Toll-like receptor 9 (TLR9) is a pattern recognition receptor that is predominantly located intracellularly in immune cells, including dendritic cells, macrophages, natural killer cells, and other antigen-presenting cells (APC). The primary ligands for TLR9 receptors are unmethylated cytidine phosphate guanosine (CpG) oligodinucleotides (ODN). TLR9 agonists induce inflammatory processes that result in the enhanced uptake and killing of microorganisms and cancer cells as well as the generation of adaptive immune responses. Preclinical studies of TLR9 agonists suggested efficacy both as monotherapy and in combination with several agents, which led to clinical trials in patients with advanced cancer. In these studies, intravenous, intratumoral, and subcutaneous routes of administration have been tested; with anti-tumor responses in both treated and untreated metastatic sites. TLR9 agonist monotherapy is safe, although efficacy is minimal in advanced cancer patients; conversely, combinations appear to be more promising. Several ongoing phase I and II clinical trials are evaluating TLR9 agonists in combination with a variety of agents including chemotherapy, radiotherapy, targeted therapy, and immunotherapy agents. In this review article, we describe the distribution, structure and signaling of TLR9; discuss the results of preclinical studies of TLR9 agonists; and review ongoing clinical trials of TLR9 agonists singly and in combination in patients with advanced solid tumors.

Immunotherapy targeting co-inhibitory receptors has been highly successful in treating a wide variety of malignancies, however only a subset of patients exhibits durable responses. The first FDA-approved immunotherapeutics targeting co-inhibitory receptors PD1 and CTLA4, alone or in combination, significantly improved survival but were also accompanied by substantial toxicity in combination. The third FDA-approved immune checkpoint inhibitor targets LAG3, a co-inhibitory receptor expressed on activated CD4+ and CD8+ T cells, especially in settings of long-term antigenic stimulation, such as chronic viral infection or cancer. Mechanistically, LAG3 expression limits both the expansion of activated T cells and the size of the memory pool, suggesting that LAG3 may be a promising target for immunotherapy. Importantly, the mechanism(s) by which LAG3 contributes to CD8+ T cell exhaustion may be distinct from those governed by PD1, indicating that the combination of anti-LAG3 and anti-PD1 may synergistically enhance antitumor immunity. Clinical studies evaluating the role of anti-LAG3 in combination with anti-PD1 are underway, and recent phase III trial results in metastatic melanoma demonstrate both the efficacy and safety of this combination. Further ongoing clinical trials are evaluating this combination across multiple tumor types and the adjuvant setting, with accompanying translational and biomarker-focused studies designed to elucidate the molecular pathways that lead to improved antitumor T cell responses following dual blockade of PD1 and LAG3. Overall, LAG3 plays an important role in limiting T cell activation and has now become part of the repertoire of combinatorial immunotherapeutics available for the treatment of metastatic melanoma.

The use of immunotherapy in the treatment of advanced and high-risk melanoma has led to a striking improvement in outcomes. Although the incidence of melanoma has continued to rise, median survival has improved from approximately 6 months to nearly 6 years for patients with advanced inoperable stage IV disease. Recent understanding of the tumor microenvironment and its interplay with the immune system has led to the explosive development of novel immunotherapy treatments. Since the approval of the therapeutic cytokines interleukin-2 and interferon alfa-2 in the 1990s, the development of novel immune checkpoint inhibitors (ICIs), oncolytic virus therapy, and modulators of the tumor microenvironment have given way to a new era in melanoma treatment. Monoclonal antibodies directed at programmed cell death protein 1 receptor (PD-1) and its ligand (PDL-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and lymphocyte-activation gene 3 (LAG-3) have provided robust activation of the adaptive immune system, restoring immune surveillance leading to host tumor recognition and destruction. Multiple other immunomodulatory therapeutics are under investigation to overcome resistance to ICI therapy, including the toll-like receptor-9 (TLR-9) and 7/8 (TLR-7/8) agonists, stimulator of interferon genes (STING) agonists, and fecal microbiota transplantation. In this review, we focus on the recent advances in immunotherapy for the treatment of melanoma and provide an update on novel therapies currently under investigation.

BackgroundA first-in-human, randomized pilot phase II clinical trial combining vaccines targeting overexpressed, non-mutated tumor blood vessel antigens (TBVA) and tyrosine kinase inhibitor dasatinib was conducted in human leukocyte antigen (HLA)-A2+ patients with advanced melanoma.MethodsPatient monocyte-derived type-1-polarized dendritic cells were loaded with HLA-A2-presented peptides derived from TBVA (DLK1, EphA2, HBB, NRP1, RGS5, TEM1) and injected intradermally as a vaccine into the upper extremities every other week. Patients were randomized into one of two treatment arms receiving oral dasatinib (70 mg two times per day) beginning in week 5 (Arm A) or in week 1 (Arm B). Trial endpoints included T cell response to vaccine peptides (interferon-γ enzyme-linked immunosorbent spot), objective clinical response (Response Evaluation Criteria in Solid Tumors V.1.1) and exploratory tumor, blood and serum profiling of immune-associated genes/proteins.ResultsSixteen patients with advanced-stage cutaneous (n=10), mucosal (n=1) or uveal (n=5) melanoma were accrued, 15 of whom had previously progressed on programmed cell death protein 1 (PD-1) blockade. Of 13 evaluable patients, 6 patients developed specific peripheral blood T cell responses against ≥3 vaccine-associated peptides, with further evidence of epitope spreading. All six patients with specific CD8+ T cell response to vaccine-targeted antigens exhibited evidence of T cell receptor (TCR) convergence in association with preferred clinical outcomes (four partial response and two stabilization of disease (SD)). Seven patients failed to respond to vaccination (one SD and six progressive disease). Patients in Arm B (immediate dasatinib) outperformed those in Arm A (delayed dasatinib) for immune response rate (IRR; 66.7% vs 28.6%), objective response rate (ORR) (66.7% vs 0%), overall survival (median 15.45 vs 3.47 months; p=0.0086) and progression-free survival (median 7.87 vs 1.97 months; p=0.063). IRR (80% vs 25%) and ORR (60% vs 12.5%) was greater for females versus male patients. Tumors in patients exhibiting response to treatment displayed (1) evidence of innate and adaptive immune-mediated inflammation and TCR convergence at baseline, (2) on-treatment transcriptional changes associated with reduced hypoxia/acidosis/glycolysis, and (3) increased inflammatory immune cell infiltration and tertiary lymphoid structure neogenesis.ConclusionsCombined vaccination against TBVA plus dasatinib was safe and resulted in coordinating immunologic and/or objective clinical responses in 6/13 (46%) evaluable patients with melanoma, particularly those initiating treatment with both agents.Trial registration numberNCT01876212.

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