Encyclopedia
Malignant melanoma (mM) is the leading cause of death among cutaneous malignancies. While its incidence is increasing, the most recent cancer statistics show a small but clear decrease in mortality rate. This new trend reflects the introduction of novel and more effective therapeutic regimens, including the two cornerstones of melanoma therapy:particularly immunotherapies and targeted therapies. Unlike chemotherapies or radiation, in which the therapy directly induces cancer cell death, immunotherapies stimulate the patient’s immune system to control and eliminate the tumor. Advantages of immunotherapies over traditional cancer treatments include increased durability for long-term control or even cure and more precisely targeted anti-tumor activity that spares healthy tissues, many times with comparable or even reduced overall toxicity. The high immunogenicity and somatic mutation burden of melanoma likely contribute to the success of immunotherapy. Treatments combining immunotherapies with targeted therapies, which disable the carcinogenic products of mutated cancer cells, have further increased treatment efficacy and durability. Toxicity and resistance, however, remain critical challenges to the field. The. The highly immunogenic nature of melanoma makes this tumor particularly susceptible to attack by the adaptive immune system. Currently, there are three types of immunotherapies currently approved by the US Food and Drug Administration (FDA) for the treatment of advanced melanoma: (1) T-cell stimulating cytokines (i.e. interferon (IFN)-α2b and interleukin-2 (IL-2)); (2) T-cell exhaustion-mitigating immune checkpoint inhibitors (ICI); and (3) a dendritic cell (DC)-activating oncolytic virus (T-VEC). Still others, such as adoptive cell transfer (ACT), hold strong promise for the future. This article summarizes past treatments and novel therapeutic interventions and discusses current clinical trials and future directions.
- metastatic melanoma
- targeted therapy
- immunotherapy
- combined therapies
- tumor relapse
- tumor resistance
1. Early Immunotherapies
1. Introduction
1.1. Interferon-α2b
Intvasiverferon (IFN)-α2b is a recombinant form of human IFN-α with antiviral and antitumor properties. It wa melanoma accounts for only 1% of all skin cancers, yet is the first immunotherapy approved for melanoma, first as an adjuvant treatment in 1996 and then as first-line therapy in 1998. By binding to IFN receptors 1 and 2, the drug triggers multiple dose- and time-dependent immunostimulatory effects, including upregulation of major histocompatibility complex 1 (MHC1) on tumor cells, enhanced activation of anti-tumor cytotoxic T lymphocytes (CTLs), depression of T regulatory cells (Tregs), enhanced dendritic cell (DC) response, and decreased intercellular adhesion molecule (ICAM) expressionleading cause of skin cancer-related deaths. In 2022, an estimated 99,780 new cases of invasive melanoma will be diagnosed and an estimated 7,650 people will die from the disease in the United States, making it the fifth most common malignancy in both sexes [1][2][3](1). In 1996, Whigh-dose (HD) IFN-α2b became the first adjuvant therapy, approved for use in stage IIB and III le the incidence of melanoma patients following surgical resection. Initial trials demonstrated significantly improved 5-year relapse-free survival (RFS) (37% vs 26%) [4][5]. HD IFN-α2b continues to increase from prior years, mortreatment is limited by high toxicity, with studies reporting dose reductions in 28-55% of patients and toxicity attrition rates of 10-26% (6). HD IFN-α2b remained the standard adjuvant therapy for high-risk melanoma until ipilimumab approval in 2015
Plity rates have declined by ~4% per year since 2015 (1). This decline reflects significant advancements in treatmeginterferon-α2b (Peg-IFN), which has a longer half-life than IFN-α2b, was approved for adjuvant use in 2011 after demonstrating for advanced and metastatic (mM) disease, the most significant improvement in 7-year RFS compared to observation (39.1% vs 34.6%) but, like HD IFN-α2b, could not provide OS benefit [6][7].
Low dose (LD) IFN-α2b was of which are targeted therapies and immunotherapprovied as first-line therapy for stage II melanoma patients in 1998 based on a trial showing improved 5-year RFS (43% vs 51%) and a trend toward improved OS (24% vs 32%) compared to observation [8]. It s. Unlike chemotherapies or radiation, in which the therapy directly indouces not have significant clinical benefit in mM [9].
Today, IFN-α is cancer cell death, immuno longther a first-line agent for mostapies stimulate the patients; however, it may still have utility as an auxiliary immunostimulatory agent, enhancing the clinical benefits of other immunotherapies.
1.2 High Dose Interleukin-2
I’s immune system to control and eliminate the tumor. The high immunogenicity and somatic mutationte burleukin-2 (IL-2) is a T-cell growth factor that leads to cytokine production and preferential expansion of CD8+ T-cells, NK cells, and Tregs. In 1998, HD intravenous (IV) administration of IL-2 became the first FDA-approvedden of melanoma likely contribute to the success of immunotherapy (2). Advantages of immunotherapy for theies over traditional cancer treatment of metastatic melanoma (mM) [10](11). Ds include increased durabile tumor responses have been well documented in a subset of mM patients, with 5-10% of patients achieving complete response and even more achieving increased disease stabilitity for long-term control or even cure and more precisely targeted anti-tumor activity that spares healthy [10][11][12]. A recent one is of IL-2-responsive mM patients who exclusively received HD IL-2 for systemic therapy confirms prolonged clinical and survival benefits [13]ssues, many times with comparable or even reduced overall toxicity. As with IFN-α therapy, the use of HD IL-2 treatment is limited by the relatively high incidence of grade 3 and 4 toxicities, which requires the drug to be administered in an intensive inpatient settbetter understanding of the molecular landscape of melanoma has helped to refine not only our understanding [14].of Tthe efficacy of treatment is further limited by the drug’s activation of anti-inflammatory T-regs, which limit CD8+ activation and effector functions. Drugs targeting specific subunits of the IL-2 receptor, such as the recombinant IL-2 receptor βγ-biased agonist NKTR-214 (Bempegaldesleukin), have shown promise in the targeted expansion of anti-tumor T and NK cells with limited expansion of Tregs and dramatically reduced toxicity [15][16][17][18][19][20].
