**4.2. Indirect immunological strategies**

### *4.2.1. Checkpoint inhibitors*

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].

#### *4.2.1.1. CTLA-4 inhibitors*

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 cells and reduced HCV viral loads [33].

#### *4.2.1.2. PD-1 inhibitors*

The PD-L1/PD-1 pathway is another mechanism of tumor-induced immune tolerance. PD-1 expression on effecter phase CD8<sup>+</sup> T cells is increased in patients with HCC compared to no HCC cirrhotic patients [34]. Moreover, there is frequent and early disease progression in patients with HCC with higher numbers of tumor-infiltrating and circulating PD-1 + CD8<sup>+</sup> T cells post hepatic resection [35]. Therefore, a supporting great rationale exists for using PD-1 and PD-L-1 blocking antibodies against HCC. Some PD-1 inhibitors, such as nivolumab, pembrolizumab, and pidilizumab, have been investigated for cancer treatment.

The CheckMate-040 phase I/II trial studied the safety and antitumor effect of nivolumab in 48 patients with advanced HCC [36]. The target population included patients with intermediate or advanced HCC and preserved liver function (Child-Turcotte-Pugh-A cirrhosis) who were candidates for systemic therapy and had progressed or were intolerant to sorafenib. In the escalation and expansion cohorts, objective tumor responses occurred in 15 and 20% of patients, respectively. There were durable responses that lasted for a median of 17 months. An additional 45% of patients had a stable disease associated with durability, lasting 6 months at minimum. Those responses were consistent across the different HCC risks, and both in sorafenib-naïve and sorafenib-exposed patients.

Overall, frequencies of grade 3/4 treatment-related AEs were 20%. Only 3% of patients discontinued nivolumab because of treatment-related adverse events, while no treatment-related deaths occurred. Elevated transaminases was the most frequent laboratory alteration (20%). However, only 5% of the patients had grade 3 or higher. Immune-related hepatitis requiring steroid therapy. CheckMate-040 showed that nivolumab might be effective with acceptable toxicity in HCC, regardless of hepatitis status. On September 22, 2017, and based on the outcome of CheckMate-040 study, the FDA granted accelerated approval to nivolumab for the treatment of hepatocellular carcinoma (HCC) as second-line therapy in patients who have been previously treated with sorafenib.

CheckMate-459 is an ongoing phase III study, (NCT02576509) that randomizes patients with advanced HCC to either nivolumab or sorafenib in the first-line setting [37].

The efficacy and safety of pembrolizumab in HCC has been investigated. The phase I/II study KEYNOTE-224 tested pembrolizumab in 104 patients with advanced HCC who progressed on sorafenib. The overall response rate was 16.3%. Durable response was seen with 94% of responders were estimated to have a response duration of 6 months or longer. The median PFS was 4.8 months, and the median OS was not been reached. The safety profile was generally comparable to that established for pembrolizumab monotherapy in other indications, and no viral flares were seen [38].
