*3.3. Targeted Therapies*

Targeted therapies revolutionized melanoma treatment in 2011, when the first therapies were approved by FDA. They belong to the following classes:

• BRAF inhibitors: Since BRAF is the most frequently mutated oncogene in melanoma [33], its inhibitors have shown promising results in several clinical trials, with rapid regression of metastasis and positive responses in 50–60% melanoma patients [32,34]. The first drug belonging to this class that has been approved for melanoma is vemurafenib, a selective inhibitor of V600-mutant BRAF [33]. In a randomized phase III clinical trial (BRIM3), vemurafenib showed an objective response rate (ORR) of 48% versus 5% for dacarbazine, and a median progression-free

survival (PFS) of 5.3 months versus 1.6 months for dacarbazine [33,35]. The second BRAF inhibitor came soon after the first one, with similar promising results [33]. Toxicities associated with this class of therapeutic agents include rash, arthralgia, fatigue, fever (for dabrafenib only) and photosensitivity (for vemurafenib only), but also the development of secondary non-melanoma cutaneous lesions, such as squamous-cell carcinoma [36,37].

• MEK inhibitors: The development of MEK inhibitors became a priority after the success with BRAF-inhibitors, and it was led by the acknowledgement that BRAF signaling is dependent on MEK1/2 downstream activation [33,38]. Trametinib belongs to this class of new targeted therapies [32], and represents the first drug of its class to be approved by the FDA as a single agent, since in the phase III METRIC clinical trial it showed an ORR of 22% and a median PFS of 4.8 months [39]. Aside from the use of MEK inhibitors to target BRAF-mutated melanomas, there is also preclinical evidence that indicates vulnerability to MEK inhibitors in a not insignificant number of melanomas which do not present BRAF V600 mutations, called wild-type BRAF melanomas (especially in NRAS-Q61-mutant tumors), and also in BRAF/NRAS wild-type melanomas, together with melanomas harboring non-V600 BRAF mutations [33,40]. *Cancers* **2020**, *12*, x 5 of 19 MEK1/2 downstream activation [33,38]. Trametinib belongs to this class of new targeted therapies [32], and represents the first drug of its class to be approved by the FDA as a single agent, since in the phase III METRIC clinical trial it showed an ORR of 22% and a median PFS of 4.8 months [39]. Aside from the use of MEK inhibitors to target BRAF-mutated melanomas, there is also preclinical evidence that indicates vulnerability to MEK inhibitors in a not insignificant number of melanomas which do not present BRAF V600 mutations, called wild-type BRAF melanomas (especially in NRAS-Q61-mutant tumors), and also in BRAF/NRAS wild-type melanomas, together with melanomas harboring non-V600 BRAF mutations [33,40].

A translational investigation led to evidence of a possible synergistic relationship between MEK and BRAF inhibitors. Since then, many combinatorial approaches of these two types of inhibitors have been investigated in clinical trials. The combination of vemurafenib and cobimetinib in a phase I study not only resulted in ORR and median PFS values that were very promising, but showed that the incidence of cutaneous hyperproliferative manifestations was substantially lower compared to BRAF inhibitor monotherapy [41]. The combination of BRAF and MEK inhibitors now forms the backbone of advanced BRAF-mutated melanoma treatment [33]. A translational investigation led to evidence of a possible synergistic relationship between MEK and BRAF inhibitors. Since then, many combinatorial approaches of these two types of inhibitors have been investigated in clinical trials. The combination of vemurafenib and cobimetinib in a phase I study not only resulted in ORR and median PFS values that were very promising, but showed that the incidence of cutaneous hyperproliferative manifestations was substantially lower compared to BRAF inhibitor monotherapy [41]. The combination of BRAF and MEK inhibitors now forms the backbone of advanced BRAF-mutated melanoma treatment [33].

#### **4. Cancer Immunotherapy 4. Cancer Immunotherapy**

*4.1. Immune Checkpoint Inhibitors (ICIs)*

2) [43].

The goal of cancer immunotherapy is the stimulation or activation of immune responses against tumor cells, with the ultimate aim of eradicating cancer from the patient's body (Figure 1). In the following sections, we discuss therapeutic treatments falling under the umbrella of the cancer immunotherapy field. The goal of cancer immunotherapy is the stimulation or activation of immune responses against tumor cells, with the ultimate aim of eradicating cancer from the patient's body (Figure 1). In the following sections, we discuss therapeutic treatments falling under the umbrella of the cancer immunotherapy field.