Whildisease but also to propose novel targets or target combinations for selective therapeutic intervention. As technologies around genetic sequencing, modification, and bioinformatics become more advanced and accessible, immunotherapies may become rarely used as a single or first-line agent today, HD IL-2 remains a second- or third-line option that provides a possible survival benefit to patients who have failed treatmentven more effective by personalization and optimized immunotherapy has the potential to become even more effective with first-line agentsthe advent [21].of Many tperials combining HD or low-dose IL-2 therapy with additional therapies are ongoingsonalized agents and regimens.
2. Immune Checkpoint Inhibitors
2. Early Therapies for Malignant Melanoma
2.1. Chemotherapy
2.2. Interferon-α2b
2.3. High-Dose Interleukin-2
3. Targeted Therapies
3.1. Tyrosine Kinase Inhibitors
3.2. MEK Inhibitors
3.3. Combination BRAF and MEK Inhibitor Therapy
3.4. Other Targeted Therapies
2
4. Immune Checkpoint Inhibitors
4.1. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) inhibitors
CTLA4 is an immune-inhibitory molecule expressed on the surface of activated T-cells. Together with its immune-activating counterpart, CD28, CTLA4 creates a critical immune checkpoint that must be overcome to achieve a durable immune response [22](82). CTLA4 is naturally upregulated in situations of chronic T-cell stimulation to prevent uncontrolled immune reactions and inappropriate development of autoimmunity. In the TME, however, this system backfires: chronic presentation of tumor antigens to T-cells inhibits the immune system from mounting an anti-tumor immune response and contributes to the immune evasion that allows continued tumor growth [23][24][25][26](83-86).
Ipilimumab was approved as the first immune checkpoint inhibitor (ICI) in 2011, the same year that vemurafenib was approved to block BRAF-mediated growth signaling. Ipilimumab is an anti-CTL4 human IgG antibody. By preventing the interaction of CTLA4 and its ligands, the drug allows T-cells to bypass the inhibitory immune checkpoint and mount a response against tumor antigens. Phase III trials of previously-treated mM patients demonstrated improved OS compared to gp100, a melanoma antigen immunostimulant with limited anti-tumor effects (10.1 vs 6.4 months, p = 0.0026) [27](26). A metanalysis of pooled data from nearly 2,000 mM patients treated with ipilimumab (both pre-treated and treatment-naïve) reported an increase in the 3-year OS rate to 22% (95% CI [20, 24%]), a dramatic increase from ~5% achieved by previous standard-of-care therapies [28](87). Perhaps even more importantly, the OS survival curve plateaued after 3 years, maintaining the ~20% OS rate for the entirety of the 10+ year follow-up [28](87). Thus, ipilimumab became both the first therapy to provide an OS benefit in advanced melanoma and the first to demonstrate that long-term durable mM disease control is possible with systemic therapy [29](88).
While responses to ipilimumab are durable, the response rates are low, ranging from 5-10%. Clinical trials have provided little insight into possible biomarkers of response. Attempts to improve response rates by adding ipilimumab to dacarbazine therapy were somewhat successful (15% vs 10%) and demonstrated a survival benefit over dacarbazine alone (OS 11.2 vs 9.1 months, p<0.001). However, these benefits came at the cost of high toxicity (rate of grade 3/ 4 AEs: 56.3% vs 27.5%, p < 0.001) [30](89). Even as a monotherapy, ipilimumab is relatively toxic with immune-related toxicities occurring in 60-80% of patients, 10-26% of which are grade 3/4 reactions [31](90). Perhaps unsurprisingly, severe AEs, which are often immune-related AEs (irAE), were found to be associated with improved ORRs [32](91).
Ipilimumab is still the only approved CTLA4 inhibitor for mM, after thouge initially promising drug tremelimumab failed to demonstrate improved OS (p=0.127) or RR over standard-of-care chemotherapy, ipidespite a longer average response duration (35.8 vs 13.7 mo; p=0.0011) (92). Today, ipilimumab monotherapy is not a first-line therapy by ASCO guidelines [33](71).
2
4.2. Programmed cell death protein 1 (PD-1) and PD-1 ligand (PD-L1) inhibitors
Like CTLA4, PD-1 is an inhibitory immune checkpoint receptor expressed by activated T cells. When PD-1 binds its receptors, PD-L1 and PD-L2, signaling through the SHP1/2 pathway downregulates the transcription factors necessary for T-cell effector functions, growth, and survival [34](93). In healthy tissues, PD-L1 is broadly expressed and upregulated in response to proinflammatory cytokines [35](94). Melanoma tumor and TME cells upregulate PD-L1 in response to tumor-infiltrating lymphocytes (TIL), suggesting that PD-L1 expression is used as a mechanism of immune evasion by the cancerous cells [36][37][38](95-97).
In 2014, two anti-PD-1 monoclonal antibodies, Pembrolizumab and nivolumab, were approved for treatment-resistant mM after demonstrating superiority over ipilimumab. Early trials of pembrolizumab monotherapy demonstrated improved 6- and 12-month PFS and RR (6-month PFS: pembrolizumab=47% vs ipilimumab=26%; 12-month PFS: P=74-68% vs I=58%; RR= P=33%, I=12%) [39][40](42, 98). Two and five-year follow-up studies and real-world findings of pembrolizumab monotherapy confirm its superior OS and durable antitumor immune activity for both treatment naïve and pre-treated mM patients [41][42][43][44][45](31, 99-102). Similarly, the CheckMate 067 trial first demonstrated that nivolumab monotherapy confers a significantly greater PFS compared to ipilimumab in treatment-naïve mM patients (nivolumab: 6.9 months 95%[4.3, 9.5]; ipilimumab: 2.9 months 95%[2.8, 3.4] [46](34). Follow-up data from 2019 then demonstrated superior 5-year OS rates (nivolumab = 44%, ipilimumab = 26%) [47](33). Nivolumab has proven to be effective in a range of melanoma tumor subtypes, including both treatment-naïve and pre-treated tumors with either WT or mutant BRAF status [48][49](30, 103).
The two PD-1 inhibitors differ by epitope binding location and target affinity strength but are equally effective as monotherapies by OS (pembrolizumab = 22.6 mo, nivolumab = 23.9 mo, p = 0.91) and time to next-line therapy or death (pembrolizumab = 15.7 mo, nivolumab = 10.8 mo, p = 0.16) [50][51](104, 105). Both are also relatively well-tolerated with lower rates of grade 3/4 toxicity (14% with pembrolizumab and 4% with nivolumab) than chemotherapy, ipilimumab, and most targeted therapies [52][50](104, 105). AEs during nivolumab therapy areis associated with improved ORRs [50](105).
