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We evaluated the prognostic value of hypoalbuminemia in context of various biomarkers at baseline, including clinical, genomic, transcriptomic, and blood-based markers, in patients with metastatic melanoma treated with anti-PD-1 monotherapy or anti-PD-1/anti-CTLA-4 combination therapy (n = 178). An independent validation cohort (n = 79) was used to validate the performance of hypoalbuminemia compared to serum LDH (lactate dehydrogenase) levels. Pre-treatment hypoalbuminemia emerged as the strongest predictor of poor outcome for both OS (HR = 4.01, 95% CI 2.10–7.67, Cox P = 2.63e−05) and PFS (HR = 3.72, 95% CI 2.06–6.73, Cox P = 1.38e−05) in univariate analysis. In multivariate analysis, the association of hypoalbuminemia with PFS was independent of serum LDH, IFN-γ signature expression, TMB, age, ECOG PS, treatment line, treatment type (combination or monotherapy), brain and liver metastasis (HR = 2.76, 95% CI 1.24–6.13, Cox P = 0.0131). Our validation cohort confirmed the prognostic power of hypoalbuminemia for OS (HR = 1.98, 95% CI 1.16–3.38; Cox P = 0.0127) and was complementary to serum LDH in analyses for both OS (LDH-adjusted HR = 2.12, 95% CI 1.2–3.72, Cox P = 0.00925) and PFS (LDH-adjusted HR = 1.91, 95% CI 1.08–3.38, Cox P = 0.0261). In conclusion, pretreatment hypoalbuminemia was a powerful predictor of outcome in ICI in melanoma and showed remarkable complementarity to previously established biomarkers, including high LDH. [ABSTRACT FROM AUTHOR]

Simple Summary: The melanoma-conditioned microenvironment promotes immune escape and tumor progression, contributing to resistance to anti-melanoma immunotherapy in a large group of treated patients. While two-dimensional cultures lack tissue-context, animals poorly predict human immune responses. Thus, a clear need exists for more physiological human models. We previously described an in vitro human melanoma-in-skin (Mel-RhS) model with the SK-MEL-28 cell line mirroring features of invasive melanoma. Here, we further investigated the tumor-induced microenvironment of this model, generated five additional Mel-RhS models (A375, COLO829, G361, MeWo, and RPMI-7951), and performed in-depth analysis of invasion, immune modulation, angiogenesis, and their respective mediators. We present three-dimensional models allowing for the study of diverse tumor-intrinsic properties that could be used to investigate efficacy of therapeutic interventions. By mimicking different stages of melanoma and related features, each Mel-RhS can be further individually tailored to obtain a physiologically evermore relevant model to study specific mechanisms underlying melanomagenesis and progression. Invasion, immune modulation, and angiogenesis are crucial in melanoma progression. Studies based on animals or two-dimensional cultures poorly recapitulate the tumor-microenvironmental cross-talk found in humans. This highlights a need for more physiological human models to better study melanoma features. Here, six melanoma cell lines (A375, COLO829, G361, MeWo, RPMI-7951, and SK-MEL-28) were used to generate an in vitro three-dimensional human melanoma-in-skin (Mel-RhS) model and were compared in terms of dermal invasion and immune modulatory and pro-angiogenic capabilities. A375 displayed the most invasive phenotype by clearly expanding into the dermal compartment, whereas COLO829, G361, MeWo, and SK-MEL-28 recapitulated to different extent the initial stages of melanoma invasion. No nest formation was observed for RPMI-7951. Notably, the integration of A375 and SK-MEL-28 cells into the model resulted in an increased secretion of immune modulatory factors (e.g., M-CSF, IL-10, and TGFβ) and pro-angiogenic factors (e.g., Flt-1 and VEGF). Mel-RhS-derived supernatants induced endothelial cell sprouting in vitro. In addition, observed A375-RhS tissue contraction was correlated to increased TGFβ release and α-SMA expression, all indicative of differentiation of fibroblasts into cancer-associated fibroblast-like cells and reminiscent of epithelial-to-mesenchymal transition, consistent with A375′s most prominent invasive behavior. In conclusion, we successfully generated several Mel-RhS models mimicking different stages of melanoma progression, which can be further tailored for future studies to investigate individual aspects of the disease and serve as three-dimensional models to assess efficacy of therapeutic strategies. [ABSTRACT FROM AUTHOR]

