*4.2.2. Oncolytic immunotherapy*

of the liver; "Tolerogenic Liver"; that helps evade the immune response. The liver's pathway to immune tolerance is multifactorial. T-cells are stimulated through a dual signaling pathway that requires the interaction of T cell receptors (TCR) with major histocompatibility complex (MHC)/peptide complexes on antigen presenting cells (APCs) and expression of co-stimulatory molecules on T cells and APCs. Down-regulation of MHC class I molecules on tumor cells induces impairment of tumor antigen processing and presentation [25]. In addition, a reduced expression of co-stimulatory molecules, such as B7-1 and B7-2, in HCC leads to T cell anergy [26]. Programmed cell death protein-1 (PD-1) overexpression in tumors promotes immune evasion and tumor growth by suppressing T-cell response [27]. PD-L1 is not the only immunosuppressive factor in the tumor microenvironment. HCC immune evasion can also be achieved through overexpression of MHC class II molecules in tumor cells, which leads to

 T cell anergy in the absence of co-stimulatory molecules (CMs) on T cells and APCs. A better understanding of the antigenic profile of HCC and tumor microenvironment has helped

Checkpoint Inhibitors play critical roles in cancer immunology. Blockading the PD-1/PD-L1 pathway could modulate the tumor microenvironment, reactive T-cell and prime the endogenous antitumor immune responses. Treatment with checkpoints inhibitors have shown benefits in clinical trials of HCC. Common immune checkpoint proteins include cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), PD-1, programmed cell death ligand one (PD-L1), lymphocyte activation gene three protein (LAG-3), B and T lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin-domain-containing (TIM-3), VISTA and OX40 [29, 30].

CTLA-4 is constitutively expressed in activated T cells and NK cells [31]. CTLA-4 inhibitors prevent the binding of CTLA-4 to B7-1 and B7-2, thereby actively encourage the activation of T cells. CTLA-4 was the first checkpoint studied in HCC. Tremelimumab, an anti-CTLA-4 monoclonal antibody, was tested in a phase II study in a 21 patients with advanced HCC and hepatitis C. The disease control rate was (76.4%), median OS and PFS were 7.5 and 6.4 months respectively. Moreover, viral loads of HCC were significantly reduced. Although a shortlived remarkable rise in serum transaminases was observed after the first dose, no patients experienced immune-related adverse events or serious hepatotoxicity [32]. In another noncomparative clinical trial involving patients with advanced HCC, a combination therapy of tremelimumab and radiofrequency ablation increased the number of intratumoral CD8<sup>+</sup> T

The PD-L1/PD-1 pathway is another mechanism of tumor-induced immune tolerance. PD-1

T cells is increased in patients with HCC compared to

to develop a refined immunotherapeutic strategies in treatment of HCC [28].

CD4<sup>+</sup>

164 Liver Cancer

**4.2. Indirect immunological strategies**

cells and reduced HCV viral loads [33].

expression on effecter phase CD8<sup>+</sup>

*4.2.1. Checkpoint inhibitors*

*4.2.1.1. CTLA-4 inhibitors*

*4.2.1.2. PD-1 inhibitors*

Targeting tumor vasculature by oncolytic viruses (OVs) is an attractive strategy that offers several advantages. Oncolytic viruses are wild-type or engineered viruses that selectively target and replicate in cancer cells and cause lysis without harming normal tissues [39]. The underlying mechanism of the antitumor activity for oncolytic viruses involves direct killing of tumor cells by expanding in the cells and causing cell lysis. Different from normal cell, viruses can expand in cancer cells considerably due to the impairment of the tumor's defense mechanisms against viral infection. [40–43]. In addition,, OVs can initiate antitumor immune response by triggering key signals through oncolysis to dendritic cells (DCs) and other antigen-presenting cells (APCs) [44]. OVs have some advantages over other treatment modalities, those include: the low probability of generating resistance as OVs often target multiple oncogenic pathways; OVs replicate in a tumor-selective fashion with minimal systemic toxicities; and virus dose in the tumor increases over time due to in situ virus amplification, as opposed to classical drug pharmacokinetics that decreases with time [45]. The efficacy of an evolutionary cancerfavoring engineered vaccinia virus (CVV) was investigated in an animal model of metastatic HCC. In this animal study, the subjects were randomized into sorafenib, CVV, or sorafenib with CVV. Metastatic regions were interestingly rare in the CVV-treated groups (i.e., CVV or sorafenib with CVV) whereas metastatic regions existed in the sorafenib-treated group [46].