Both PD-1 inhibitors are also effective in the adjuvant setting. A five-year onestudy on adjuvant pembrolizumab demonstrated significant increased RFS, decreased risk of distant metastasis or death (HR 0.60 95%[0.49,0.73]), and sustained treatment effect compared to placebo [51][53](32, 41). Interestingly, adjuvant pembrolizumab also proved efficacious in patients with PD-L1-negative and undetermined tumors [39][53](41, 42). In pre-treated stage IV melanoma patients with no evidence of residual disease, adjuvant nivolumab alone or in combination with ipilimumab proved similarly effective in increasing RFS compared to placebo [54](106). These drugs are the current first-line adjuvant therapy for resected WT melanoma. Patients with resected BRAF-mutant melanomas may choose between pembrolizumab, nivolumab, or dabrafenib-trametinib combination therapy as first-line adjuvant therapy [33](71).
Optimal utilization of ICIs is hindered by several major challenges, including resistance and poor predictability of patient response. Approximately 30% of melanoma patients have an innate resistance to PD-1 inhibitors and 25% of responders acquire resistance during treatment [55][56][57](107-109). CTLA4 inhibitors face a similar challenge [27](26). Mechanisms of resistance likely include specific tumor cell genetics (loss-of-function mutations in JAK1/2 [58](110)), differing expression levels of tumor cell surface proteins (for example,.g. MHC I expression [59](111), alternate ICIs [60](112), and epigenetic T-cell changes limiting effector function and memory [61](113)). Efforts to increase durability by combining ICIs with auxiliary agents such as PEG-IFN [62](114) or hydroxychloroquine(115) [63] have had mixed results, with none providing a clear clinical benefit. Unfortunately, increased toxicity often outweighs any benefit to durability or RR that auxiliary agents provide.
A few studies have identified markers associated with more successful clinical outcomes. For example, independent biomarkers associated with favorable OS of mM patients treated with pembrolizumab include high relative eosinophil count (>1.5%), high relative lymphocyte count (>17.5%), and absence of non-soft tissue or lung metastasis. Patients meeting none of these criteria have a poor prognosis with pembrolizumab [64](116). Others have identified that the occurrence of immune-mediated AEs may be associated with better ORR, OS, and PFS with nivolumab and ipilimumab monotherapies but not with pembrolizumab [65](117). Another—albeit much smaller (n n= 40)—it wasthe study also found that PD-L1 expression on circulating tumor cells may also be a predictive biomarker for PD-1 inhibitor response, suggesting that liquid biopsy may provide clinically relevant information during treatment selection [66](118). However, subgroup analyses have demonstrated the PD-1 blockade still provides clinical benefits in PD-1 negative tumors [46](34). Conflicting evidence on the subject makes using tumor PD-L1 expression as a predictive marker for PD-L1 inhibitor response or overall prognosis for mM controversial [67][68](119, 120).
2
4.3. Combination ICI Therapy
Combining CTLA4 and PD-1 blockade is more effective than either class in monotherapy [69][70](121, 122), yet carries a significantly higher risk of severe toxicity. As monotherapies, nivolumab and ipilimumab have grade 3/4 AE rates of 16-27% and 27%, respectively. When used together, this rate increases to 55-71% [46][54](34, 106). Reducing toxicity while maintaining the clinical benefit of combination therapy may be possible with alternative dosing strategies. Regimens of standard-dose pembrolizumab (200 mg) with either 150mg or 50 mg reduced-dose ipilimumab show a meaningful reduction in toxicity (grade 3-5 toxicity rate <26%) without a significant reduction in ORR (PEM200+IPI50: ORR, 55%, and CR, 16%; PEM200+IPI100: ORR, 61%, and CR, 25% ). In fact, 12-month PFS and OS rates are actually higher with these regimens (12-mo PFS: 65% for PEM200+IPI50; 82% for PEM200+IPI100; OS: >90% for both) compared to standard dosage and previous alternative dosages (12-mo PFS: 46-53% with standard dosing, 47% and 68% with alternative dosing; 12-mo OS: 73-89%) [71][72][73][74][75](123-127). Larger trials are still necessary.
2.4. Novel immune checkpoint inhibitors
4.4. Novel immune checkpoint inhibitors
The second generation of PD1 and CTLA4 ICIs are emerging. TheseHX008, a new agents include anti-PD1nti-PD1 antibody, demonstrated equal efficacy and reduced toxicity compared to nivolumab in pre-clinical trials (128) and hantibody s been well-tolerated in early clinical trials (129). HX008 [75],in combinanti-PDtion with the novel PD-L1 monoclonal antibody, LP002 , is entering clinical trials as a potential synergistic regimen for overcoming PD1 resistance in previously-treated mM patients (NCT04756934),. New agents may also soon emerge for CTLA4 blockade. Recent work suggests that separate mechanisms mediate the clinical efficacy and the toxicity of CTLA4 antibodies. This has allowed for the creation of an anti-CTLA4 antibody (ONC-392) [76]that is highly selective for the immunotherapeutic mechanism. In preclinical data, this antibody maintains selective depletion of Tregs while preserving CTLA4 recycling, the mechanism responsible for irAEs (130). Initial phase I trials were promising and demonstrated no irAEs (131). Phase I/II trials are currently recruiting (NCT04140526) (132).
Lymphocyte activation gene 3 (LAG3) is another T-cell inhibitory checkpoint receptor, the upregulation of which may be a resistance mechanism to PD1 inhibition therapy [77](133). An ongoing phase III study (RELATIVITY-047; NCT03743766) of the new anti-LAG3 antibody relatimab has demonstrated promising initial results. Patients receiving combination relatimab and nivolumab achieve significantly higher rates of 12-month PFS (47 vs 36%, p=0.0055) than those receiving nivolumab alone (134). Rates of severe toxicity are higher in the combination group (19% vs 9.7%), but are lower than the toxicity rates previously reported with combination CTLA4/PD-1 inhibition (34, 106, 134). Further investigation is needed to determine the effects on response rates and overall survival.
