Targeted delivery of a vaccine protein to Langerhans cells in the human skin via the C-type lectin receptor Langerin.

Human skin is a preferred vaccination site as it harbors multiple dendritic cell (DC) subsets, which display distinct C-type lectin receptors (CLR) that recognize pathogens. Antigens can be delivered to CLR by antibodies or ligands to boost antigen-specific immune responses. This concept has been established in mouse models but detailed insights into the functional consequences of antigen delivery to human skin DC in situ are sparse. In this study, we cloned and produced an anti-human Langerin antibody conjugated to the EBV nuclear antigen 1 (EBNA1). We confirmed specific binding of anti-Langerin-EBNA1 to Langerhans cells (LC). This novel LC-based vaccine was then compared to an existing anti-DEC-205-EBNA1 fusion protein by loading LC in epidermal cell suspensions before coculturing them with autologous T cells. After restimulation with EBNA1-peptides, we detected elevated levels of IFN-γ- and TNF-α-positive CD4+ T cells with both vaccines. When we injected the fusion proteins intradermally into human skin explants, emigrated skin DC targeted via DEC-205-induced cytokine production by T cells, whereas the Langerin-based vaccine failed to do so. In summary, we demonstrate that antibody-targeting approaches via the skin are promising vaccination strategies, however, further optimizations of vaccines are required to induce potent immune responses.

Specific Protein Antigen Delivery to Human Langerhans Cells in Intact Skin.

Immune modulating therapies and vaccines are in high demand, not least to the recent global spread of SARS-CoV2. To achieve efficient activation of the immune system, professional antigen presenting cells have proven to be key coordinators of such responses. Especially targeted approaches, actively directing antigens to specialized dendritic cells, promise to be more effective and accompanied by reduced payload due to less off-target effects. Although antibody and glycan-based targeting of receptors on dendritic cells have been employed, these are often expensive and time-consuming to manufacture or lack sufficient specificity. Thus, we applied a small-molecule ligand that specifically binds Langerin, a hallmark receptor on Langerhans cells, conjugated to a model protein antigen. Via microneedle injection, this construct was intradermally administered into intact human skin explants, selectively loading Langerhans cells in the epidermis. The ligand-mediated cellular uptake outpaces protein degradation resulting in intact antigen delivery. Due to the pivotal role of Langerhans cells in induction of immune responses, this approach of antigen-targeting of tissue-resident immune cells offers a novel way to deliver highly effective vaccines with minimally invasive administration.

Skin dendritic cells in melanoma are key for successful checkpoint blockade therapy.

BACKGROUND: Immunotherapy with checkpoint inhibitors has shown impressive results in patients with melanoma, but still many do not benefit from this line of treatment. A lack of tumor-infiltrating T cells is a common reason for therapy failure but also a loss of intratumoral dendritic cells (DCs) has been described. METHODS: We used the transgenic tg(Grm1)EPv melanoma mouse strain that develops spontaneous, slow-growing tumors to perform immunological analysis during tumor progression. With flow cytometry, the frequencies of DCs and T cells at different tumor stages and the expression of the inhibitory molecules programmed cell death protein-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells were analyzed. This was complemented with RNA-sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR) analysis to investigate the immune status of the tumors. To boost DC numbers and function, we administered Fms-related tyrosine 3 ligand (Flt3L) plus an adjuvant mix of polyI:C and anti-CD40. To enhance T cell function, we tested several checkpoint blockade antibodies. Immunological alterations were characterized in tumor and tumor-draining lymph nodes (LNs) by flow cytometry, CyTOF, microarray and RT-qPCR to understand how immune cells can control tumor growth. The specific role of migratory skin DCs was investigated by coculture of sorted DC subsets with melanoma-specific CD8+ T cells. RESULTS: Our study revealed that tumor progression is characterized by upregulation of checkpoint molecules and a gradual loss of the dermal conventional DC (cDC) 2 subset. Monotherapy with checkpoint blockade could not restore antitumor immunity, whereas boosting DC numbers and activation increased tumor immunogenicity. This was reflected by higher numbers of activated cDC1 and cDC2 as well as CD4+ and CD8+ T cells in treated tumors. At the same time, the DC boost approach reinforced migratory dermal DC subsets to prime gp100-specific CD8+ T cells in tumor-draining LNs that expressed PD-1/TIM-3 and produced interferon γ (IFNγ)/tumor necrosis factor α (TNFα). As a consequence, the combination of the DC boost with antibodies against PD-1 and TIM-3 released the brake from T cells, leading to improved function within the tumors and delayed tumor growth. CONCLUSIONS: Our results set forth the importance of skin DC in cancer immunotherapy, and demonstrates that restoring DC function is key to enhancing tumor immunogenicity and subsequently responsiveness to checkpoint blockade therapy.