**Figure 1.** The cancer immunity cycle, modified from [42]. **Figure 1.** The cancer immunity cycle, modified from [42].

the presence of co-stimulatory signals to overcome the barrier of negative inhibition. Checkpoint inhibitors (CPIs) are very important to prevent continuous occurrence of immune reactions (Figure

Immune checkpoint inhibitors are a new class of cancer therapeutics that have the physiological

The two most important immune checkpoints that have been studied in immunotherapy are the cytotoxic-T lymphocytes antigen 4 (CTLA-4) and the programmed cell death protein 1 (PD-1) [44,45]. CTLA-4 is a receptor and a member of the immunoglobulin superfamily CD28:B7 [46]. It can be found

#### *4.1. Immune Checkpoint Inhibitors (ICIs) Cancers* **2020**, *12*, x 6 of 19

Immune checkpoint inhibitors are a new class of cancer therapeutics that have the physiological purpose to negatively regulate the activation of T cells. These checkpoints make it more difficult for T cells to activate, as they need both the interaction with the epitope through the MHC I class, and the presence of co-stimulatory signals to overcome the barrier of negative inhibition. Checkpoint inhibitors (CPIs) are very important to prevent continuous occurrence of immune reactions (Figure 2) [43]. on the surface of both effector T cells and Treg cells, as its function is to regulate the extent of the early stage activation of these two types of immune cells. CTLA-4 binds CD80 and CD86 with higher affinity than CD28 does and blocks the amplification signal that the co-stimulatory binding is supposed to send, in order to trigger T cells expansion. In tumors, CTLA-4 is overexpressed to suppress the activation of immune cells which could have been successful in reaching the tumor site (generally referred to as tumor infiltrating lymphocytes—TILs) [47].

**Figure 2.** Timeline of immuno-oncology (IO) agents approved for cancer therapies. **Figure 2.** Timeline of immuno-oncology (IO) agents approved for cancer therapies.

PD-1 is another co-inhibitory molecule expressed in stimulated T cells, Treg cells, B-activated cells and NK cells, and it exerts its function once it is bound to its two ligands, PD-L1 and PD-L2. PD-L1 is expressed more and is found on antigen presenting cells (APCs), dendritic cells (DCs), macrophages and B cells, but it is also expressed in tumor cells which are able to abrogate the lymphocyte response [5]. These two immune checkpoints have been investigated as a target for several monoclonal antibodies, which are already being exploited in cancer therapy for their ability in binding a specific antigen. The first monoclonal antibody against immune checkpoints to be discovered was ipilimumab, an anti-CTLA-4 antibody that has been firstly approved as a first-line treatment of metastatic The two most important immune checkpoints that have been studied in immunotherapy are the cytotoxic-T lymphocytes antigen 4 (CTLA-4) and the programmed cell death protein 1 (PD-1) [44,45]. CTLA-4 is a receptor and a member of the immunoglobulin superfamily CD28:B7 [46]. It can be found on the surface of both effector T cells and Treg cells, as its function is to regulate the extent of the early stage activation of these two types of immune cells. CTLA-4 binds CD80 and CD86 with higher affinity than CD28 does and blocks the amplification signal that the co-stimulatory binding is supposed to send, in order to trigger T cells expansion. In tumors, CTLA-4 is overexpressed to suppress the activation of immune cells which could have been successful in reaching the tumor site (generally referred to as tumor infiltrating lymphocytes—TILs) [47].

melanoma [48]. In the anti-PD-1 group there are other two common ICIs, pembrolizumab and nivolumab, both with indications for metastatic melanoma. Pembrolizumab has been the first anti-PD-1 monoclonal antibody that has been discovered, and with clinical trials KEYNOTE-001, KEYNOTE-002 and KEYNOTE-006 it has gained the first-line therapy indication for metastatic melanoma [49]. In particular, in trials KEYNOTE 006 AND KEYNOTE-002, which both presented comparative arms, patients treated with pembrolizumab significantly improved their progressionfree survival (PFS), overall survival (OS) and overall response rates (ORR) relative to ipilimumab in PD-1 is another co-inhibitory molecule expressed in stimulated T cells, Treg cells, B-activated cells and NK cells, and it exerts its function once it is bound to its two ligands, PD-L1 and PD-L2. PD-L1 is expressed more and is found on antigen presenting cells (APCs), dendritic cells (DCs), macrophages and B cells, but it is also expressed in tumor cells which are able to abrogate the lymphocyte response [5]. These two immune checkpoints have been investigated as a target for several monoclonal antibodies, which are already being exploited in cancer therapy for their ability in binding a specific antigen.