Another promising ICI target is TIM-3 (T-cell immunoglobulin and mucin domain 3). TIM-3 blockade restores anti-tumor functions in ex vivo studies of previously exhausted NK and effector T-cells [78](135) and enhances cancer vaccine-induced antitumor responses in murine melanoma models [79](136). A bispecific anti-PD-1 and TIM-3 antibody (RO7121661/RG7769) demonstrated superior anti-tumor TIL activity, IFN-γ secretion, and tumor growth control compared to the monospecific PD-1 antibody in mouse models [80](137). The agent has recently entered phase I human trials (NCT03708328).
3
5. Oncolytic Virus Therapy
Cancer cells achieve a neoplastic phenotype by genetic and epigenetic mutations. These mutations, however, impair signaling pathways (RAS, WNT, PTEN, RB1, TP53) that are essential for the intracellular antiviral response [67]. Recent advances in genetic modification, such as CRISPR, have allowed researchers to create antineoplastic viruses that exploit the vulnerability of mutated cells to viral infection while sparing healthy cells [68]. Talimogene laherparepvec (T-VEC), an oncolytic human herpes simplex virus 1 (HSV-1), is the first and only oncolytic virus (OV) approved for metastatic and unresectable melanoma. When T-VEC is injected directly into the tumor site, it promotes the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) to activate DCs and increase tumor antigen presentation to T cells. In the phase 3 OPTiM trial, 64% of directly injected and 34% of uninjected nonvisceral lesions decreased in size by >50%. Complete resolution of lesions occurred in 47% of injected lesions, 22% of noninjected nonvisceral lesions, and 9% of noninjected visceral lesions. When compared to recombinant GM-CSF administration, T-VEC demonstrated a higher durable RR (16% vs. 2.1%, p = 0.001), ORR (26% vs. 5.7%), and OS (23.3 months, p = 0.051). Severe toxicity rates were only 2% [69]. Laboratory evidence showed that T-VEC has increased efficacy in melanomas with INFγ–JAK–STAT pathway mutations [70]. Since dysregulation of INFγ is a common mechanism of resistance to ICI therapy, ongoing trials are investigating T-VEC as a salvage or combination therapy (NCT04330430, NCT04068181). Systemic administration of OV therapy is also being explored. However, maintaining viral titers capable of generating an antitumor response after systemic administration has proved challenging to systemic OV monotherapy [71][72]. Trials are also investigating their role as sensitizing agents or within combination immunotherapies. Systemic OVs may still have a role as priming agents or within combination therapy (NCT04152863). Promising new antimelanoma oncolytic viruses are in various stages of development. A targeted, inflammation-inducing vesicular stomatitis virus (VSV-GP) has shown promise in animal models [73]. A CD40 ligand and IFN-beta dual-transgene-armed adenovirus (MEM-288) and a tumor-selective TNF-alpha and IL-2-expressing adenovirus (TILT-123) are in stage I clinical trials (NCT05076760, NCT04217473) [74][75]. A promising intralesional Coxsackievirus A21 V937 may soon move to phase III trials [76]. Other OVs, such as ONCOS-102, have limited efficacy as monotherapy, but may provide clinical benefit by enhancing the effects of coadministered immunotherapies [77]. Personalized OVs, targeting each patient’s unique constellation of neoantigens, may be the future of OV therapy. A live-attenuated Listeria monocytogenes (ADXS-NEO) that secretes a personalized viral-neoantigen fusion has already demonstrated effective activation of antitumor T cells and inhibition of TME myeloid-derived suppressor cells and Tregs in preclinical studies [78][79]. Human trials have since been approved for initiation (NCT03265080).6. Melanoma Vaccines
The five major categories of melanoma vaccines currently in development include: (1) melanoma-cell-targeted vaccines, (2) dendritic cell (DC) vaccines, (3) peptide-based vaccines, (4) vector-based vaccines, and (5) mRNA or DNA vaccines. Unlike preventative immunizations, cancer vaccines are therapeutic, activating the patient’s immune system to incite an antitumor response against a known cancer or to prevent disease recurrence in the adjuvant setting. Whole cell vaccines use modified melanoma cells to activate a complex immune response to the cell’s melanoma antigens. In theory, using a whole-cell approach exposes the immune system to many potential antigens, circumventing the need to identify the most immunogenic antigens for each tumor [80]. Most of the cells used in these vaccines are also genetically modified to express immunostimulatory molecules because irradiated tumor cells are not typically immunogenic on their own [80]. In the past, most whole-cell vaccines were made using allogenic cells. Once technical challenges are addressed, we may be able to use autologous tumor cells to create individuated vaccines for each patient. While several melanoma cell vaccines have shown promise in the laboratory, clinical efficacy has so far been limited [81]. DC vaccines are used to directly inject activated or modified DCs into the tumor site to increase antitumor T-cell activation. While these vaccines have shown some immunogenicity in murine models, efficacy has not yet been demonstrated in human trials, likely due to the immunosuppressive nature of the tumor microenvironment [82][83]. Peptide vaccines supply tumor-specific or tumor-associated antigen (i.e., gp100, MART-1/MelanA, tyrosinase) fragments that can be presented by professional APCs to induce effector T-cell activation. Despite promising laboratory data, short-peptide vaccines have shown limited clinical efficacy, even with adjuvant immunostimulatory agents. Long-peptide vaccines with adjuvant immunostimulatory agents are currently under investigation [84]. In 2018, a phase I trial of eight high-risk melanoma patients demonstrated the safety and immunogenicity of a personal neoantigen long-peptide vaccine administered in the adjuvant setting [85]. Four years later, and despite several relapses, all maintained neoantigen-specific T-cell responses and showed evidence of tumor infiltration by neoantigen-specific, cytotoxic T-cell clones [86]. A study evaluating the feasibility of a similar personal neoantigen vaccine in combination with anti-PD1 therapy as a first- or second-line therapy is ongoing (NCT03715985). Vector vaccines use recombinant viral vectors to deliver tumor antigen transgenes directly to APCs. Within the APCs, the transgenes are expressed to produce high concentrations of tumor antigens that can be presented on both MHCI and MHCII for enhanced T-cell activation. The simultaneous expression of viral proteins by the delivered vectors boosts the immunogenicity of the vaccine [87]. Vaccines that use mRNA have garnered significant excitement as therapeutics after the advancements and efficacy demonstrated in the COVID-19 pandemic response. While still in the early stages of research, mRNA vaccines may have the potential to induce the targeted expression of nearly any protein. Using an mRNA approach avoids safety concerns associated with DNA and viral vector vaccines. Therapeutic mechanisms under investigation include enhancing expression of tumor-specific antigens in DCs, mRNA-mediated delivery of specific antitumor or anti-ICI antibodies, and programming cancer cells to express suicidal intracellular proteins [88][89][90]. Challenges include mRNA instability, low in vivo translation rates, and autoimmune reactions [90].7. Photodynamic Therapy