study with 30 patients with advanced HCC stratified into mature autologous DCs pulsed, and other control group received supportive treatment. The result demonstrated an improvement in overall survival with two patients (13.3%) partial radiological response was observed, and nine patients (60%) has stable disease. The study concludes using tumor antigen-pulsed DCs vaccine can be effective adjuvant therapy with other treatment modalities of HCC or palliative treatment option in advanced HCC where other treatment options are not applicable [53]. In addition, the safety and tolerance of DC vaccines have been confirmed in patients with HCC [54].

Biologic and Immunotherapy Developments in Advanced Hepatocellular Carcinoma

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

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Adoptive cell therapy (ACT) is an immunotherapeutic approach that attacks cancer cells using genetically engineered patients' lymphocytes. It functions by stimulating or loading autologous lymphocytes with cytokines or tumor antigens, cultivating them ex vivo and then re-infusing them into the patient [55–57]. Adoptive immunotherapy for HCC includes cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, and chimeric antigen receptor (CAR) T cells. The effectiveness and safety of ACT in patients with HCC have

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].

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

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

characterized, mainly due to difficulties in purifying and expanding them.

been studied in many experiments, which paved the road for its clinical implication.

**4.3. Direct immunological strategy**

*4.3.1.1. Cytokine induced killer cells (CIK)*

*4.3.1.2. Tumor infiltrating lymphocytes (TILs)*

*4.3.1.3. Natural killer cells (NKCs)*

*4.3.1. Adoptive cell therapy*

JX-594 is a thymidine kinase gene-inactivated oncolytic vaccinia virus engineered for the expression of transgenes encoding human granulocyte-macrophage-colony-stimulating factor (GM-CSF) and β-galactosidase, which increases antitumor immune responses [39, 47–49] This virus is safe in humans and extremely toxic to cancer cells.

Oncolytic viruses have produced enough therapeutic efficacy with great optimism in the future trials. Although the initial concerns of clinical investigators were for safety like a risk of viral infection or introduce oncogenic mutation, these have proven not to be a significant issue in these trials.

#### *4.2.3. HCC vaccines*

Cancer vaccination is performed by utilizing antigenic substances to stimulate tumor-specific immune responses that can remove cancer cells and prevent recurrences. HCC vaccines include cancer cells, antigen peptides, DCs, and DNA-based.

#### *4.2.3.1. Antigen peptide vaccines*

Peptide-based tumor-associated antigens (TAAs), such as alpha-fetoprotein (AFP), GPC3, SSX-2, NY-ESO-1, human telomerase reverse transcriptase (hTERT), HCA587, and melanoma antigen gene-A (MAGE-A), are excellent vaccine targets for the treatment of HCC [50].

AFP which normally originates from embryonic liver cells, can be overexpressed on HCC cell surfaces. However, immune responses to AFP are limited due to acquired immune tolerance during the development of the immune system. To overcome this tolerance, a research group investigated the use of a recombinant rat AFP to induce cross-reactions between xenografts and endogenous molecules in animals and observed modest cellular and humoral immune responses [51]. In a phase II trial of GPC3-derived peptide vaccine for HCC, 25 patients received 10 vaccinations over 1 year after surgery. The recurrence rate in patients who underwent both surgery and vaccination was significantly lower than the rate in 21 patients who underwent surgery only (24% vs. 48 and 52.4% vs. 61.9% at 1 and 2 years, *p* = 0 047 and 0.387, respectively), demonstrating the efficacy of the GPC3-derived vaccine [52].

#### *4.2.3.2. Dendritic cell (DC) vaccines*

DCs, were found to be the most powerful APCs in the body's immune system, and capable of stimulating naïve T cells and driving primary immune responses. A phase I/IIa comparative study with 30 patients with advanced HCC stratified into mature autologous DCs pulsed, and other control group received supportive treatment. The result demonstrated an improvement in overall survival with two patients (13.3%) partial radiological response was observed, and nine patients (60%) has stable disease. The study concludes using tumor antigen-pulsed DCs vaccine can be effective adjuvant therapy with other treatment modalities of HCC or palliative treatment option in advanced HCC where other treatment options are not applicable [53]. In addition, the safety and tolerance of DC vaccines have been confirmed in patients with HCC [54].
