotherapy and immunotherapy is the basis of treatment for squamous cell carcinoma of the head and neck [28]. However, tumor recurrence and metastasis remain at a high level with these treatment modalities. There is growing evidence that chimeric antigen receptor T (CAR-T) cells have promising antitumor effects in the treatment of HNSCC [29,30].

Currently, investigators are approaching the treatment of squamous cell carcinoma of the head and neck through three main areas: preclinical studies, clinical studies, and the search for potential targets [30,31] (Table.1).

Table 1: The treatment of squamous cell carcinoma of the head and neck.

In preclinical studies, investigators found that HER2 expression was detectable in 0-47% of HNSCC patient tissues and that HER2 overexpression was often associated with a poorer prognosis for HNSCC patients [44,45]. Warren et al. developed specific CAR-T cells targeting HER2 for the treatment of HNSCC and their results showed that anti-HER2 CAR-T cells resulted in a 56% reduction in tumor size, suggesting that HER2 may be a potential target for CAR-T cells to treat HERpositive HNSCC [38,46,47]. Similarly, overexpression of CD70, a ligand for tumor necrosis factor, which is highly expressed in HNSCC, is associated with a reduction in CD8+ T cells and can induce immunosuppression TME [48]. Park et al. analysed nine proteins highly expressed in HNSCC cells as potential CAR-T cell targets and demonstrated that compared to the untreated group, anti-CD70 CAR-T cells could effectively eradicate HNSCC cells [38,49]. In addition to this, EGFR was overexpressed in hypopharyngeal cancer, which accounts for about 5% of HNSCC [50]. Thus, Dong et al. developed CAR-T cells targeting EGFR to limit the growth of EGFR-positive hypopharyngeal cancer cells. The results showed that the cytokine secretion and lysis rates of hypopharyngeal cancer cells were significantly increased after coculture of hypopharyngeal cancer cells with CAR-T cells [32].

For clinical studies in the treatment of HNSCC, Sophie Papa et al. conducted a phase I clinical trial for the treatment of HNSCC in combination with intra-tumor injection of the binary lysing adenovirus CAdVEC to investigate the safety and cytotoxic effects of CAR-T cell immunotherapy targeting HER2 in the treatment of HNSCC [30].

In conclusion, in the treatment of HNSCC, researchers have been searching for its new targets to enhance the specificity of CAR-T cells, reduce the side effects of CAR-T cells, and continuously promote the transformation of CAR-T cell immunotherapy to clinical applications.

CAR-T cell immunotherapy for breast cancer

Breast cancer is the leading cancer in women. Approximately 20-30% of breast cancer patients develop invasive or metastatic disease after radical surgical resection and eventually die [51,52]. Despite advances in chemotherapy, endocrine therapy and molecular targeted therapy, there are still some breast cancer patients who are less sensitive to treatment. Treatment resistance and tumor recurrence or metastasis also develop as treatment proceeds [53]. Therefore, investigators need to explore more favourable treatment strategies. Chimeric antigen receptormodified T cells have made some progress in immunotherapy for breast cancer by exploring breast cancer-related targets [5456]. For example, for HER-2-positive breast cancer, a subtype that affects approximately one quarter of breast cancer patients, HER-2 gene amplification or protein overexpression underlies HER-2-positive breast cancer [57,58]. HER-2 can be used as a recognition molecule for constructing HER-2-specific CAR-T cells for immunotherapy of HER2-positive breast cancer [59]. li et al. used CAR-T cells targeting HER-2 cells in combination with PD-1 blockers to kill Herceptin-resistant breast cancer cells [60]. seyedmirzaei et al. used HER-2-specific CAR-T cells to eliminate tumors and improve survival of trastuzumab-resistant breast tumor cells in mice [61]. meili et al. prepared chA21 scFV-based HER-2specific CAR-T cells and it was shown to recognize and kill HER2-positive breast cancer cells in vitro, as well as to induce breast cancer regression in vivo [62].

In addition, CAR-T cell immunotherapy is being further investigated by researchers in triple-negative breast cancer (TNBC) and other types of breast cancer (Table 2).

Table 2: CAR-T cell immunotherapy in TNBC and other types of breast cancer.

CAR-T cell immunotherapy for HIV

CAR-T cell immunotherapy has been applied in clinical trials for a variety of cancers with promising results, which have demonstrated the safety, feasibility, efficacy and durability of CAR-T cell immunotherapy [63,64]. Based on these results, the HIV field has begun to view CAR-T cell immunotherapy as a promising HIV cure strategy [65,66]. Unlike other strategies for treating HIV, CAR-T cell immunotherapy would be a targeted, curative tool designed to completely eliminate the HIV reservoir from the patient’s organism [67-69]. Table 3 summarizes the clinical trials of investigators using CAR-T cell immunotherapy for HIV treatment over the last two decades.

Table 3: CAR-T cell immunotherapy for HIV treatment.

CAR-T cell immunotherapy for ovarian cancer

Ovarian cancer (OC), one of the most common gynecologic malignancies, has a poor prognosis and high mortality rate [72]. Most patients are diagnosed at advanced stages (stage III or IV), with a global 5-year survival rate of 25% to 47%. Surgical resection and first-line chemotherapy are the main treatment modalities for OC [73]. However, due to resistance to chemotherapy, patients usually relapse within a few years of initial treatment. Cell-based therapy, chimeric antigen receptor T (CAR-T) cell therapy, represents an alternative immunotherapeutic approach with great potential for the immunotherapy of ovarian cancer [74,75]. Ovarian tumors typically lack TSA, and therefore, the target of immunotherapy is usually one or more tumor-associated antigens [7]. Researchers are currently constructing CAR-T cells that target different targets on the surface of ovarian cancer to achieve ovarian cancer immunotherapy [5,19]. For example, erb-b2 receptor tyrosine kinase 2 (ERBB2) [62,76], programmed cell death-ligand 1 (CD274) [73,77], programmed cell death 1 (PDCD1) [78-80], epithelial cell adhesion molecule (Ep-CAM) [81], anti-Müllerian hormone receptor type 2 (AMHR2) [82], annexin A2 (ANXA2) [83], trophoblast glycoprotein (TPBG) [74], folate receptor alpha ( FOLR1) [84,85], mesothelin (MSLN) [72,8688], mucin 16 (MUC16) [89,90] and CD24 [75], among others (Figure 2).

Figure 2: Constructing CAR-T cells that target different targets on the surface of ovarian cancer to achieve ovarian cancer immunotherapy.