A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens.

Cancer cells effectively evade immune surveillance, not only through the well-known PD-1/PD-L1 pathway but also alternative mechanisms that impair patient response to immune checkpoint inhibitors. We present a novel co-culture model that pairs a reporter T-cell line with different melanoma cell lines that have varying immune evasion characteristics. We developed a scalable high-throughput lentiviral arrayed CRISPR interference (CRISPRi) screening protocol to conduct gene perturbations in both T-cells and melanoma cells, enabling the identification of genes that modulate tumor immune evasion.

Neoantigen-targeted dendritic cell vaccination in lung cancer patients induces long-lived T cells exhibiting the full differentiation spectrum.

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here, we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients.

A bispecific Clec9A-PD-L1 targeted type I interferon profoundly reshapes the tumor microenvironment towards an antitumor state.

Despite major improvements in immunotherapeutic strategies, the immunosuppressive tumor microenvironment remains a major obstacle for the induction of efficient antitumor responses. In this study, we show that local delivery of a bispecific Clec9A-PD-L1 targeted type I interferon (AcTaferon, AFN) overcomes this hurdle by reshaping the tumor immune landscape.Treatment with the bispecific AFN resulted in the presence of pro-immunogenic tumor-associated macrophages and neutrophils, increased motility and maturation profile of cDC1 and presence of inflammatory cDC2.

High-resolution and quantitative spatial analysis reveal intra-ductal phenotypic and functional diversification in pancreatic cancer.

A 'classical' and a 'basal-like' subtype of pancreatic cancer have been reported, with differential expression of GATA6 and different dosages of mutant KRAS. We established in situ detection of KRAS point mutations and mRNA panels for the consensus subtypes aiming to project these findings to paraffin-embedded clinical tumour samples for spatial quantitative analysis. We unveiled that, next to inter-patient and intra-patient inter-ductal heterogeneity, intraductal spatial phenotypes exist with anti-correlating expression levels of GATA6 and KRAS .

Selective IL-1 activity on CD8 T cells empowers antitumor immunity and synergizes with neovasculature-targeted TNF for full tumor eradication.

Background: Clinical success of therapeutic cancer vaccines depends on the ability to mount strong and durable antitumor T cell responses. To achieve this, potent cellular adjuvants are highly needed. Interleukin-1β (IL-1β) acts on CD8 T cells and promotes their expansion and effector differentiation, but toxicity and undesired tumor-promoting side effects hamper efficient clinical application of this cytokine.

Targeting IFN activity to both B cells and plasmacytoid dendritic cells induces a robust tolerogenic response and protection against EAE.

Type I Interferon (IFN) was the very first drug approved for the treatment of Multiple Sclerosis (MS), and is still frequently used as a first line therapy. However, systemic IFN also causes considerable side effects, affecting therapy adherence and dose escalation. In addition, the mechanism of action of IFN in MS is multifactorial and still not completely understood.

Small-scale manufacturing of neoantigen-encoding messenger RNA for early-phase clinical trials.

Messenger RNA (mRNA) has become a promising tool in therapeutic cancer vaccine strategies. Owing to its flexible design and rapid production, mRNA is an attractive antigen delivery format for cancer vaccines targeting mutated peptides expressed in a tumor-the so-called neoantigens. These neoantigens are rarely shared between patients, and inclusion of these antigens in a vaccine requires the production of individual batches of patient-tailored mRNA.

Dendritic Cell-Based Immunotherapy in Lung Cancer.

Lung cancer remains the leading cause of cancer-related death worldwide. The advent of immune checkpoint inhibitors has led to a paradigm shift in the treatment of metastatic non-small cell and small cell lung cancer. However, despite prolonged overall survival, only a minority of the patients derive clinical benefit from these treatments suggesting that the full anti-tumoral potential of the immune system is not being harnessed yet.

mRNA in cancer immunotherapy: beyond a source of antigen.

mRNA therapeutics have become the focus of molecular medicine research. Various mRNA applications have reached major milestones at high speed in the immuno-oncology field. This can be attributed to the knowledge that mRNA is one of nature's core building blocks carrying important information and can be considered as a powerful vector for delivery of therapeutic proteins to the patient.

The Opposing Effect of Type I IFN on the T Cell Response by Non-modified mRNA-Lipoplex Vaccines Is Determined by the Route of Administration.

mRNA-lipoplex vaccines are currently being explored in phase II clinical trials for the treatment of patients with advanced solid tumors. Mechanistically, these mRNA-lipoplex vaccines are characterized by the induction of type I interferon (IFN) centered innate responses. Earlier studies have identified type I IFNs as major regulators of the T cell response instigated by mRNA-lipoplex vaccines.

Specific targeting of IL-1β activity to CD8 T cells allows for safe use as a vaccine adjuvant.

Annual administration and reformulation of influenza vaccines is required for protection against seasonal infections. However, the induction of strong and long-lasting T cells is critical to reach broad and potentially lifelong antiviral immunity. The NLRP3 inflammasome and its product interleukin-1β (IL-1β) are pivotal mediators of cellular immune responses to influenza, yet, overactivation of these systems leads to side effects, which hamper clinical applications.