ipilimumab-naive patients (KEYNOTE 006), and significantly improved PFS and ORR, but not OS (although OS data are immature), relative to chemotherapy in ipilimumab-refractory patients, who had also received BRAF/MEK inhibitor therapy if BRAF-mutation positive (KEYNOTE 002) [50]. Pembrolizumab can to be administered as the first line therapy (BRAF wildtype melanoma) or after treatment with ipilimumab, in a combination with anti-CTLA-4 or in patients with BRAF mutations after treatment BRAF inhibitor such as vemurafenib, sorafenib and dabrafenib. Atezolizumab in combination with cobimetinib and vemurafenib is also used for the patients with BRAF V600 mutation-positive unresectable or metastatic melanoma (first line therapy) (Figure 3) (IMspire150, NCT02908672) [51–55]. Therefore, it is reasonable to suppose that atezolizumab could bring some new advantages if compared to the targeting of PD-1 exerted by pembrolizumab, such as The first monoclonal antibody against immune checkpoints to be discovered was ipilimumab, an anti-CTLA-4 antibody that has been firstly approved as a first-line treatment of metastatic melanoma [48]. In the anti-PD-1 group there are other two common ICIs, pembrolizumab and nivolumab, both with indications for metastatic melanoma. Pembrolizumab has been the first anti-PD-1 monoclonal antibody that has been discovered, and with clinical trials KEYNOTE-001, KEYNOTE-002 and KEYNOTE-006 it has gained the first-line therapy indication for metastatic melanoma [49]. In particular, in trials KEYNOTE 006 AND KEYNOTE-002, which both presented comparative arms, patients treated with pembrolizumab significantly improved their progression-free survival (PFS), overall survival (OS) and overall response rates (ORR) relative to ipilimumab in ipilimumab-naive patients (KEYNOTE 006), and significantly improved PFS and ORR, but not OS (although OS data

the preservation of PD-L2 interactions with PD-1 which carries out the immune checkpoint functions

1

are immature), relative to chemotherapy in ipilimumab-refractory patients, who had also received BRAF/MEK inhibitor therapy if BRAF-mutation positive (KEYNOTE 002) [50].

Pembrolizumab can to be administered as the first line therapy (BRAF wildtype melanoma) or after treatment with ipilimumab, in a combination with anti-CTLA-4 or in patients with BRAF mutations after treatment BRAF inhibitor such as vemurafenib, sorafenib and dabrafenib. Atezolizumab in combination with cobimetinib and vemurafenib is also used for the patients with BRAF V600 mutation-positive unresectable or metastatic melanoma (first line therapy) (Figure 3) (IMspire150, NCT02908672) [51–55]. Therefore, it is reasonable to suppose that atezolizumab could bring some new advantages if compared to the targeting of PD-1 exerted by pembrolizumab, such as the preservation of PD-L2 interactions with PD-1 which carries out the immune checkpoint functions that avoids autoimmune reactions during therapy, allowing for a more tolerable safety profile for this immunotherapeutic new drug [56]. *Cancers* **2020**, *12*, x 7 of 19 that avoids autoimmune reactions during therapy, allowing for a more tolerable safety profile for this immunotherapeutic new drug [56].

**CPI refractory melanoma - treatment scheme overview**

**Figure 3.** CPI refractory melanoma-treatment scheme overview. **Figure 3.** CPI refractory melanoma-treatment scheme overview.