Despite the recent impressive clinical success of immunotherapy against melanoma, development of primary and adaptive resistance against immune checkpoint inhibitors remains a major issue in a large number of treated patients. This highlights the need for melanoma models that replicate the tumor's intricate dynamics in the tumor microenvironment (TME) and associated immune suppression to study possible resistance mechanisms in order to improve current and test novel therapeutics. While two‐dimensional melanoma cell cultures have been widely used to perform functional genomics screens in a high‐throughput fashion, they are not suitable to answer more complex scientific questions. Melanoma models have also been established in a variety of experimental (humanized) animals. However, due to differences in physiology, such models do not fully represent human melanoma development. Therefore, fully human three‐dimensional in vitro models mimicking melanoma cell interactions with the TME are being developed to address this need for more physiologically relevant models. Such models include melanoma organoids, spheroids, and reconstructed human melanoma‐in‐skin cultures. Still, while major advances have been made to complement and replace animals, these in vitro systems have yet to fully recapitulate human tumor complexity. Lastly, technical advancements have been made in the organ‐on‐chip field to replicate functions and microstructures of in vivo human tissues and organs. This review summarizes advancements made in understanding and treating melanoma and specifically aims to discuss the progress made towards developing melanoma models, their applications, limitations, and the advances still needed to further facilitate the development of therapeutics. [ABSTRACT FROM AUTHOR]

Preclinical studies show that locoregional CTLA-4 blockade is equally effective in inducing tumor eradication as systemic delivery, without the added risk of immune-related side effects. This efficacy is related to access of the CTLA-4 blocking antibodies to tumor-draining lymph nodes (TDLNs). Local delivery of anti–CTLA-4 after surgical removal of primary melanoma, before sentinel lymph node biopsy (SLNB), provides a unique setting to clinically assess the role of TDLN in the biological efficacy of locoregional CTLA-4 blockade. Here, we have evaluated the safety, tolerability, and immunomodulatory effects in the SLN and peripheral blood of a single dose of tremelimumab [a fully human immunoglobulin gamma-2 (IgG2) mAb directed against CTLA-4] in a dose range of 2 to 20 mg, injected intradermally at the tumor excision site 1 week before SLNB in 13 patients with early-stage melanoma (phase 1 trial; NCT04274816). Intradermal delivery was safe and well tolerated and induced activation of migratory dendritic cell (DC) subsets in the SLN. It also induced profound and durable decreases in regulatory T cell (Treg) frequencies and activation of effector T cells in both SLN and peripheral blood. Moreover, systemic T cell responses against NY-ESO-1 or MART-1 were primed or boosted (N = 7), in association with T cell activation and central memory T cell differentiation. These findings indicate that local administration of anti–CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, our data demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and support TDLN-targeted delivery methods. Under the skin: Although systemic immune checkpoint blockade (ICB) displays therapeutic efficacy in cancers, it induces immune-related adverse events (irAEs). Avoiding irAEs is crucial for effective ICB. Van Pul et al. performed a phase 1 clinical trial testing the effects of an intradermal injection of anti–CTLA-4 at the site of primary tumor excision in patients with early-stage melanoma. Seven of 13 patients immunologically responded to the treatment, with little to no irAEs. Responders had more tumor antigen–specific T cells in the blood, increased migratory DC activation in the sentinel lymph node (SLN), and decreased Tregs in both the SLN and blood. Thus, intradermal anti–CTLA-4 after primary tumor excision in melanoma induced a favorable immune response and has promise as a treatment option for patients with early-stage melanoma. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors such as anti-PD-1 have revolutionized the field of oncology over the past decade. Nevertheless, the majority of patients do not benefit from them. Virotherapy is a flexible tool that can be used to stimulate and/or recruit different immune populations. T-cell enabling virotherapy could enhance the efficacy of immune checkpoint inhibitors, even in tumors resistant to these inhibitors. The T-cell potentiating virotherapy used here consisted of adenoviruses engineered to express tumor necrosis factor alpha and interleukin-2 in the tumor microenvironment. To study virus efficacy in checkpoint-inhibitor resistant tumors, we developed an anti-PD-1 resistant melanoma model in vivo. In resistant tumors, adding virotherapy to an anti-PD-1 regimen resulted in increased survival (p=0.0009), when compared to anti-PD-1 monotherapy. Some of the animals receiving virotherapy displayed complete responses, which did not occur in the immune checkpoint-inhibitor monotherapy group. When adenoviruses were delivered into resistant tumors, there were signs of increased CD8 T-cell infiltration and activation, which - together with a reduced presence of M2 macrophages and myeloid-derived suppressor cells - could explain those results. T-cell enabling virotherapy appeared as a valuable tool to counter resistance to immune checkpoint inhibitors. The clinical translation of this approach could increase the number of cancer patients benefiting from immunotherapies. [ABSTRACT FROM AUTHOR]

