2.1 T cell receptor-modified T cells

TCR-Ts are generated by a gene-engineered method to change an autologous or allogenic T cell such that it expresses a particular tumor antigen and causes the lysis of cancer cells. The main goal of TCR-T treatments is to guide CD8+ T cells, the main agents of antitumor immune responses, to human leukocyte antigen (HLA) class I restricted sites [5]. Endogenously processed linear peptide epitopes, presented by major histocompatibility (MHC) molecules, are recognized by the TCR, so that both surface-bound proteins and intracellular targets are covered [6]. TCR-Ts elicit lower peak cytokine levels at high antigen densities. However, certain CAR-T cells can cause major immunological toxicities that may result in the cytokine release syndrome (CRS), which can be fatal [3]. Additionally, the use of a particular TCR in immunotherapy is always dependent on the presence of the patient's matching HLA molecule and the level of MHC class I molecule expression in the target cells. However, MHC class I can be downregulated in viral infections, and its expression is low in a number of tumors that induce immune evasion [7].

2.2 Chimeric antigen receptor T cells

T cells have the capacity to identify tumor antigens without the aid of HLA because of CAR-Ts. The critical component of CAR-T therapies is the CAR, which is composed of four essential parts: an extracellular hinge domain, a trans-membrane domain, an intracellular signal domain, and a binding domain specific to the tumor-associated antigen (TAA). This binding domain is typically derived from the scFv fragment of the antigen-binding region in monoclonal antibodies [8]. Most aberrant cancer proteins are present within cells and only presented by MHC complexes after proteasomal degradation. CAR-Ts only recognize surface TAAs, making it difficult to discover appropriate antigens in many cancers [3].

Four generations of CAR-T therapy have been produced, and these mainly differ in the co-stimulatory domain. In the first generation of CAR, the tyrosine activation motif on the CD3ζ chain without a co-stimulatory domain activates T cells. In the second generation of CAR, a second co-stimulatory domain, often CD28 or 4-1BB (CD137) moieties, was included to enhance the ability of T cells to survive and proliferate. In the third generation of CAR-T, an additional co-stimulatory domain (CD28 and 4-1BB or TLR2) was introduced to significantly increase the effectiveness of infused CAR-T cells. The addition of the intracellular region of the cytokine receptor to the CAR was included in the fourth generation of CAR and significantly aids in the promotion of T cell growth [5]. The antigen-binding site retained its distinctive property, allowing CARs to recognize their target in a manner similar to that of an antibody by binding molecules in a conformation-dependent manner, without the need for intracellular antigen processing and MHC pathway presentation. This increases the application of CAR-T cells but also has a drawback. The use of CARs is restricted to molecules that are present on the cell surface, encompassing only approximately 30% of all known proteins. Additionally, soluble versions of the target in the peripheral circulation can interfere with CAR-T cells. A concerning possibility is that these soluble antigens might “capture” CAR-T cells and prevent them from traveling to their target location. Furthermore, they might cause CAR-T cells to spontaneously activate off-target and cause unfavorable side effects [6].

4.1 Hepatitis B virus

HBV mainly causes human HCC. Despite the fact that the global immunization campaign has reduced the infection incidence of HBV in children under the age of five to 1%, a World Health Organization (WHO) research study indicated that there are still an estimated 257 million chronic HBV carriers worldwide. Furthermore, according to WHO's report in 2020, as of 2015, the mortality from this virus was over 8.8 million, mostly from liver cirrhosis and HCC [17]. Several HBV-specific TCRs were produced after the creation of a CAR that was specific for the HBV envelope protein; this strategy served as the foundation for the use of T cell immunotherapy for HBV infection [6]. The nucleoside analogs (NA) used in current HBV antiviral treatments prevent the synthesis of HBV-DNA but have no effect on the generation of antigen from HBV-RNA templates. Because they display HBV antigens similar to untreated infected cells, HBV-infected hepatocytes treated with NA can be specifically targeted by CAR/TCR-T cells [8]. HCC cells produce a peptide chain that can attach to major MHC molecules, making it possible for T lymphocytes to identify the cells. HCC cells often have integrated HBV-DNA fragments. For CAR/TCR-T cell treatment, HBsAg may be a possible target [16]. In vitro recognition of HBV-positive cell lines and HBsAg particles by HBsAg-specific CAR-T cells was assessed in a preclinical investigation. However, in cytotoxicity experiments, HBsAg-specific CAR-T cells failed to kill HBV-positive cell lines. When HBsAg-CART cells were adoptively transferred into HBV-infected humanized mice, the levels of HBsAg and HBV-DNA in the liver significantly dropped in comparison with those in control animals. Notably, after treatment with HBsAg-specific CAR-T cells, the proportion of HBV core–positive hepatocytes among all human hepatocytes significantly decreased, suggesting noncytopathic viral elimination [18].