Photodynamic therapy (PDT) involves the administration and subsequent activation of tumor-cell-targeted photosensitizers (PS) (i.e., porphyrin, phthalocyanines, chlorins, porphycenes). When these photosensitizers are activated by light of a specific wavelength, cytotoxic and immunostimulatory reactive oxygen species (ROS) are generated in tumor tissues and tumor-associated blood vessels [91]. Since ROS are only produced in the presence of the both the photosensitizer and the light source, this therapy has the potential to induce localized tumor destruction with limited off-target effects. Preclinical studies of PDT have demonstrated enhanced antitumor immune responses in lung and nonmelanoma skin cancer [92]. Early human clinical trials in head and neck cancers and basal cell carcinomas suggest a therapeutic benefit [93][94]. In melanoma mouse models, there is some evidence of survival benefit after PDT, as well as synergism with PD1 inhibitor therapy, suggesting a potential for use in the adjuvant setting in humans [91][95][96][97]. Despite promising preclinical results, PDT faces significant limitations for use in mM. Melanoma cells are innately resistant to PDT due to pigmentation and enhanced ROS tolerance [98]. Furthermore, light sources reach deeper tumor cells at significantly lower levels, if at all. Photosensitizing agents have also yet to be consistently optimized for efficient delivery and specificity in order to avoid off-target effects. Studies are ongoing to address these issues, with particular interest in nanoparticle delivery systems and nanoemulsions [99]. One such study found that using acai oil in a nanoemulsion (NanoA) as a photosensitizer induced apoptosis in 85% of melanoma tumor cells, with 82% reduction in tumor volume while maintaining high viability in healthy cells in in vitro and in vivo murine models [100].8. OncTolytic virus therapy
Cancer ce
ll-like receptor agonists
Toll-ls achieve a neoplastic phenotype by genetic and epigenetic mutations. These mutations, however, impair signaling pathways (RAS, WNT, PTEN, RB1, TP53) that aike receptor (TLR) activation and the resulting pre essential for the intra-cellular antiviral respons-inflammatory cytokine [81]. Rreclent advances in genetic modification, such as CRISPR, have allowed researchers to create anti-neoplastic viruses that exploit the vulnerability of mutated cells to viral infection while sparing healthy cellsase is a critical step in the induction of both the innate [82].
Talimogene laherparepvec (T-VEC), an oncolytic human herpes simplex virus 1d adaptive immune response (HSV-1170), . is the first and only oncolytic virus (OV) approved for metastatic and unresectable melanoma. When T-VEC is injected directly into the tumor site, it promotes the secretion of granulocyte-macrophage colony-stimulating factor (GM CSF) to activate DCs and increase tumor antigen presentation to T-cells. In the phase 3 OPTiM trial, 64% of directly injected and 34% of uninjected non-visceral lesions decreased in size by >50%. Complete resolution of lesions occurred in 47% of injected lesions, 22% of non-injected non-visceral lesions, and 9% of non-injected visceral lesions. When compared to reAs poor immunogenicity continues to limit the efficacy of melanoma immunotherapies in some patients, TLRs are a logical ancillary agent that provides pro-inflammatory modulation of the tumor microenvironment. Small combinant GM-CSF administration, T-VEC demonstrated higher durable RR (16% vs 2.1%, p = 0.001), ORR (26% vs 5.7%),tion studies have so far and OS (23.3 months, p = 0.051). Severe emonstoxicity rates were only 2% [83]. Laboratory evidence shows that T-VEC has increased efficacy in melanomas with INFγ-JAK-STAT pathway mutations [84]. SincLR agonists induce palliative dysregulation of INFγ is a common mechanism of resistance to ICI therapy, ongoing trials are investigating T-VEC as a salvage or combination therapy (NCT04330430, NCT04068181).
Systocal disease control for inoperablemic administration of OV therapy is also being explored. However, maintaining viral titers capable of generating an anti-tumor response aftermM when combined with intralesional IL-2 (171), systemic administration has proved challenging toncreased local and systemic OV monotherapy [85][86]. Trials are also ianvestigating their role as sensitizing agents or within combination-melanoma immunotherapies. Systemic OVs may still have a role as priming agents or within combination therapyity with monobenzone (NCT04152863172).
4. The Future of Melanoma Immunotherapy
Over, the past 8 years, immunotherapy has revolutionized the treatment of mM, offering patients more treatment options with higher efficacy and less toxicity. Two-year overall survival rates have risen dramatically from ~10% to ~60% [47]. Fnd increased total and vaccine-specific CD8+ T-cells with a murlther into the identification ofi-peptide melanoma neoantigensvaccine (173). aAnd their immunogenic potentials is essential for the advancement of the field. The ability to create individualized therapies specific to each patient’s tumor and immune landscape has the potential to revolutionize melanoma therapy. However, significant advances in rapid tumor-cell sequencing and vaccine production must first be achieved. In the shorter term, combination therapy and melanomaother clinical trial demonstrated increased plasmacytoid DC activation and increased tumor regression when the TLR7/8 agonist Resiquimod was co-administered with a peptide vaccines show promiseation (174). for Climproving the efficacy, response rates, and durability of current first-line immunotherapies.
4.1 Sequencing and combining therapies
Usingnical trials further combined therapies to treat mM may be the easiest way to achieve longer-lasting disease control, overcome innate resistance, evade adaptive resistance to immunotherapy, and optimize clinical response. There is significant interest in finding the best combinations of the two most effective approved vestigating the immunostimulatory properties of TLR agonists in the context of melanoma immunotherapy classes—targeted and ICI therapy (NCT02631447are ongoing (NCT03276832, NCT032352454072900, NCT02902029394132, NCT02224781). Such may also address the two major roadblocks in the deployment of these therapies: rapid resistance development a3684785). Laboratory evidence also suggests that TLR agonists may boost the efficacy of targeted small molecule inhibitors (175). Ind mouse modest response rates.
