*4.3.1.1. Cytokine induced killer cells (CIK)*

CIK cells are a heterogeneous MHC independent cell population which are able to both recognize tumor antigens and kill cancer cells directly [58, 59]. In a phase III study of adjuvant CIK therapy after radical resection for HCC, patients were randomized to receive four cycles of CIK therapy or no treatment. The median time to recurrence (TTR) was 13.6 months in the CIK group and 7.8 months in the control group (p = 0.01), All adverse events were grade 1 or 2. There were no significant differences in incidence between the two groups, indicating the safety and efficacy with respect to prolonging TTR of CIK therapy in patients with HCC. However, there were no statistically significant differences between the groups in disease-free survival (DFS) and overall survival (OS) [60]. In addition, a meta-analysis of 693 patients with HCC demonstrated that a combination of dendritic cell- (DC-) CIK cells and TACE improves 1-and 2-year OS, overall response rate (ORR), disease control rate (DCR), and the quality of life [61].

### *4.3.1.2. Tumor infiltrating lymphocytes (TILs)*

TILs are autologous tumor-infiltrating lymphocytes (TIL), which are derived from tumor tissues and are cultured and induced using IL-2 and anti-CD3 antibodies ex vivo [62–64]. Reinfusion of autologous TILs, which possess tumor-specific immunity, may target multiple tumor antigens. Low toxicity of autologous TILs was verified in a phase I study involving patients with HCC, suggesting a novel treatment option [65]. To date, TILs have not been well characterized, mainly due to difficulties in purifying and expanding them.

#### *4.3.1.3. Natural killer cells (NKCs)*

NK cells are component of innate immune system and can directly kill tumor cells and infected cells without preliminary sensitization or MHC restriction [66]. However, they lack the ability to target tumor cells and can injure normal liver tissues. In a previous series of experiments, the cytotoxicity of NK cells against HCC cells was enhanced by first generating a new hepatoma cell line, K562-mb15-41BBL, which achieved a more efficient stimulation of NK cells in vitro [67]. Furthermore, HCC cells exposed to 5 μmol/L sorafenib for 48 h showed high sensitivity to NK cells. Finally, NKG2D, an engineered NK-cell-activating receptor, was tested in vitro and in mice. All of the outcomes were positive in increasing the cytotoxicity of NK cells, providing the possibility of further clinical trials in HCC.

Checkpoint inhibitors combinations have also been studied, as a way to improve synergy and overcome resistance. PD-L1 is not the only immunosuppressive factor in the tumor microenvironment. The regulatory T cells (Treg) stands out among the immunosuppressive cells of the tumor microenvironment. Anti-CTLA-4 agents deplete tumor-associated Treg via an FccR dependant mechanism in preclinical models and have promising result in malignant

Biologic and Immunotherapy Developments in Advanced Hepatocellular Carcinoma

http://dx.doi.org/10.5772/intechopen.79872

169

Historically, traditional chemotherapeutic agents have not shown great efficacy in the treatment of HCC when used in the advanced disease stage, in particular in case of progression after locoregional therapy. Moreover, conventional cytotoxic chemotherapies have not provided a clinical benefit or prolonged survival for patients with advanced HCC. There are limited data supporting the use of cytotoxic chemotherapies in unresectable disease, and it

Advanced HCC remains a deadly disease with limited systemic treatment options. The advent of sorafenib as first-line treatment ignited a plethora of trials testing various targeted and immunotherapeutic approaches. Currently, both regorafenib and nivolumab are FDA approved for second-line treatment among patients with advanced HCC who progressed

> **Line of treatment**

No Second or

List of abbreviations: MOA: mechanism of action, Ref: references, DCR: disease control rate, FGFR: fibroblast growth factor receptor, McA: monoclonal antibody, MKI: multi-tyrosine kinase inhibitor, OS: overall survival, PFS: progression free survival, PDGFR: platelet-derived growth factor receptor, PD-1: programmed death-1, TKI: trosine kinase inhibitor,

third line

Anti-PD-1 Yes Second line CheckMate-040 DCR, OS,PFS [36]

**Trial Positive** 

Yes First line SHARP OS, PFS [3]

Yes Second line RESORCE OS, DCR [13]

No First line REFLECT Non-inferior

**outcome**

to sorafenib

CELESTIAL OS [15]

**Ref.**

[11]

**Approved**

melanoma [77].