CD73 contributes to anti-inflammatory properties of afferent lymphatic endothelial cells in humans and mice.

CD73 is an important ectoenzyme responsible for the production of extracellular adenosine. It is involved in regulating inflammatory responses and cell migration and is overexpressed in various cancers. The functions of CD73 in blood endothelial cells are understood in detail, but its role on afferent lymphatics remains unknown. Moreover, anti-CD73 antibodies are now used in multiple clinical cancer trials, but their effects on different endothelial cell types have not been studied. This study reveals that a previously unknown role of CD73 on afferent lymphatics is to dampen immune responses. Knocking it out or suppressing it by siRNA leads to the upregulation of inflammation-associated genes on lymphatic endothelial cells and a more pro-inflammatory phenotype of interacting dendritic cells in vitro and in vivo. In striking contrast, anti-CD73 antibodies had only negligible effects on the gene expression of lymphatic- and blood-endothelial cells. Our data thus reveal new functions of lymphatic CD73 and indicate a low likelihood of endothelial cell-related adverse effects by CD73 targeting therapeutic antibodies.

Notch-Mediated Generation of Monocyte-Derived Langerhans Cells: Phenotype and Function.

Langerhans cells (LCs) in the skin are a first line of defense against pathogens but also play an essential role in skin homeostasis. Their exclusive expression of the C-type lectin receptor Langerin makes them prominent candidates for immunotherapy. For vaccine testing, an easily accessible cell platform would be desirable as an alternative to the time-consuming purification of LCs from human skin. Here, we present such a model and demonstrate that monocytes in the presence of GM-CSF, TGF-ß1, and the Notch ligand DLL4 differentiate within 3 days into CD1a+Langerin+cells containing Birbeck granules. RNA sequencing of these monocyte-derived LCs (moLCs) confirmed gene expression of LC-related molecules, pattern recognition receptors, and enhanced expression of genes involved in the antigen-presenting machinery. On the protein level, moLCs showed low expression of costimulatory molecules but prominent expression of C-type lectin receptors. MoLCs can be matured, secrete IL-12p70 and TNF-α, and stimulate proliferation and cytokine production in allogeneic CD4+ and CD8+ T cells. In regard to vaccine testing, a recently characterized glycomimetic Langerin ligand conjugated to liposomes demonstrated specific and fast internalization into moLCs. Hence, these short-term in vitro‒generated moLCs represent an interesting tool to screen LC-based vaccines in the future.

A TLR7 agonist strengthens T and NK cell function during BRAF-targeted therapy in a preclinical melanoma model.

Therapeutic success of targeted therapy with BRAF inhibitors (BRAFi) for melanoma is limited by resistance development. Observations from preclinical mouse models and recent insights into the immunological effects caused by BRAFi give promise for future development of combination therapy for human melanoma. In our study, we used the transplantable D4M melanoma mouse model with the BRAFV600E mutation and concomitant PTEN loss in order to characterize alterations in tumor-infiltrating effector immune cells when tumors become resistant to BRAFi. We found that BRAFi-sensitive tumors displayed a pronounced inflammatory milieu characterized by high levels of cytokines and chemokines accompanied by an infiltration of T and NK cells. The tumor-infiltrating effector cells were activated and produced high levels of IFN-γ, TNF-α and granzyme B. When tumors became resistant and progressively grew, they reverted to a low immunogenic state similar to untreated tumors as reflected by low mRNA levels of proinflammatory cytokines and chemokines and fewer tumor-infiltrating T and NK cells. Moreover, these T and NK cells were functionally impaired in comparison to their counterparts in BRAFi-sensitive tumors. Their effector cell function could be restored by additional peritumoral treatment with the TLR7 agonist imiquimod, a clinically approved agent for nonmelanoma skin cancer. Indeed, resistance to BRAFi therapy was delayed and accompanied by high numbers of activated T and NK cells in tumors. Thus, combining BRAFi with an immune stimulating agent such as a TLR ligand could be a promising alternative approach for the treatment of melanoma.