4.2 Neoadjuvant therapy
Nels, co-administratioadjuvant therapy is typically used to reduce tumor burden and allow for less extensive surgeries. Before with targeted BRAF inhibitors preserves immunotherapy, neoadjuvant systemic therapy was not standard-of-care for mM genicity and delays treatment, likely because the r resistance (176).
RIG1-lisks of delaying surgery outweighed the limited benefits these therapies could provide. However, preclinical data suggest that this may not be true for immunotherapy [87], ee (retinoic acid-inducible gene-1) receptor (RLR) agonists and melanoma differentiation-asspeocially for therapies targeting T-cell function and proliferation. Theoretically, initiating immunotherapy while the major tumor mass is still present may induce a stronger anti-tumor T-cell response. Indeed, a small feasibility one confirmedted antigen 5 (MDA-5) agonists are two other potential immunostimulatory targets for use in combination therapy. In mouse models, these results, demonstrating that patients receiving neoadjuvant and then adjuvant treatment had significantly more expansion of tumor-resident T-cell clones than patients whopro-apoptotic molecules have proven capable of overcoming the characteristic resistance of melanoma cells to apoptosis (177).
9. Adoptive cell transfer with tumor-infiltrating lymphocytes
In rmeceived the same treatment courses exclusively as adjuvant [88]. Neolanoma, the presence of tumor-infiltradjuvant immunotherapy also seems to outperform adjuvant therapy in comparative studiesing lymphocytes (TILs) is associated with event-free survival benefitmore favorable OS, RFS, and DSS/MSS [89](178). HPrognowever, the sample size was small sis improves as TIL cell count and the toxicity profile of the neoadjuvant arm was disappointing. Larger trials are currently underway to investigate neoadjuvant regimens that preserve efficacy while limiting toxicityphysical area of infiltration increases, likely because these metrics reflect an effective anti-tumor immune response (NCT02977052178). with promising initial resultsAs discussed [90].
4.3 Adoptive cell transfer with tumor-infiltrating lymphocytes
In melanboma, the presence of tumor-infiltrating lymphocytes (TILs) is associated with more favorable OS,ve, high-dose IL-2 therapy is used to expand melanoma TILs RFS, and DSS/MSS [91]in vivo. Adoptive cell transfer (ACT) of TILs is the process of expanding autologous lymphocytes in vitro, usually aided by IL-2, IL-7, IL-15, and/or IL-21, followed by re-infusion to the patient [92](179). This strategy circumvents many limitations of other immunotherapies. For example, in vitro TIL culture allows for the selective expansion of lymphocytes with the strongest effector function and the highest tumor-antigen affinity. Using autologous cells from resected tumor specimens avoids issues of rejection and allows each treatment to be uniquely targeted to the patient’s specific tumor antigens [93](180). Since expansion and activation occur without the suppressive effects of the TME, higher numbers of activated lymphocytes (>1011 TILs) can be achieved. This also allows for pretreatment manipulation of the patient’s immune system to optimize the efficacy of ACT or other planned immunotherapies without compromising the anti-tumor response. Greater response rates have been achieved when lymphodepletion proceeds and IV IL-2 follows ACT, both of which promote T-cell homeostatic cytokine production [94][95][96](181-183).
Since TIL-ACT regimens are not yet standardized, the degree of treatment efficacy reported in clinical trials has varied. Disease progression and overall survival after ACT-TIL are dependent on the expansion of neoepitope-specific CD8+ T-cells [97](184). ORRs typically range from 28% - 45% (179). The highest ORRs achieved using TIL-ACT were 49%, 52%, and 72% using lymphodepleting chemotherapy alone or [92]combined with 2 or 12 Gy TBI, respectively, in heavily pretreated mM patients. Complete response was initially observed in 22% of patients (185). Five-year follow-up found notable durability and suggests curative potential. Of the 22% CRs, all but one remained disease-free after 3 years, resulting in 100% 3-year and 95% 5-year survival rates [98](185). It is especially exciting that these results occurred in challenging mM cases, in which patients had a median of 3 metastatic sites and had all failed first-line treatments, including 20% who had failed ICI therapy.
O However, it should be noted thalt TBI is no longer used in patient preparation for ACT (182). In another longitudinal ACT study, patients 46 of 48 single-dose ACT complete responders maintained ongoing responses for at least 7.5 years and had a 10-year melanoma-specific survival of 96% (186).
Patients who receive TIL-ACT after failing ICI treatment have lower ORRs (56% vs 24%) and OS (28.5 vs 11.6 months) than ICI-naïve patients [99](186). The same is true for patients with BRAF V600E/K mutations who failed prior targeted therapy (ORR: 21% vs 60% if naïve; OS 9.3 vs 50.7 months) [99](186). This is likely because the poor immunogenicity and complex resistance mechanisms that allow tumors to evade ICIs also limit the efficacy of TIL-ACT [92](179). However, an ongoing onestudy of TIL-ACT in treatment-resistant mM has demonstrated an 80% disease control rate. Considering the higher toxicity rates and similar response rates of other second-line treatments, such as nivolumab or ipilimumab, TIL-ACT may be the best option for some patients resistant to alternative treatments [100][101](187, 188).
Limitations to ACT-TIL are similar to those of other immunotherapies. As discussed above, resistance remains a central issue. Similarly, target identification, predictability of immunogenicity, and anti-tumor specificity (sparing healthy tissues) are essential for ACT-TIL success, but solutions remain in the early stages of development. Protocols for TIL expansion, antigen identification, pre-treatment immunodepletion, and post-infusion TIL maintenance (for example,e.g. IL-2 dosing) must be optimized for time, cost, efficacy, and safety in order to make this therapy feasible on a larger scale.
4.4 ACT with T-CARs
A novel study aimed to improve ACT-TIL durability is investigating whether ex vivo treatment of TIL with a novel Myc-TAT (trans-activator transcription) fusion protein can prolong survival and promote proliferation of these cells after patient re-infusion (NCT03385486), potentially limiting the need for IL-2 infusions, which must be administered at high acuity centers.