Whereas TLR9 agonists are recognized as powerful stimulators of antitumor immunity, GM-CSF has had mixed reviews. In previously reported randomized trials we assessed the effects of local immune modulation in early-stage melanoma with CpG-B alone or with GM-CSF. Here we discuss the added value of GM-CSF and show sex-related differences. [ABSTRACT FROM AUTHOR]

Lymph nodes draining the primary tumor are essential for the initiation of an effective anti-tumor T-cell immune response. However, cancer-derived immune suppressive factors render the tumor-draining lymph nodes (TDLN) immune compromised, enabling tumors to invade and metastasize. Unraveling the different mechanisms underlying this immune escape will inform therapeutic intervention strategies to halt tumor spread in early clinical stages. Here, we review our findings from translational studies in melanoma, breast, and cervical cancer and discuss clinical opportunities for local immune modulation of TDLN in each of these indications. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFNγ generates mature DCs that produce IL-12 and polarize CD4+ T cells towards a Th1 phenotype. Here, we extended these observations by showing that the DCs generated with the clinical grade maturation cocktail of MPLA/IFNγ induce superior tumour antigen-specific CD8+ CTL responses compared to the cytokine cocktail matured DCs that are currently used in the clinic. MPLA/IFNγ DCs can induce CTL responses in healthy individuals as well as in melanoma patients. The CTL induction was mainly dependent on the IL-12 produced by the MPLA/IFNγ DCs. The high amounts of induced CTLs are functional as they produce IFNγ and lyse target cells and this cytolytic activity is antigen specific and HLA restricted. Furthermore, the CTLs proved to kill tumour cells expressing endogenous tumour antigen in vitro. Therefore, MPLA/IFNγ DCs are very promising for the use in future cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Simple Summary: The incidence of metastatic melanoma has been increasing over the past years with current therapies showing limited efficacy to cure the disease. Therefore, other options are being investigated, such as adoptive cell therapy (ACT) where activated immune cells are infused into a patient to attack melanoma. Natural killer (NK) cells are part of the innate immune system and extremely suitable for this kind of therapy since they show minimal toxicities in the clinical setting. In this review, we focus on current strategies for NK cell therapy and the development of new approaches that hold great promise for the treatment of advanced melanoma. Adoptive cell therapy (ACT) represents a promising alternative approach for patients with treatment-resistant metastatic melanoma. Lately, tumor infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR)-T cell therapy have shown improved clinical outcome, compared to conventional chemotherapy or immunotherapy. Nevertheless, they are limited by immune escape of the tumor, cytokine release syndrome, and manufacturing challenges of autologous therapies. Conversely, the clinical use of Natural Killer (NK) cells has demonstrated a favorable clinical safety profile with minimal toxicities, providing an encouraging treatment alternative. Unlike T cells, NK cells are activated, amongst other mechanisms, by the downregulation of HLA class I molecules, thereby overcoming the hurdle of tumor immune escape. However, impairment of NK cell function has been observed in melanoma patients, resulting in deteriorated natural defense. To overcome this limitation, "activated" autologous or allogeneic NK cells have been infused into melanoma patients in early clinical trials, showing encouraging clinical benefit. Furthermore, as several NK cell-based therapeutics are being developed for different cancers, an emerging variety of approaches to increase migration and infiltration of adoptively transferred NK cells towards solid tumors is under preclinical investigation. These developments point to adoptive NK cell therapy as a highly promising treatment for metastatic melanoma in the future. [ABSTRACT FROM AUTHOR]