Initially, immunotherapy was employed in melanoma treatment with administration of interferon and interleukin cytokines, such as IFN-α and IL-2, which were approved by the FDA with melanoma indications in 1996 and 1998, respectively [32]. Unfortunately, this approach did not show notable benefits for patients, due to the severe side effects associated with systemic administration and to the much poorer therapeutic effects that came with other routes of administration, like the subcutaneous one [32,57]. A modern approach to the immunotherapy of melanoma has grown from elucidations on the role of specific immunomodulatory molecules, and led to a goal shift directed to the enhancement of cell-mediated immunity [33]. To do this, some of the aforementioned ICIs (Figures 2 and 3) have been investigated and were subsequently approved for melanoma therapy: Initially, immunotherapy was employed in melanoma treatment with administration of interferon and interleukin cytokines, such as IFN-α and IL-2, which were approved by the FDA with melanoma indications in 1996 and 1998, respectively [32]. Unfortunately, this approach did not show notable benefits for patients, due to the severe side effects associated with systemic administration and to the much poorer therapeutic effects that came with other routes of administration, like the subcutaneous one [32,57]. A modern approach to the immunotherapy of melanoma has grown from elucidations on the role of specific immunomodulatory molecules, and led to a goal shift directed to the enhancement of cell-mediated immunity [33]. To do this, some of the aforementioned ICIs (Figures 2 and 3) have been investigated and were subsequently approved for melanoma therapy:

 Ipilimumab (anti-CTLA-4): Gained regulatory approval by the FDA to treat melanoma after a series of phase III clinical trials (CA184-002 as a single agent, CA184-024 in combination with dacarbazine). The tumor responses according to the Response Criteria in Solid Tumors (RECIST) criteria varied from 5.7% to 11.0% in the anti-CTLA-4 treatment arms. The median overall survival (OS) was improved to 10 months for the anti-CTLA-4 monotherapy arm as compared to 6.4 months for the peptide vaccine-alone arm (HR 0.68; *p* < 0.001 [58], CA184-002, NCT00094653). The five-year survival rate was 18.2% (95% CI, 13.6% to 23.4%) for patients treated with anti-CTLA-4 + dacarbazine vs. 8.8% (95% CI, 5.7% to 12.8%) for patients treated with placebo plus dacarbazine (*p* = 0.002, CA184-024, NCT00324155) [59]. Toxicity associated with ipilimumab includes immune-related symptoms such as dermatitis, colitis, diarrhea and, • Ipilimumab (anti-CTLA-4): Gained regulatory approval by the FDA to treat melanoma after a series of phase III clinical trials (CA184-002 as a single agent, CA184-024 in combination with dacarbazine). The tumor responses according to the Response Criteria in Solid Tumors (RECIST) criteria varied from 5.7% to 11.0% in the anti-CTLA-4 treatment arms. The median overall survival (OS) was improved to 10 months for the anti-CTLA-4 monotherapy arm as compared to 6.4 months for the peptide vaccine-alone arm (HR 0.68; *p* < 0.001 [58], CA184-002, NCT00094653). The five-year survival rate was 18.2% (95% CI, 13.6% to 23.4%) for patients treated with anti-CTLA-4 + dacarbazine vs. 8.8% (95% CI, 5.7% to 12.8%) for patients treated with placebo plus dacarbazine (*p* = 0.002, CA184-024, NCT00324155) [59]. Toxicity associated with ipilimumab includes

they are selective for another receptor which is usually expressed on immune T cell surface— PD-1. Phase III clinical trial reported the median overall survival which has not been reached in the nivolumab-plus-ipilimumab group and was 37.6 months in the nivolumab group, as compared with 19.9 months in the ipilimumab group (hazard ratio for death with nivolumab plus ipilimumab vs. ipilimumab, 0.55 [*p* < 0.001]; hazard ratio for death with nivolumab vs.

less commonly, hepatitis, uveitis and hypophysitis [60].

immune-related symptoms such as dermatitis, colitis, diarrhea and, less commonly, hepatitis, uveitis and hypophysitis [60].

• Pembrolizumab and nivolumab (anti-PD1): After the ipilimumab proof of concept that a checkpoint blockade could actually be an effective strategy to treat melanoma, pembrolizumab and nivolumab were investigated for the same indication, even if (or maybe especially because) they are selective for another receptor which is usually expressed on immune T cell surface—PD-1. Phase III clinical trial reported the median overall survival which has not been reached in the nivolumab-plus-ipilimumab group and was 37.6 months in the nivolumab group, as compared with 19.9 months in the ipilimumab group (hazard ratio for death with nivolumab plus ipilimumab vs. ipilimumab, 0.55 [*p* < 0.001]; hazard ratio for death with nivolumab vs. ipilimumab, 0.65 [*p* < 0.001]). The overall survival rate at 3 years was 58% in the nivolumab-plus-ipilimumab group and 52% in the nivolumab group, as compared with 34% in the ipilimumab group (NCT01844505) [33,61–63].