Eight patients with advanced HBV-HCC were included in a phase I clinical trial (NCT03899415) that used adoptively transplanted short-lived autologous T cells that had HBV-specific TCR. One of the patients experienced a partial recovery that lasted for 27.7 months. Importantly, following HBV-TCR-T cell infusion, the majority of the patients showed a decrease in circulating HBsAg and HBV DNA levels or their stability, demonstrating on-target effects [18]. In another phase I clinical trial (NCT04677088), autologous T cells that expressed specific TCRs encoded by the incorporation of HBV-DNA into the host DNA were introduced. A small HBV peptide, rather than the whole HBV antigen, is encoded by the HBV-DNA integration. By transfecting mRNA into cells, autologous T cells were made to express the selected TCRs, and these TCR T cells were adoptively transplanted into two patients in increasing quantities (1 × 10⁴–10 × 10⁶ TCR-T cells/kg) weekly for 112 days or 1 year, with no negative side effects. Five of six lung metastases in one patient showed volume reduction during the first year of the T-cell treatment [15]. The few permanently infected hepatocytes that are present in these individuals may be the only target for CAR/TCR T cells [8]. Research has indicated that immunotherapeutic IL-2 is capable of rescuing CD8+ T lymphocytes that have been rendered dysfunctional by hepatocellular priming. Therefore, when anti-progressive disease (PD)-L1 therapy is not immediately effective, IL-2-based techniques should be taken into account for the combination treatment of chronic HBV infection with CAR/TCR T cells [19].

4.2 Hepatitis C virus

The HCV, which affects 180 million individuals globally, is one of the main causes of chronic liver disease [20]. With over 90% cure rates, direct antiviral medicines have changed patient care [21]. However, some patients are still unable to completely eliminate the HCV, and therefore, a novel therapy is urgently needed for these patients. In preclinical ACT research, hepatocytes infected with HCV derived from cell culture (HCVcc) were destroyed by HCV-specific TCR-Ts targeting the HCV E2 protein. Target cells expressing HCV/E2 of various genotypes and subtypes may be identified by anti-HCV CAR-T cells, and these cells can then be killed. HCV/E2-transfected and HCVcc-infected target cells exhibited cytotoxic activity when exposed to anti-HCV CAR-T cells. Additionally, T cell degranulation and the release of pro-inflammatory and antiviral cytokines occurred concurrently with antigen detection [21]. This preclinical study indicated that engineered E2-specific T cells may be a powerful treatment for HCV-associated HCC.

4.3 High risk human papillomavirus

HPV, the most common oncogenic virus, is the cause of 30% of all malignancies caused by infectious agents [9]. HPV-18 and HPV-16 make up most of the carcinogenic HPV subpopulation. Recent data from in vitro and in vivo experiments have raised the possibility that HPV-positive epithelial malignancies might benefit from TCR T cells made to destroy HPV-infected cells by focusing on HLA-A*02:01 linked to a portion of the HPV-16 protein E7. The HPV E7 antigen, an alluring therapeutic target, is constitutively expressed by HPV+ malignancies but not by healthy tissues [22]. In a preclinical study, T cells that were genetically modified to target HPV-16 E7 caused regression of HPV-16+ human malignancies in an animal model. A preclinical foundation for an ongoing clinical study of E7 TCR T cells treating patients with metastatic HPV+ malignancies was supported by these findings [23]. Nagarsheth and colleagues conducted a first-in-human, phase 1 clinical trial of T cells engineered with a TCR targeting HPV-16 E7 for the treatment of metastatic human papilloma virus–associated epithelial cancers (NCT02858310) [23]. Six of the 12 patients who received the therapy had objective tumor responses, providing preliminary proof of the effectiveness of this treatment. One or more tumors completely regressed in three individuals. In some patients, cancer regression was extensive with durable regression of some tumors. One patient developed retroperitoneal, pelvic, thigh, and more than 80 lung metastases as a result of metastatic vulvar squamous cell carcinoma (SCC). She had previously received seven systemic anticancer medications. Following therapy, she had a partial response (PR) lasting 8 months, with 25 lung tumors completely regressing and being undetectable on imaging 8 months later. Another patient with metastatic SCC had more than 90 metastatic tumors affecting the thorax, retroperitoneum, bones, and kidney. Chemoradiation and PD-1-based treatment had been previously used to treat this patient. He had a PR lasting 9 months, with 80 tumors completely regressing and being undetectable on imaging 14 months following therapy. These findings highlight immune editing as a constraint on the curative potential of cellular treatment and perhaps other immunotherapies in advanced epithelial cancer. Furthermore, the results show that viral antigen–specific engineered T cells can induce the regression of common carcinomas.