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated apoptotic tumor cell-derived extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8+ T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer. [ABSTRACT FROM AUTHOR]

To mount a strong anti-tumor immune response, non T cell inflamed (cold) tumors may require combination treatment encompassing vaccine strategies preceding checkpoint inhibition. In vivo targeted delivery of tumor-associated antigens (TAA) to dendritic cells (DCs), relying on the natural functions of primary DCs in situ, represents an attractive vaccination strategy. In this study we made use of a full-length MART-1 expressing C/B-chimeric adenoviral vector, consisting of the Ad5 capsid and the Ad3 knob (Ad5/3), which we previously showed to selectively transduce DCs in human skin and lymph nodes. Our data demonstrate that chimeric Ad5/3 vectors encoding TAA, and able to target human DCs in situ, can be used to efficiently induce expansion of functional tumor-specific CD8+ effector T cells, either from a naïve T cell pool or from previously primed T cells residing in the melanoma-draining sentinel lymph nodes (SLN). These data support the use of Ad3-knob containing viruses as vaccine vehicles for in vivo delivery. "Off-the-shelf" DC-targeted Ad vaccines encoding TAA could clearly benefit future immunotherapeutic approaches. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors have advanced the treatment of melanoma. Nevertheless, a majority of patients are resistant, or develop resistance, to immune checkpoint blockade, which may be related to prevailing immune suppression by myeloid regulatory cells in the tumor microenvironment (TME). ORCA-010 is a novel oncolytic adenovirus that selectively replicates in, and lyses, cancer cells. We previously showed that ORCA-010 can activate melanoma-exposed conventional dendritic cells (cDCs). To study the effect of ORCA-010 on melanoma-conditioned macrophage development, we used an in vitro co-culture model of human monocytes with melanoma cell lines. We observed a selective survival and polarization of monocytes into M2-like macrophages (CD14+CD80−CD163+) in co-cultures with cell lines that expressed macrophage colony-stimulating factor. Oncolysis of these melanoma cell lines, effected by ORCA-010, activated the resulting macrophages and converted them to a more proinflammatory state, evidenced by higher levels of PD-L1, CD80, and CD86 and an enhanced capacity to prime allogenic T cells and induce a type-1 T cell response. To assess the effect of ORCA-010 on myeloid subset distribution and activation in vivo, ORCA-010 was intratumorally injected and tested for T cell activation and recruitment in the human adenovirus nonpermissive B16-OVA mouse melanoma model. While systemic PD-1 blockade in this model in itself did not modulate myeloid or T cell subset distribution and activation, when it was preceded by i.t. injection of ORCA-010, this induced an increased rate and activation state of CD8α+ cDC1, both in the TME and in the spleen. Observed increased rates of activated CD8+ T cells, expressing CD69 and PD-1, were related to both increased CD8α+ cDC1 rates and M1/M2 shifts in tumor and spleen. In conclusion, the myeloid modulatory properties of ORCA-010 in melanoma, resulting in recruitment and activation of T cells, could enhance the antitumor efficacy of PD-1 blockade. [ABSTRACT FROM AUTHOR]

