2.1. Concept of CAR/TCR Engineered T Cells
After decades of relatively low success rates when trying to convert immunological concepts in efficacious immunotherapeutic tools—with the possible exception of allogeneic hematopoietic cell transplantation that was empirically developed as a cellular immunotherapy to treat mostly hematological malignancies—recent years have witnessed the introduction of several practices changing medicinal products. In particular, the remarkable success rates and improvement in outcome seen with the introduction of immune-checkpoint inhibitors for the treatment of malignant melanoma and lung cancers has heralded a rush among biotech and pharma companies to develop new tools to activate or expand the abilities of the patient immune system to control tumor growth.
Further progress in the engineering of monoclonal antibodies lead to the development of BITE®
or bispecific T cell engager; the first BITE®
to reach the market was blinatunomab that targets CD19 and is indicated for the treatment of relapsed/refractory (r/r) adult acute lymphoblastic leukemia (ALL) since 2015. BITE© antibodies have two arms, one that binds a membrane antigen expressed at the surface of the targeted (tumor) cell such as CD19 and the other that binds T cells leading to their activation and cytotoxic effect in the close vicinity of the tumor cells [27
Another important and more recent avenue is the development of hematopoietic cellular therapies (Figure 1
) manufactured from or made of immune effector cells (IECs), the most publicized of which being CAR-T Cells [28
]. CAR stands for ‘Chimeric Antigen Receptor’ a synthetic protein encoded by a DNA sequence that juxtaposes the extracellular domain of a single chain immunoglobulin, the intracellular domain of the Zeta chain of the T-Cell Receptor (TCR) with a hinge region and one or several domains from costimulatory molecules such as CD28 or 4.1 BB in between [30
]. The extracellular domain targets a membrane antigen expressed at the surface of targeted cells such as CD19 for lymphoid malignancies (ALL, non-Hodgkin’s lymphoma, etc.) or B Cell Maturation Antigen (BCMA) expressed on malignant plasma cells in patients affected with multiple myeloma. Recognition of the target antigen is not restricted by the Major Histocompatibility Complex (MHC) molecules, allowing for wide clinical applications. Binding of the cognate ligand triggers T-cell activation and cytotoxicity through the TCR domain. The nature of the co-stimulatory domain(s) has important implications for in vivo amplification and persistence of CAR-T cells after their infusion.
Currently, three autologous CAR-T cells—all targeting CD19—have been approved by health authorities in the USA and Europe, as well as in many other countries: these include tisagenlecleucel for the treatment of relapsed/refractory ALL under the age of 25 [31
] as well as for the treatment of relapsed/refractory diffuse large B-cell Lymphomas (DLBCL) [32
], axicabtagene ciloleucel for the treatment of r/r DLBCL and primary mediastinal NHL [34
] and brexucabtagene autoleucel for the treatment of r/r mantle cell NHL [35
]. Two autologous CAR-T Cells targeting BCMA, idecabtagene vicleucel [36
] and ciltacabtagene autoleucel [37
], are likely to be soon approved by the FDA, the EMA, and Chinese health authorities for the treatment of patients affected with advanced multiple myeloma.
Numerous developments are underway with the evaluation of novel tumor targets to treat new categories of diseases such as Hodgkin’s disease [38
], myeloid malignancies [39
] or solid tumors, strategies to overcome resistance, largely due to the loss of the targeted tumor antigen [40
], strategies to mitigate side-effects associated with CAR-T cells administration such as the cytokine release syndrome (CRS) or immune effector cells associated neurological syndromes (ICANS) [41
] improved and more complex CAR structures designed to counteract the immune suppressive environment that characterizes many tumor types, support in vivo persistence of CAR-T cells and recruit endogenous immune effectors [44
]. In addition, CAR-technologies are now combined with gene editing as a substitute to retroviral or lentiviral vector transduction [45
] with the use of allogeneic cells that hold the promise of off-the-shelf medicines [46
] and the genetic engineering of other immune cell subsets such as natural killer (NK) cells [47
], γ/δ T cells, or macrophages. The field is thus blooming with expectations.