A Specific, Glycomimetic Langerin Ligand for Human Langerhans Cell Targeting.

Langerhans cells are a subset of dendritic cells residing in the epidermis of the human skin. As such, they are key mediators of immune regulation and have emerged as prime targets for novel transcutaneous cancer vaccines. Importantly, the induction of protective T cell immunity by these vaccines requires the efficient and specific delivery of both tumor-associated antigens and adjuvants. Langerhans cells uniquely express Langerin (CD207), an endocytic C-type lectin receptor. Here, we report the discovery of a specific, glycomimetic Langerin ligand employing a heparin-inspired design strategy and structural characterization by NMR spectroscopy and molecular docking. The conjugation of this glycomimetic to liposomes enabled the specific and efficient targeting of Langerhans cells in the human skin. We further demonstrate the doxorubicin-mediated killing of a Langerin+ monocyte cell line, highlighting its therapeutic and diagnostic potential in Langerhans cell histiocytosis, caused by the abnormal proliferation of Langerin+ myeloid progenitor cells. Overall, our delivery platform provides superior versatility over antibody-based approaches and novel modalities to overcome current limitations of dendritic cell-targeted immuno- and chemotherapy.

A Liposomal Platform for Delivery of a Protein Antigen to Langerin-Expressing Cells.

The skin is an attractive site for vaccination and harbors a dense network of Langerhans cells that are the prime target for antigen delivery approaches in the epidermis. While specific targeting of Langerhans cells has been shown to elicit the necessary T-cell response using antibody-based delivery approaches, the targeted administration of particulate antigens in the form of nanoparticle-based vaccine formulations has been challenging. We previously reported on a specific targeting ligand for human Langerin, a C-type lectin expressed on Langerhans cells. This ligand is presented on liposomes and renders them highly specific for the uptake by Langerhans cells. Here we show a detailed study of the uptake and intracellular routing of the particles in model cell lines by confocal and live cell imaging as well as flow cytometric assays. Liposomes are internalized into early endosomal compartments and accumulate in late endosomes and lysosomes, shortly followed by a release of the cargo. Furthermore, we show the encapsulation of protein antigens and their delivery to cell lines and primary human Langerhans cells. These data further support the applicability of the targeted liposomal particles for protein vaccine applications.

Application of interferon modulators to overcome partial resistance of human ovarian cancers to VSV-GP oncolytic viral therapy.

Previously, we described an oncolytic vesicular stomatitis virus variant pseudotyped with the nonneurotropic glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, which was highly effective in glioblastoma. Here, we tested its potency for the treatment of ovarian cancer, a leading cause of death from gynecological malignancies. Effective oncolytic activity of VSV-GP could be demonstrated in ovarian cancer cell lines and xenografts in mice; however, remission was temporary in most mice. Analysis of the innate immune response revealed that ovarian cancer cell lines were able to respond to and produce type I interferon, inducing an antiviral state upon virus infection. This is in stark contrast to published data for other cancer cell lines, which were mostly found to be interferon incompetent. We showed that in vitro this antiviral state could be reverted by combining VSV-GP with the JAK1/2-inhibitor ruxolitinib. In addition, for the first time, we report the in vivo enhancement of oncolytic virus treatment by ruxolitinib, both in subcutaneous as well as in orthotopic xenograft mouse models, without causing significant additional toxicity. In conclusion, VSV-GP has the potential to be a potent and safe oncolytic virus to treat ovarian cancer, especially when combined with an inhibitor of the interferon response.