10. ACT with T-CARs
Another approach to ACT utilizes autologous T-cells modified ex vivo with cell-surface chimeric antigen receptors (CAR-T cells). The extracellular component of the CAR is a variable region of a synthetic antibody. It is attached to a T-cell signaling moiety and co-stimulatory domains, which allows MHC-independent T-cell activation [102](189). CAR-T cells can thus target tumors cells that have downregulated MHCs as an immune-escape mechanism [103](190).
Success with CAR-T ACT for the treatment of hematologic malignancies sparked thean investigation into the therapy for solid malignancies. However, success in mM clinical trials has been limited
4.5 Melanoma Vaccines
Th. An early trial five major categories of mewith vascular endothelial growth factor receptor-2 (VEGFR-2) targeted CAR-Ts was terminated due to limited objective response (191) (NCT01218867). A phase I trial of anoma vaccines currently in development include ti-GD2 CAR-T was completed in 2019 but has not reported any conclusive results (NCT02107963). High rates of toxicity and evidence of rapid TME resistance to the therapy were discouraging (1192, 193). melanoma cell-targeted vaccines,However, recent advances in avoiding tumor resistance and limiting toxicity have renewed interest in CAR-T technologies (2191). dendritic cell (DC) vaccines, (3) peptide-based vaccines, Phase I trials of CAR-T cells targeting IL13Rα2 (NCT04119024), CD20 (NCT03893019), and B7-H3 (NCT05190185) are currently recruiting. Pre-clinical studies have identified several promising potential future targets, including CD126 (4194), vector-based vaccineschondroitin sulfate proteoglycan 4 (CSPG4) also known as MCSP (195), tand em CD70 and B7-H3 (5196), mRNAand αvβ3 integrin (197).
11. Radiotherapy
For DNAmany years, vaccines. Unlike preventative immunizations, cancer vaccines areradiation was considered an immunosuppressive therapy (198). However, recent studies have demonstrated that radiotherapies induce complex immunologic effects. Locally, radiation therapeutic, activating the patient’s immune system to incite an anti-tumor response against a known cies modulate the TME by increasing inflammatory cytokine production, APC and CD8+ T-cell activation, and sensitizing tumor-supporting stromal cells to T-cell-mediated destruction (199-202). Radiation also induces direct cancer cell DNA damage, MHC I expression, and Fas cell death ligand expression by cancer cells, increasing both the recognition and susceptibility of cancer or to prevent disease recurrence in the adjuvant setting.
Wcells to effector T- and NK cells (203-208). Immunotherapies achieve higher response rates and efficacy in immune-activated TMEs (209). Taken together, thole-cell vaccines use modified melanoma cells to simultaneously expose these effects contribute to enhanced innate and adaptive antitumor immunity and suggest synergistic potential with immune system to many potential melanoma antigens, circumventing the need to identiotherapies. The combination may also enhance the effects of RT, specifically by increasing the incidence of the “abscopal effect,” a rare immune-mediated response to radiation that results in tumor regression at non-radiated metastatic sites (210, 211).
However, befyore the mostadvent of immunogenic antigens for each tumore checkpoint inhibitors, neither local nor whole-body radiotherapy provided a consistent additional [104]benefit. DC vaccines are used to directly inject activated or modified DCs into the tumor site to increase anti-tumor T-cell activationombining IFN-α2b with fractionated radiotherapy has been studied many times in mM patients but has been complicated by high rates of toxicity without clear long-term benefits (212-214). Despite the suggestion by a phase I trial that stereotactic body radiation therapy (SBRT) and subsequent HD IL-2 in patients with mM demonstrated improved response rates compared to historical HD IL-2 monotherapy data (71% vs ~15%) (25, 215), phase II trials have not yet demonstrated significant benefit over IL-2 alone (216).
Combining Ptargeted immunotheptide vaccines supply tumor-specific or tumor-associaterapies with RT, however, has demonstrated more promising results. Preclinical models strongly support the synergistic efficacy of RT, anti-CTLA4, and antigen (i.e. gp100, MART-1/MelanA, tyrosinase) fragments that can be presented by professional APCs to induce effector T-cell activation. Vector vaccines u-PD-L1 antibody triple therapy (205, 217-219). The combination also seems safe, with multiple trials demonstrating that concurrent anti-PD-L1 or anti-CTLA-4 therapy with focal palliative RT, whole-body radiotherapy (WBRT), or stereotactic radiosurgery are well tolerated without significantly increased rates of grade 3/4 toxicities (220, 221). Efficacy, however, is controversial. Two recent multivariate analyses including 451 and 936 melanoma patients found improved survival in those receiving combinant viral vectors to deliver tumor antigen transgenes directly to APCs. Within the APCs, the transgenes are expressed to produce high concentrations of tumor antigens that can be presented on both MHCI and MHCII for enhanced T-cell activationtion therapy compared to either RT or immunotherapy alone (222, 223). Another single study demonstrated that concurrent anti-PD-1 therapy and fractionated RT resulted in significantly higher ORR (64.7%% vs 33.3%, p=0.02) without significant differences in 6-month disease-free survival and OS (224, 225). However, a high number of mM patients with brain metastases were included in all three of these studies. This may be a significant confounder in these studies since combination therapy may be particularly effective in this historically difficult population to treat (226-228). Further study is also needed to determine the best immunotherapeutic agents, order and time intervals at which to administer each treatment, radiation dosing and fractionation, and site of irradiation. Over 50 clinical trials are currently underway to evaluate RT and immunotherapy in various stages of mM treatment (229).
12. Future outlook
Over Tthe simultaneous expression of viral proteins by tpast 8 years, immunotherapy has revolutionized the treatment of mM, offering patients more treatment options with higher efficacy and less toxicity. Two-year overall survival rates have risen dramatically from ~10% to ~60% (33). Further delivered vectors boosts theresearch into the identification of melanoma neoantigens and their immunogenicity of the vaccin potentials is essential for the advancement of the [105]field. The ability to crapeutic mRNA vaccines have garneredeate individualized therapies specific to each patient’s tumor and immune landscape has the potential to revolutionize melanoma therapy. However, significant excitement after the advancements and effadvances in rapid tumor-cell sequencing and vaccine production must first be achieved. In the shorter term, combination therapy and melanoma vaccines show promise for improving the efficacy, response rates, and durability of current first-line immunotherapies.