In addition to the excitement raised by the first approved CAR-T Cells, the field of IECs is also pursuing developments with TCR transgenic T-Cells (also called engineered T-cells). In this context, recognition of the targeted tumor cells is not limited to membrane antigens but allows for the recognition of MHC-restricted peptides and may thus be more adapted to the treatment of solid tumors (Figure 1
). Nevertheless, MHC restriction limits the application to subsets of patients that share the most frequent HLA types in a population of common ancestry. Editing of the endogenous TCR to be replaced by the transgenic TCR is likely to improve biological activity in the future, but similar to CAR-T cells, the issues of T-cell exhaustion in an immune suppressive tumor micro-environment and of trafficking of the genetically modified T-cells to the tumor site needs to be tackled before consistent clinical efficacy can be demonstrated and the first medicinal products in this category are approved and reach the market. Both for TCR-T cells and CAR-T cells, the choice and validation of the target antigen is of utmost importance for optimal clinical efficacy and minimization of on-target/off-tumor side-effects.
All currently available and investigational IECs represent a new category of medicinal products that require a very specific organization for the manufacturing process in the context of newly defined regulatory frameworks, as well as a very specific organization for hospitals that provide access to these treatments [48
]. The complex, sophisticated, and largely manual logistics—that involves shipment of viable cells over long distances—partly explains the high price tag of these innovative gene therapy or cell therapy medicinal products. It also implies a significant turnaround time before the (autologous) therapy becomes available to the candidate patient, raising significant issues in terms of disease control during this period, with the need for bridging therapy in a proportion of patients, and patients with fast progressive tumors remaining ineligible for such approaches. Despite these uncertainties, the field is quickly moving forward and the potential for combinations with other forms of immunotherapies such as immune checkpoint inhibitors or with targeted therapies/chemotherapies fuels high expectations in the patients’ community and their families. Thorough evaluation of the safety profile and efficacy profile of these medicinal products that are mostly authorized on the basis of phase I/II registration trials, will require the collection of data over extended period of time in real-world conditions in the post-authorization era [49
]. This will also help define the role of these IECs in the treatment of various categories of neoplastic diseases, in particular in comparison with other immunotherapeutic agents such as BITE© [50
2.2. Targets in Hepatocellular Carcinoma Adoptive Cell Transfer
Like other immunogenic tumors, subjects undergoing hepatic resection for HCC with prominent lymphocyte infiltration are associated with reduced recurrence and better prognosis as compared with those without prominent lymphocyte infiltration [51
]. Moreover, recurrence after liver transplantation for HCC is related to immunosuppression [53
] as well as the presence of T regulatory cells (Tregs) in the infiltrate [52
In the next section, we are listing the main targets used in ACT for HCC. None of these antigen are tumor-specific antigens (expressed by the tumor with minimal to no expression in normal tissue) [54
]. They mainly belong to three categories of tumor antigens: (i) tumor-associated antigens: antigens whose expression is enriched but not specific to cancer cells (e.g., AFP, GPC-3); (ii) cancer–testis antigens: antigens whose expression is limited to cancer cells and reproductive tissues but not adult somatic tissue (e.g., NY-ESO-1, MAGE); (iii) viral-derived cancer antigens: antigens expressed by cancer cells derived from an oncogenic viral origin (VHB, VHC).
(1) Alpha-fetoprotein (AFP) is a 70-KDa glycoprotein found in serum of early mammalian embryos, synthesized at the site of embryonal hematopoiesis: the yolk sac [55
]. After birth, the levels drop off rapidly, and by the second year only trace amounts are detectable in serum. The normal adult levels typically range between 1 and 40 ng/mL. Reappearance or high serum levels are observed in several conditions: pregnancy, hepatic disorders, and malignancies such as hepatocellular carcinomas, germ cell tumors (especially with yolk sac tumor components), breast, esophagus, cervical, pancreatic, endometrial, gastric, lung, and rectum cancers [56
]. Up to 50% of HCC tumors express AFP [57
]. Tumor AFP expression generally correlates with serum AFP, although this correlation is not absolute. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC [58
]. Pre-clinical studies demonstrated the potential of AFP for cellular immunotherapies [59
]. It has been reported that malignant liver cells produce AFP-L3, even when HCC is at its early stages, and especially when the tumor mass is supplied by the hepatic artery.