4.4 Merkel cell polyomavirus

Merkel cell carcinoma (MCC), which has neuroendocrine characteristics, is an uncommon yet extremely aggressive skin cancer [24]. The disease-associated mortality of MCC has been calculated to be as high as 46% [24]. In 80% of MCC cancers, the MCV, which was identified in 2008, is clonally integrated. Several UV-related mutations are present at high frequencies in the remaining 20% of MCC cancers [25]. At least 60% of all MCC is caused by clonal integration of MCV DNA into the tumor genome with sustained production of viral T antigens [26]. The maintenance of MCC cell lines in vitro as well as virus-mediated carcinogenesis depend on MCV-encoded T-antigens (TAgs) [27]. Thus, TAgs may be a viable target for MCC treatment using modified T cells. Preclinical research identified naturally occurring MCV TAg epitopes and isolated TAg-specific TCRs. HLA-A2-positive cells that were loaded with the cognate peptide or cells that consistently expressed MCV TAgs were able to activate T cells that expressed these TCRs. TCR therapy resulted in the regression of existing tumors in a mouse model and proved the cytotoxic capacity of T cells modified to express these TCRs in vitro [27]. Patients with MCC that spread to other areas of the body were examined in a phase I/II clinical trial (NCT02584829) to determine the side effects and effectiveness of targeted radiation therapy, recombinant interferon beta, and avelumab with or without MCV TAg-specific polyclonal autologous CD8+ T cells. However, the lack of financing forced the end of the investigation. The limited results demonstrated that in the MCV TAg-specific polyclonal autologous CD8+ T cell treatment group, two patients had complete response, one had PR, three had PD, and one had partial complete response. In the 1-year follow-up period, four of the seven patients treated with MCV TAg-specific polyclonal autologous CD8+ T cells developed new metastases. Three of the seven patients who received MCV TAg-specific polyclonal autologous CD8+ T cell therapy died of all-cause mortality. The cellular treatment group did not show significant adverse events, but six of the seven patients had experienced other moderate adverse events, such as fever, skin infection, decreased lymphocyte count, skin ulceration, and hypertension. The outcome of the clinical trial is promising. Only one clinical trial is in progress, and thus more research is required to determine the effectiveness and safety of the MCV-TAg-specific engineered T cell immunotherapy.

4.5 Epstein-Barr virus

EBV is associated with the development of a broad range of malignancies, including Burkitt's lymphoma, Hodgkin lymphoma (HL), B-, T-, and natural killer (NK)-cell lymphomas, post-transplant lymphoproliferative disorder, nasopharyngeal carcinoma (NPC), and gastric carcinoma [28]. Over 90% of people worldwide are infected with EBV, but there are still no effective treatments or vaccinations [29]. These EBV-associated cancers exhibit viral antigens such as leader protein, latent membrane protein (LMP)-1, LMP-2, EBNA-1, EBNA-2, and EBNA-3A/B/C; the various tumor types have varied expression patterns [30]. The first proof that LMP-1₁₆₆-TCR engineered T-cells allow efficient recognition to display potent cytotoxicity toward engineered LMP-1 overexpressing tumor cells in vitro and in vivo was presented in a preclinical study that demonstrated a novel HLA-A2-restricted TCR that specifically recognizes the LMP-1₁₆₆ epitope [31]. The EBV-induced Natural T cell (EBViNT) clinical investigation examined autologous EBV/LMP2A-specific CD8+ T cells in patients with relapsed/refractory EBV-positive malignancies. Of the 11 recruited patients, 8 patients (4 with NPCs, 1 with HL, 2 with extranodal NK/T lymphomas, and 1 with diffuse large B-cell lymphoma) received a single infusion of EBViNT. All patients responded favorably to a single infusion of EBViNT, and three had demonstrable antitumor effects. This study demonstrated the safety of EBViNT therapy and suggests that the therapy may represent a new alternative for treating EBV-positive cancer patients with recurrent disease who have not responded to standard treatment [32]. A clinical trial (NCT04509726) for the LMP2-specific TCR-T in combination with IL-12 for the treatment of EBV-positive metastatic/refractory nasopharyngeal cancer has been filed; however, no participants have been recruited. The LMP2 antigen–specific high-affinity TCR-transduced autologous T-cell treatment for recurrent and metastatic nasopharyngeal cancer is being tested in another open-label, single-center, phase I clinical study (NCT03925896) and is now accepting participants. The high affinity TCR targeting the aforementioned EBV antigen was screened from healthy donors using the sorting and single-cell cloning approach in a phase II clinical study (NCT03648697) that focused on the NPC highly expressed EBV antigens such as LMP1, LMP2, and EBNA1. The TCR gene is then transferred to autologous T cells using lentivirus. In this trial, NPC patients who have previously received treatment but have progressed or relapsed will be examined for the safety and tolerability of EBV-TCR-T cell therapy.