12.1. Sequencing and combining therapies
Using cacy demonstrated in the COVID-19 pandemicombined therapies to treat mM may be the easiest way to achieve longer-lasting disease control, overcome innate resistance, evade adaptive resistance to immunotherapy, and optimize clinical response. While still in the early stages, mRNA vaccinThere is significant interest in finding the best combinations of the two most effective approved therapy classes—targeted and ICI therapy (NCT02631447, NCT03235245, NCT02902029, NCT02224781). Such studies may have the potential to induce the targeted expression of nearly any pr also address the two major roadblocks in the deployment of these therapies: rapid resistance development and modest response rates. Other studies are investigating combinations of approved and experimental therapies, including combinations of immunotherapy plus KIT inhibitors (198-200), VEGF inhibitors (NCT02681549, NCT01950390, NCT00790010) PI3K-AKT-mTOR patein. Ushway inhibitors (NCT02646748, NCT03131908), cyclin-dependent kinase (CDK) inhibitors (NCT02791334)(201, 202), FGFR1-3 inghibitors (NCT02159066), propranolol (NCT03384836), an mRNA apd even fecal microbiota transplant (203). Interesting experoach avoids safety concerns associated with DNAimental therapeutics beginning to be explored in human trials include a small molecule that selectively kills cancer cells that express elevated levels of specific proteins (NCT04809805) (204) and a viral vector vaccines. Therapeutic mechanisms udevice that removes soluble tumor necrosis factor receptors (sTNFR) during apheresis to reduce tumor-induced immunosuppression (NCT04142931) (205).
Consider investigationation of quality-of-life markers must also be include enhancing the expression of tumor-specific antigens in DCs, mRNA-mediated delivery of specific d when determining optimal therapy regimens as some highly effective immunotherapies carry high rates of toxicity that may become further amplified in certain contexts. For example, a 2019 trial demonstrated that while dabrafenib, trametinib, and pembrolizumab triple therapy may increase PFS compared to dabrafenib + trametinib doublet therapy, it also increases AEs from 26.7% to 58.3%, an unacceptable toxicity profile for many patients (206).
12.2 Neoadjuvant therapy
Neoadjuvanti- therapy is typically used to reduce tumor or anti-ICI antibodies, and programming cancer cells to express suiburden and allow for less extensive surgery. Before immunotherapy, neoadjuvant systemic therapy was not standard-of-care for mM treatment, likely because the risks of delaying surgery outweighed the limited benefits these therapies could provide. However, preclinical data suggest that this may not be true for immunotherapy (207), especidal intracellular proteinslly for therapies targeting T-cell function and proliferation. [106][107][108].
4.6 Toll-like receptor agonists
Theoll-like receptor (TLR) activation and theretically, initiating immunotherapy while the major tumor mass is still present may induce a stronger anti-tumor T-cell response. Indeed, a small feasibility study confirmed these resulting pro-inflammatory cytokine releass, demonstrating that patients receiving neoadjuvant and then adjuvant treatment had significantly more expansion of tumor-resident T-cell clones than patients who received the same treatment courses exclusively as adjuvant (208). Neoadjuvant is a critical stmmunotherapy also seems to outperform adjuvant therapy in comparative studies with event-free survival benefit (209). However, the samp in the induction of both the innate and adaptive immune responsle size was small and the toxicity profile of the neoadjuvant arm was disappointing. Larger trials are currently underway to investigate [109].neoadjuvant Aregimens poor immunogenicity continuthat preserve efficacy while limiting toxicity (NCT02977052) with promising initial results (210).
Response to limit the efficacy of melanomadata from these early studies also suggest that neoadjuvant therapy provides helpful markers for prognosis. A pathological complete response to neoadjuvant immunotherapies in some pay, for example, correlates with significantly improved RFS and 5-year OS (210-212). The extients, TLRs are a of neoadjuvant response observed can also inform a more effective choice for adjuvant therapy. Pathological ancillary agent thatspecimens may also inform adjuvant therapy choice by provides pro-inflammatory modulation of theing insight into each patient’s specific tumor adaptations, mechanisms of resistance, and biomarkers, such as post-surgical tumor microenvironment
4.7 Predictive markers and personalized medicine
utational burden and IFN-γ score (210).
12.3. Predictive markers and personalized medicine
Predicting response rates, toxicity, and durability present a major challenge to current mM immunotherapies. The melanoma and immune -oncology oneresearch communities are investing significant resources to identify predictive biomarkers [110](213) that would allow treatments to be better optimized for each patient’s therapeutic goals. While predictive response markers are still limited, computation algorithms have been developed to provide individualized predictions of response to pembrolizumab in mM patients with some success (214). Several independent predictive markers of outcome, including tumor-mutational burden, neoantigen load, and pretreatment CD8+ TIL count, have also been identificaed across multiple immunotherapies (215-218). With the hion ofgh rates at which mM develops therapy resistance, an investigation into how immunotherapies affect tumor neosignaling, tumor pathology, and the TME must also be considered.
Identificationt of tumor neoantigens and predictability of immunogenicity poses another issue. There are over 16,200 distinct class I HLA alleles, each with distinct peptide-binding preferences. Predicting which epitopes will likely be presented by each patient’s APCs is key to the future of immunotherapies such as ACT, OVs, and melanoma vaccines as thise epitope-APC interaction ultimately determines the immunogenicity of a given neoantigen. Some recent progress has been made: The HLAthena model can predict endogenous HLA-binding peptides with >75% accuracy [111](219). The Tumor Neoantigen Selection Alliance (TESLA) developed a bioinformatic-informed model of tumor epitope immunogenicity capable of filtering out 98% of non-immunogenic peptides with a precision of over 0.70 [112](220). However, no tool currently exists that can accurately predict if a specific neoantigen-HLA combination will be recognized by an individual’s TCRs. More informed models will require a larger and more diverse data set. The accessibility and affordability of next-generation molecular and functional diagnostics may one day allow each patient to receive personalized immunotherapy, optimized specifically to their tumor and goals.
13. Conclusions
While current therapeutic regimens demonstrate clear efficacy in many patients with advanced melanoma, some patients either relapse or do not respond to these regimens. Thus, the development of alternative strategies is still necessary.
We have summarized the current status of the melanoma treatment landscape and outlined results from past and ongoing clinical trials. Optimization of current therapies through rational combinations and optimized selection of alternative drug targets are the next frontier in melanoma treatment, with the ultimate overarching goal of sustained efficacy in the context of a more personalized treatment plan.