(2) Glypican-3 (GPC-3) is a member of the heparan sulfate proteoglycan family controlling cell division and growth regulation. GPC-3 is an antigen expressed in over 70% of HCCs but rarely in non-malignant tissues. Indeed, GPC-3 positive immunostaining can differentiate hepatocellular carcinoma (HCC) from dysplastic changes in cirrhotic livers. Recent studies demonstrated that greater GPC-3 expression in tumor cells was associated with a worse prognosis for HCC [60
]. Glypican-3 antibodies are investigated as a therapeutic option for HCC, either alone or as a drug carrier [61
]. Numerous pre-clinical studies support the evidence of GPC-3 targeting with adoptive cell therapies [64
(3) Melanoma antigen gene family (MAGE) consists of 12 members and is expressed almost exclusively in cancer tissues in a wide variety of malignant tumors [66
]. In RNA expression in HCC, MAGE-1 and -3 were expressed in approximately 68% of the tumors; MAGE-8 was expressed in 46%; and MAGE-2, -6, -10, -11, and -12 were expressed in approximately 30% [70
]. Several MAGE peptides have been shown to induce a strong cytotoxic T-lymphocyte (CTL) response in patients with melanoma [72
(4) New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a protein consisting of 180 amino acids. As a member of the cancer testis antigen (CTA) family, NY-ESO-1 has been shown to be expressed in spermatogonia, primary spermatocytes, oogonia, and placenta and in a variety of cancers, such as melanoma, ovarian cancer, cervical cancer, gastric cancer, and HCC. [74
]. In Nakamura et al., NY-ESO-1 mRNA was detected in 18 of 41 (43.9%) hepatocellular carcinomas [75
(5) Human telomerase reverse transcriptase (hTERT) plays a key role in conferring immortality to cancer cells through the regulation of telomere length. It has been reported that 80% to 90% of hepatocellular carcinomas (HCCs) express hTERT [76
]. Additionally, peptides containing hTERT epitopes are able to induce hTERT-specific cytotoxic lymphocytes [77
(6) NK group 2 member D ligand (NKG2DL) is a type II transmembrane-anchored C-type lectin-like protein receptor expressed on natural killer (NK) cells, CD8+
T cells, subsets of γδ T cells, and some autoreactive CD4+
T cells. The Cancer Genome Atlas and microarrays of HCC samples showed NKG2DL are generally absent on the surface of normal cells but are overexpressed on malignant cells, offering good targets for CAR-T therapy. [78
]. Recently, in vitro studies reported that NKG2D CAR-T cells efficiently killed the HCC cell lines.
(7) Epithelial cell adhesion molecule (EpCAM) is a type I membrane protein of 314 amino acids (aa) of which only 26 aa are facing the cytoplasm [79
]. EpCAM has oncogenic potential and is activated by release of its intracellular domain, which can signal into the cell nucleus by engagement of elements of the wnt pathway [81
]. EpCAM was found to be frequently over-expressed in a wide variety of carcinomas, including HCC, colon, gastric, pancreas, and breast cancers [82
(8) Mucin1 glycoprotein 1 (MUC1) belongs to the family of human epithelial mucins [84
] Its expression on normal cells is hidden from the immune system, and its aberrant glycosylation (large number of O-glycosylated tandem repeat) on tumors creates new epitopes recognized by the immune system [85
]. Pre-clinical studies in vitro and in xenograft models validated MUC1 target for CAR-T therapy [86
(9) Viral antigens: viral surface proteins are not controlled by available antiviral agents and are usually maintained in HCC with integrated viral genomes [88
]. In vitro and in mice [89
], CAR-T cells directed against the HBV surface proteins enabled human T cells to kill HBV-infected human hepatocytes and to eliminate viral DNA. Interestingly, TCR gene-modified T cells (T cells genetically engineered with a high-affinity, HLA-A2-restricted, HCV NS3:1406-1415-reactive TCR) mediated regression of established HCV+
HCC in xenograft model [90