**6. Conclusions**

**5. Cytotoxic chemotherapies**

**Agent Type MOA FDA** 

PDGFR, TKI

stromal TKI

AXL c-KIT

FGFR, PDGFRs, c-kit

Sorafenib MKI VEGFRs,

Lenvatinib MKI VEGFR,

Regorafenib MKI VEGFR, anti-

Cabozantinib MKI VEGFR, MET,

McA

VEGFRs: vascular endothelial growth factor receptors.

**Table 1.** Most common systemic agents for advanced HCC.

Nivolumab IgG4

should be used preferably in the context of a clinical trial [78].

#### *4.3.1.4. CAR-T cell*

Chimeric antigen receptor redirected-T cells (CAR-T cells) are genetically modified T lymphocytes that specifically target tumor-associated antigens (TAAs) and kill cancer cells in an MHCindependent manner [68, 69]. CAR-T cells have achieved inspiring outcomes in patients with B cell malignancies with great therapeutic efficacy in leukemia and lymphoma therapy. CAR T therapy is being studied for solid tumors, such as HCC [70]. In some solid tumors with a tremendous phenotypic heterogeneity, CAR T cells could target the tumor antigen and cause antigen-positive cell death, while antigen-negative cancer cells may induce tumor relapse. However, Cart T cell structure engineering has been evolved significantly. Recently, CAR T cells with a transgenic "payload or TRUCK," also called the "fourth generation" CAR T cells, were designed [71]. This CAR T cells work by releasing inducible cytokines such as IL-12 which will augment T cell activation and further activate innate immune system to kill antigen negative cancer cells. Specific Tumor-associated antigens in HCC that recognized by cytotoxic T lymphocytes (CTLs) have been investigated. GPC3, which usually correlates with poor prognosis in HCC, has been demonstrated as a promising liver cancer-specific target in multiple studies, due to its overexpression in HCC and limited expression in normal tissues [72] GPC3-targeted CAR T cells could providing promising therapeutic intervention for GPC3-positive HCC. The ability of GPC3-targeted CAR T cells to eliminate GPC3-positive HCC cells was confirmed both in vivo and in vitro, and the survival of mice with HCC xenografts was prolonged with CAR T cell therapy in vivo [73]. In another study, T cells with two complementary CARs against GPC3 and asialoglycoprotein receptor 1 (ASGR1) decreased the risk of on-target, off tumor toxicities and demonstrated potent antitumor immune responses targeting GPC3<sup>+</sup> ASGR1<sup>+</sup> HCCs both in vivo and in vitro [74]. However, to date, the related studies conducted have been predominantly basic, and more clinical trials are required to prove the efficacy of CAR T in HCC.

#### **4.4. Combination strategies**

Combination therapies include combinations of different checkpoint inhibitors with TKIs, oncolytic viruses, small molecules and ablative therapies.

Combining anti-PD-1 with sorafenib has been studied in an animal model in HCC. The results showed efficacy only with the concomitant targeting of the hypoxic and immunosuppressive microenvironment with agents such as CXCR4 inhibitors, and not when combined with sorafenib alone [75]. According to these results, a potential future approach could be by careful titration of VEGF inhibition with the aim to block the VEGF pathway and contemporarily alleviate hypoxia by vascular normalization, enhancing immunotherapy efficacy [76].

Checkpoint inhibitors combinations have also been studied, as a way to improve synergy and overcome resistance. PD-L1 is not the only immunosuppressive factor in the tumor microenvironment. The regulatory T cells (Treg) stands out among the immunosuppressive cells of the tumor microenvironment. Anti-CTLA-4 agents deplete tumor-associated Treg via an FccR dependant mechanism in preclinical models and have promising result in malignant melanoma [77].