4.6 Human T-cell lymphotropic virus-1

Adult T-cell leukemia (ATL), an aggressive form of T-cell cancer, may be induced by HTLV-1 infection. Between 5 and 10 million people were estimated to be carrying the HTLV-1 virus worldwide in 2012, with high endemicity clusters [33]. While the majority of HTLV-1-infected people remain asymptomatic carriers for the entirety of their lives, only approximately 5% eventually acquire ATL after a lengthy latency period of 50–60 years [34]. Despite an active immune response that includes cytotoxic T cells and NK cells, HTLV-1 survives in the host, indicating that the virus has created efficient defenses against host immune surveillance [35]. Tax and HBZ are two essential HTLV-1 oncoproteins; the expression of Tax varies among ATL cases, and the expression of HBZ is constant [36]. There is no preclinical or clinical trial of engineered T cell therapy targeting HTLV-1 specific oncogenic viral antigens. However, a study of the long-term survivors with ATL demonstrated that Tax-specific memory cytotoxic T-lymphocytes, together with anticancer agents, eradicated ATL cells and exhibited long-term preventive effects from relapsed ATL [37]. The study indicated that it is possible to use engineered CAR or TCR T cells specific for the HTLV-1 Tax antigen to treat ATL patients infected with HTLV-1.

4.7 Kaposi sarcoma herpesvirus

KSHV (also known as human herpesvirus 8) was identified as the pathogen of Kaposi sarcoma (KS). Immunosuppressed individuals have been reported to develop KS (iatrogenic KS). Epidemic KS (HIV1KS) refers to KS in the presence of HIV infection and AIDS. KS rates in persons living with HIV are 500 times higher than those in the general population [38]. The KSHV is also pathogenetically linked to a number of lymphoproliferative diseases, including diffuse large cell lymphoma (DLBCL), germinotropic lymphoproliferative disorder, multicentric Castleman disease, and primary effusion lymphoma (PEL)/extra cavitary-PEL [39]. In contrast to the strong T-cell responses to other human herpesviruses, T cell responses to KSHV are at a modest level [40]. When CD8+ T lymphocytes bind viral antigens expressed on MHC-I by infected cells, cytokines are secreted, cytotoxic granules are released, or the target cell is induced to undergo apoptosis. This process cannot take place if the target cell's MHC-I expression is impaired. T cell-mediated killing is thwarted by many KSHV proteins. Both KSHV K3 and K5 are viral ubiquitin ligases that promote MHC-I degradation and prevent CD8+ T lymphocytes from activating and recognizing infected cells. Pomalidomide is an immunotherapy used to treat KSHV-associated disorders that focuses on increasing MHC-I expression to improve CD8+ T cell identification of cancer cells [41]. The MHC-I molecule restricts TCR-T cell therapy functionality. TCR-T treatment for KSHV-associated disorders may be possible when combined with pomalidomide, although there is no clinical trial to support this possibility. Selecting a particular tumor-related antigen is crucial for TCR-T and/or CAR-T cell treatment for KSHV-associated malignancy. KSHV induces a variety of IFN-T cell responses; however, they are rarely as potent as those against EBV, Cytomegalovirus, and influenza. Reactions to some antigens (like K8.1) may offer more protection against viral reactivation than others (like ORF73/LANA). These findings shed light on the possible efficacy of KSHV-specific T cell immunotherapy using modified T cells to target K8.1 [40].