To increase (tumor) vaccine efficacy, there is an urgent need for phenotypic and functional characterization of human dendritic cell (DC) subsets residing in lymphoid tissues. In this study we identified and functionally tested 4 human conventional DC (cDC) subsets within skin-draining sentinel lymph nodes (SLNs) from early-stage melanoma patients. These SLNs were all tumor negative and were removed on average 44 days after excision of the primary melanoma. As such, they were considered representative of steady-state conditions. On comparison with skin-migrated cDC, 2 CD1a+ subsets were identified as most likely skin-derived CD11cint Langerhans cells (LC) with intracellular langerin and E-cadherin expression or as CD11chi dermal DCs with variable expression of langerin. Two other CD1a- LN-residing cDC subsets were characterized as CD14-BDCA3hiCD103- and CD14+BDCA3loCD103+, respectively. Whereas the CD1a+ skin-derived subsets displayed greater levels of phenotypic maturation, they were associated with lower levels of inflammatory cytokine release and were inferior in terms of allogeneic T-cell priming and IFNγ induction. Thus, despite their higher maturation state, skin-derived cDCs (and LCs in particular) proved inferior T-cell activators compared with the CD1a- cDC subsets residing in melanoma-draining LNs. These observations should be considered in the design of DC-targeting immunotherapies. [ABSTRACT FROM AUTHOR]

In situ delivery of tumor-associated antigen (TAA) genes into dendritic cells (DC) has great potential as a generally applicable tumor vaccination approach. Although adenoviruses (Ad) are an attractive vaccine vehicle in this regard, Ad-mediated transduction of DCs is hampered by the lack of expression of the Ad receptor CAR on the DC surface. DC activation also requires interaction of CD40 with its ligand CD40L to generate protective T-cell-mediated tumor immunity. Therefore, to create a strategy to target Ads to DCs in vivo, we constructed a bispecific adaptor molecule with the CAR ectodomain linked to the CD40L extracellular domain via a trimerization motif (CFm40L). By targeting Ad to CD40 with the use of CFm40L, we enhanced both transduction and maturation of cultured bone marrow-derived DCs. Moreover, we improved transduction efficiency of DCs in lymph node and splenic cell suspensions in vitro and in skin and vaccination site-draining lymph nodes in vivo. Furthermore, CD40 targeting improved the induction of specific CD8+T cells along with therapeutic efficacy in a mouse model of melanoma. Taken together, our findings support the use of CD40-targeted Ad vectors encoding full-length TAA for in vivo targeting of DCs and high-efficacy induction of antitumor immunity. [ABSTRACT FROM AUTHOR]

Abstract: Adenovirus (Ad)-based vaccines are considered for cancer immunotherapy, yet, detailed knowledge on their mechanism of action and optimal delivery route for anti-tumor efficacy is lacking. Here, we compared the anti-tumor efficacy of an Ad-based melanoma vaccine after intradermal, intravenous, intranasal or intraperitoneal delivery in the B16F10 melanoma model. The intradermal route induced superior systemic anti-melanoma immunity which was MyD88 signaling-dependent. Predominant transduction of non-professional antigen-presenting cells at the dermal vaccination sites and draining lymph nodes, suggested a role for cross-presentation, which was confirmed in vitro. We conclude that the dermis provides an optimal route of entry for Ad-based vaccines for high-efficacy systemic anti-tumor immunization and that this immunization likely involves cross-priming events in the draining lymph nodes. [Copyright &y& Elsevier]

Abstract: Cutaneous melanoma is the most aggressive type of skin cancer. Paradoxically, melanoma is also the most immunogenic tumour identified to date: tumour-reactive T cells are detectable both in the blood and in tumour-draining lymph nodes (TDLN) of melanoma patients and their frequency can be increased by specific vaccination. However, early melanoma development is accompanied by impaired immune effector functions in the initial TDLN, the sentinel lymph node (SLN). Most notably, a reduced frequency and activation state of dendritic cells (DC) interferes with the uptake and presentation of tumour-associated antigens (TAA) to specific anti-tumour cytotoxic T-lymphocytes (CTL) and T helper cells (Th). These impaired immune effector functions may contribute to the early metastatic events that are associated with this tumour type. Since complete surgical excision at an early stage remains the only curative treatment option (adjuvant therapy options are limited and show no survival benefits), immunopotentiation of the SLN to jump-start or boost tumour specific immunity in early stage melanoma may be a valuable adjuvant treatment option that can be generally applied with minimal discomfort to the patient. Early clinical studies indicate that local Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) or Cytosine-phosphate-Guanine (CpG) administration leads to activation of different DC subsets and conditions the SLN microenvironment to be more conducive to the generation of T-cell-mediated anti-tumour immunity. [Copyright &y& Elsevier]