Safe eradication of large established tumors using neovasculature-targeted tumor necrosis factor-based therapies.

Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo.

A novel leptin receptor antagonist uncouples leptin's metabolic and immune functions.

Leptin links body energy stores to high energy demanding processes like reproduction and immunity. Based on leptin's role in autoimmune diseases and cancer, several leptin and leptin receptor (LR) antagonists have been developed, but these intrinsically lead to unwanted weight gain. Here, we report on the uncoupling of leptin's metabolic and immune functions based on the cross talk with the epidermal growth factor receptor (EGFR).

Targeting interferon activity to dendritic cells enables in vivo tolerization and protection against EAE in mice.

Type I Interferon (IFN) is widely used for multiple sclerosis (MS) treatment, but its side effects are limiting and its mechanism of action still unknown. Furthermore, 30-50% of MS patients are unresponsive, and IFN can even induce relapses. Fundamental understanding of the cellular target(s) of IFN will help to optimize treatments by reducing side effects and separating beneficial from detrimental effects.

Treatment with mRNA coding for the necroptosis mediator MLKL induces antitumor immunity directed against neo-epitopes.

Cancer immunotherapy can induce durable antitumor responses. However, many patients poorly respond to such therapies. Here we describe a generic antitumor therapy that is based on the intratumor delivery of mRNA that codes for the necroptosis executioner mixed lineage kinase domain-like (MLKL) protein.

Delivering Type I Interferon to Dendritic Cells Empowers Tumor Eradication and Immune Combination Treatments.

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects.

Type I Interferons Interfere with the Capacity of mRNA Lipoplex Vaccines to Elicit Cytolytic T Cell Responses.

Given their high potential to evoke cytolytic T cell responses, tumor antigen-encoding messenger RNA (mRNA) vaccines are now being intensively explored as therapeutic cancer vaccines. mRNA vaccines clearly benefit from wrapping the mRNA into nano-sized carriers such as lipoplexes that protect the mRNA from degradation and increase its uptake by dendritic cells in vivo. Nevertheless, the early innate host factors that regulate the induction of cytolytic T cells to mRNA lipoplex vaccines have remained unresolved.

Intratumoral Delivery of TriMix mRNA Results in T-cell Activation by Cross-Presenting Dendritic Cells.

Modulating the activity of tumor-infiltrating dendritic cells (TiDC) provides opportunities for novel cancer interventions. In this article, we report on our study of the uptake of mRNA by CD8α(+) cross-presenting TiDCs upon its intratumoral (i.t.

Intratumoral delivery of mRNA: Overcoming obstacles for effective immunotherapy.

The immunosuppressive tumor microenvironment (TME) is a major obstacle in cancer immunotherapy. Therefore, it has gained attention as a target site. mRNA emerged as a versatile drug class for cancer therapy.

Targeting the tumor microenvironment to enhance antitumor immune responses.

The identification of tumor-specific antigens and the immune responses directed against them has instigated the development of therapies to enhance antitumor immune responses. Most of these cancer immunotherapies are administered systemically rather than directly to tumors. Nonetheless, numerous studies have demonstrated that intratumoral therapy is an attractive approach, both for immunization and immunomodulation purposes.

The ReNAissanCe of mRNA-based cancer therapy.

About 25 years ago, mRNA became a tool of interest in anticancer vaccination approaches. However, due to its rapid degradation in situ, direct application of mRNA was confronted with considerable skepticism during its early use. Consequently, mRNA was for a long time mainly used for the ex vivo transfection of dendritic cells, professional antigen-presenting cells known to stimulate immunity.

The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy.

Dendritic cell (DC)-based cancer vaccines, where the patient's own immune system is harnessed to target and destroy tumor tissue, have emerged as a potent therapeutic strategy. In the development of such DC vaccines, it is crucial to load the DCs with tumor antigens, and to simultaneously activate them to become more potent antigen-presenting cells. For this, we report on microbubbles, loaded with both antigen mRNA as well as immunomodulating TriMix mRNA, which can be used for the ultrasound-triggered transfection of DCs.

Optimized dendritic cell-based immunotherapy for melanoma: the TriMix-formula.

Since decades, the main goal of tumor immunologists has been to increase the capacity of the immune system to mediate tumor regression. In this regard, one of the major focuses of cancer immunotherapy has been the design of vaccines promoting strong tumor-specific cytotoxic T lymphocyte responses in cancer patients. Here, dendritic cells (DCs) play a pivotal role as they are regarded as nature's adjuvant and as such have become the natural agents for antigen delivery in order to finally elicit strong T cell responses (Villadangos and Schnorrer in Nat Rev Immunol 7:543-555, 2007; Melief in Immunity 29:372-383, 2008; Palucka and Banchereau in Nat Rev Cancer 12:265-277, 2012; Vacchelli et al.